Novel capping enzyme

Abstract

The present invention relates to an RNA capping enzyme comprising or consisting of an amino acid sequence having high identity with any of the sequences of SEQ ID NO: 1-28, which can be expressed in vitro, in human cells, in mouse cells or in yeast cells, and which has the activities of RNA 5 '-triphosphatase (RT), guanylate transferase (GT) and RNA guine-N7 methyltransferase (RNMT) at the same time. The invention also relates to a mutant obtained by carrying out substitution, addition and / or deletion of amino acid on any sequence in SEQ ID NO: 1-28, a nucleotide sequence for coding the capping enzyme and / or the mutant thereof, and an expression construct or a host cell containing the nucleotide sequence. The invention also relates to a composition comprising the capping enzyme as well as a method and a reaction system for capping RNA by using the capping enzyme, a mutant or a composition thereof.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering. The present invention specifically relates to an RNA capping enzyme and a mutant thereof, a nucleic acid sequence encoding the capping enzyme and / or its mutant, an expression construct or a host cell comprising the nucleic acid sequence. The present invention also relates to a composition comprising the capping enzyme and a method and a reaction system for capping RNA using the capping enzyme, its mutant or its composition. Background Art

[0002] After transcription in eukaryotic organisms, mRNA needs to be capped at the 5' end. The cap structure is mainly an inverted N7-methylated guanosine added to the RNA head, which is connected to the first nucleotide of RNA through a triphosphate bond. The cap structure plays an important role in the stability, nuclear export and translation of mRNA, and can reduce its immunogenicity in cells. In mRNA therapy and mRNA vaccine technology, capping of exogenous mRNA is very important for its effective expression in mammalian cells.

[0003] Capping enzyme is an effective enzyme that can catalyze the formation of a cap structure, thereby improving the capping effect of mRNA. Capping enzyme plays a very important role in the preparation of mRNA vaccines, and can improve the effectiveness and safety of vaccines. At present, the main capping enzyme used in industrial production processes such as mRNA vaccines is the Vaccinia Capping Enzyme, but it is composed of two subunits, D1 and D12, with low expression and purification efficiency and high price, which limits its application in the industrial production of mRNA vaccines. At the same time, most of the previous patents were optimized for poxvirus capping enzymes, such as mutations or the establishment of fusion proteins, and the sequence diversity was relatively single. To this end, exploring and mining more capping enzymes with higher capping efficiency, compact and complete functions, and easier scale and industrial production is of great significance for mRNA vaccines, gene therapy, etc.

[0004] In order to successfully express in host cells, many viruses have their own RNA 5' triphosphatase (RT), guanylate transferase (GT) and RNA guanine-N7 methyltransferase (RNMT). These enzymes are different in source, mechanism of action and sequence, and their activities are also uneven. Therefore, it is urgent to obtain a variety of capping enzymes from natural sequences in this field, and to obtain capping enzymes with higher capping activity, more stable capping effect, higher capping rate, and more conducive to large-scale and industrial production through mutation and screening. For this reason, the technical solution of the present invention is proposed. Summary of the invention

[0005] Specifically, the present invention solves the technical problems existing in the prior art through the following technical solutions:

[0006] 1. A capping enzyme, comprising or consisting of:

[0007] (i) an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 1-28; or

[0008] (2) An amino acid sequence encoded by a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the nucleotide sequence shown in any one of SEQ ID NOs: 29-56.

[0009] 2. The capping enzyme according to item 1, which is expressed in (a) in vitro, (b) in human cells, (c) in mouse cells or (d) in yeast cells, and has the activities of RNA 5' triphosphatase (RT), guanylate transferase (GT) and RNA guanine-N7 methyltransferase (RNMT) at the same time.

[0010] 3. The capping enzyme according to item 1 or 2, wherein the capping enzyme comprises amino acid substitution, addition and / or deletion of any one of SEQ ID NOs: 1-28 or the capping enzyme is a mutant obtained by amino acid substitution, addition and / or deletion of any one of SEQ ID NOs: 1-28.

[0011] 4. The capping enzyme according to any one of items 1 to 3, comprising a deletion of any one of SEQ ID NOs: 1-28 or being a mutant obtained by deleting any one of SEQ ID NOs: 1-28, wherein the deletion is an N-terminal truncation and / or a C-terminal truncation.

[0012] 5. The capping enzyme according to item 3 or 4, wherein the deletion is a deletion of residues 1-6 of SEQ ID NO: 4, a deletion of residues 1-24 of SEQ ID NO: 7, or a deletion of residues 813-1003 of SEQ ID NO: 5.

[0013] 6. The capping enzyme according to any one of items 1 to 5, comprising a substitution of any one of SEQ ID NOs: 1-28 or being a mutant obtained by substituting any one of SEQ ID NOs: 1-28, wherein the substitution is selected from the group consisting of motif shuffling and site-directed mutagenesis.

[0014] 7. The capping enzyme according to item 6, wherein the motif rearrangement is to rearrange the motifs in SEQ ID NOs: 1-3, 7-9, 12-13, 16 and 21.

[0015] 8. The capping enzyme according to item 6 or 7, wherein the motif rearrangement is to rearrange the following motifs:

[0016] (a) a motif selected from the group consisting of amino acids 37-41, 108-110, 355-361, 443-445, 495-497, 532-541, 649-653, and 677-684 in SEQ ID NO: 2;

[0017] (b) a motif in SEQ ID NO:8 selected from the group consisting of amino acids 37-41, 108-110, 355-361, 443-445, 495-497, 532-541, 650-654, and 678-685;

[0018] (c) a motif selected from the group consisting of amino acids 37-41, 109-111, 356-362, 444-446, 496-498, 533-542, 651-655, and 679-686 in SEQ ID NO:9;

[0019] (d) a motif selected from the group consisting of amino acids 62-66, 133-135, 380-386, 468-470, 520-522, 557-566, 675-679, and 703-710 in SEQ ID NO:7;

[0020] (e) a motif selected from the group consisting of amino acids 38-42, 110-112, 357-363, 445-447, 497-499, 534-543, 651-655, and 679-686 in SEQ ID NO: 1;

[0021] (f) a motif selected from the group consisting of amino acids 38-42, 109-111, 356-362, 444-446, 499-501, 536-545, 653-657, and 681-688 in SEQ ID NO:3;

[0022] (g) a motif in SEQ ID NO:16 selected from the group consisting of amino acids 63-71, 263-269, 452-459, and 759-763;

[0023] (h) a motif selected from the group consisting of amino acids 63-71, 263-269, 452-459, and 759-763 of SEQ ID NO: 12;

[0024] (i) a motif selected from the group consisting of amino acids 63-71, 263-269, 452-459, and 759-763 in SEQ ID NO: 13; or

[0025] (j) a motif in SEQ ID NO:21 selected from the group consisting of amino acids 63-71, 263-269, 452-459 and 759-763.

[0026] 9. The capping enzyme according to any one of items 6-8, wherein the motif is rearranged as follows:

[0027] (a) performing a motif rearrangement on SEQ ID NO:2 selected from the group consisting of:

[0028] Replace amino acids 37-41 with DRHQR, ERHQN, GKHKS or ERQQV,

[0029] Replace amino acids 108-110 with ERS, ERN or DRN,

[0030] Replace amino acids 355-361 with QEAKVIA, QSAHIIS or EELGIVS,

[0031] Replace amino acids 443-445 with AHQ, AEQ, ENL or AEL,

[0032] Replace amino acids 495-497 with ENS or EHQ,

[0033] Replace amino acids 532-541 with IKTVLSVWGD, VKTVLSVWAN, STTVVSVWND or MTTVLSVWGN,

[0034] Substitution of amino acids 649-653 with VDMAS, VDFVS, MDAAS, VDAST, or VDLSS, and

[0035] Amino acids 677-684 are replaced with SLSRVGAL, SLSKNGAM, ALLETGSM, LLLEKEAV or SLSKQGSL;

[0036] (b) performing a motif rearrangement on SEQ ID NO:8 selected from the group consisting of:

[0037] Replace amino acids 37-41 with DRHQR, DRHQK, ERHQN or ERQQV,

[0038] Replace amino acids 108-110 with ERS, DRS or ERN,

[0039] Replace amino acids 355-361 with QEAKVIA, QEARVIA or EELGIVS,

[0040] Replace amino acids 443-445 with AHQ, ADQ, AEQ or AEL,

[0041] Replace amino acids 495-497 with SHP or EHQ,

[0042] Replace amino acids 532-541 with IKTVLSVWGD, VKTVLSVWGN, VKTVLSVWAN or MTTVLSVWGN,

[0043] Replace amino acids 650-654 with VDMAS, VDFSS, VDFVS, MDAAS or VDLSS,

[0044] Amino acids 678-685 are replaced with SLSRVGAL, SMSSDKAM, SLSKNGAM, ALLETGSM or SLSKQGSL;

[0045] (c) rearranging SEQ ID NO:9 by a motif selected from the group consisting of:

[0046] Replace amino acids 37-41 with DRHQR, DRHQK, ERHQN or GKHKS,

[0047] Replace amino acids 109-111 with ERS, DRS or DRN,

[0048] Replace amino acids 356-362 with QEAKVIA, QEARVIA or QSAHIIS,

[0049] Replace amino acids 444-446 with AHQ, ADQ, AEQ or ENL,

[0050] Replace amino acids 496-498 with SHP or ENS,

[0051] Replace amino acids 533-542 with IKTVLSVWGD, VKTVLSVWGN, VKTVLSVWAN or STTVVSVWND,

[0052] Replace amino acids 651-655 with VDMAS, VDFSS, VDFVS, MDAAS or VDAST,

[0053] Amino acids 679-686 are replaced with SLSRVGAL, SMSSDKAM, SLSKNGAM, ALLETGSM or LLLEKEAV;

[0054] (d) performing a motif rearrangement on SEQ ID NO:7 selected from the group consisting of:

[0055] Replace amino acids 62-66 with DRHQR, DRHQK, ERHQN or ERQQV,

[0056] Replace amino acids 133-135 with ERS, DRS or ERN,

[0057] Replace amino acids 380-386 with QEAKVIA, QEARVIA or EELGIVS,

[0058] Replace amino acids 468-470 with AHQ, ADQ, AEQ or AEL,

[0059] Replace amino acids 520-522 with SHP or EHQ,

[0060] Replace amino acids 557-566 with IKTVLSVWGD, VKTVLSVWGN, VKTVLSVWAN or MTTVLSVWGN,

[0061] Replace amino acids 675-679 with VDMAS, VDFSS, VDFVS, VDAST or VDLSS,

[0062] Amino acids 703-710 are replaced with SLSRVGAL, SMSSDKAM, SLSKNGAM, LLLEKEAV or SLSKQGSL;

[0063] (e) rearranging SEQ ID NO:1 by a motif selected from the group consisting of:

[0064] Replace amino acids 38-42 with DRHQK, ERHQN, GKHKS or ERQQV,

[0065] Replace amino acids 110-112 with DRS, ERN or DRN,

[0066] Replace amino acids 357-363 with QEARVIA, QSAHIIS or EELGIVS,

[0067] Replace amino acids 445-447 with ADQ, AEQ, ENL or AEL,

[0068] Replace amino acids 497-499 with ENS or EHQ,

[0069] Replace amino acids 534-543 with VKTVLSVWGN, VKTVLSVWAN, STTVVSVWND or MTTVLSVWGN,

[0070] Replace amino acids 651-655 with VDFSS, VDFVS, MDAAS, VDAST or VDLSS,

[0071] Amino acids 679-686 were replaced with SMSSDKAM, SLSKNGAM, ALLETGSM, LLLEKEAV or SLSKQGSL;

[0072] (f) rearranging SEQ ID NO:3 by a motif selected from the group consisting of:

[0073] Replace amino acids 38-42 with DRHQR, DRHQK, GKHKS or ERQQV,

[0074] Replace amino acids 109-111 with ERS, DRS or DRN,

[0075] Replace amino acids 356-362 with QEAKVIA, QSAHIIS or EELGIVS,

[0076] Replace amino acids 444-446 with AHQ, ADQ, ENL or AEL,

[0077] Replace amino acids 499-501 with ENS or EHQ,

[0078] Replace amino acids 536-545 with IKTVLSVWGD, VKTVLSVWGN, STTVVSVWND or MTTVLSVWGN,

[0079] Replace amino acids 653-657 with VDMAS, VDFSS, MDAAS, VDAST or VDLSS,

[0080] Amino acids 681-688 are replaced with SLSRVGAL, SMSSDKAM, ALLETGSM, LLLEKEAV or SLSKQGSL;

[0081] (g) performing a motif rearrangement on SEQ ID NO: 16 selected from the group consisting of:

[0082] Replace amino acids 63-71 with AIEIQSLEE, AIALQSLEE, AIEIQKIDE, AVEIQSPDE, AVEVQGIEE, AVQVDNDAE or SLIAINKDK,

[0083] Amino acids 263-269 are replaced with MNVITTI, LNVLTAI, LNVVTTI, LNVITTI, LNVITTV or CNVLSTI,

[0084] Amino acids 452-459 are replaced with TSMSDYIV, VLNDSYIV, TLKNEYVI, TLPNDYIV, KMKNEYIV, PVNNSYIV or PTN-KFVV,

[0085] Amino acids 759-763 are replaced with SDGIY, SDEVY, SDGIH, TDDVY or TDSIY;

[0086] (h) rearranging SEQ ID NO:12 by a motif selected from the group consisting of:

[0087] Replace amino acids 63-71 with AIALQSLEE, AVEIQEMDE, AIEIQKIDE, AVEIQSPDE, AVEVQGIEE, AVQVDNDAE or SLIAINKDK,

[0088] Amino acids 263-269 are replaced with LNVLTAI, LNVLTTL, LNVVTTI, LNVITTI, LNVITTV or CNVLSTI,

[0089] Amino acids 452-459 are replaced with VLNDSYIV, TLRNEYVV, TLKNEYVI, TLPNDYIV, KMKNEYIV, PVNNSYIV or PTN-KFVV,

[0090] Amino acids 759-763 are replaced with SDEIY, SDEVY, SDGIH, TDDVY or TDSIY;

[0091] (i) rearranging SEQ ID NO: 13 by a motif selected from the group consisting of:

[0092] Replace amino acids 63-71 with AIEIQSLEE, AIALQSLEE, AVEIQEMDE, AIEIQKIDE, AVEVQGIEE, AVQVDNDAE or SLIAINKDK,

[0093] Amino acids 263-269 are replaced with MNVITTI, LNVLTAI, LNVLTTL, LNVITTI, LNVITTV or CNVLSTI,

[0094] Amino acids 452-459 are replaced with TSMSDYIV, VLNDSYIV, TLRNEYVV, TLKNEYVI, KMKNEYIV, PVNNSYIV or PTN-KFVV,

[0095] Amino acids 759-763 are replaced with SDGIY, SDEIY, SDEVY, TDDVY or TDSIY; or

[0096] (j) rearranging SEQ ID NO:21 by a motif selected from the group consisting of:

[0097] Replace amino acids 63-71 with AIEIQSLEE, AIALQSLEE, AVEIQEMDE, AVEIQSPDE, AVEVQGIEE, AVQVDNDAE or SLIAINKDK,

[0098] Amino acids 263-269 are replaced with MNVITTI, LNVLTAI, LNVVTTI, LNVITTI, LNVITTV or CNVLSTI,

[0099] Amino acids 452-459 are replaced with TSMSDYIV, VLNDSYIV, TLRNEYVV, TLPNDYIV, KMKNEYIV, PVNNSYIV or PTN-KFVV,

[0100] Amino acids 759-763 are replaced with SDGIY, SDEIY, SDGIH, TDDVY or TDSIY.

[0101] 10. The capping enzyme according to item 6, wherein the site-directed mutagenesis is performed on the sequences of SEQ ID NOs: 1-4, 6-9 and 12.

[0102] 11. The capping enzyme according to item 6 or 10, wherein the site-directed mutagenesis is to mutate the following sites:

[0103] (a) a position selected from the group consisting of amino acids 21, 23, 37, 45, 100, 137, 139, 164, 180, 192, 235, 242, 255, 264, 303, 322, 354, 372, 397, 413, 417, 457, 493, 514, 567, 570, 574, 600, 603, 610, 628, 643, 653, 654, 675, 684, 710, and 759 of SEQ ID NO: 1;

[0104] (b) a position selected from the group consisting of amino acids 20, 22, 36, 44, 98, 135, 137, 162, 178, 190, 233, 240, 253, 262, 301, 320, 352, 370, 395, 411, 415, 455, 491, 512, 565, 568, 572, 598, 601, 608, 626, 641, 651, 652, 673, 682, 708, and 757 of SEQ ID NO:2;

[0105] (c) a position selected from the group consisting of amino acids 21, 23, 37, 45, 99, 136, 138, 163, 179, 191, 234, 241, 254, 263, 302, 321, 353, 371, 396, 412, 416, 456, 495, 516, 569, 572, 576, 602, 605, 612, 630, 645, 655, 656, 677, 686, 712, and 761 of SEQ ID NO:3;

[0106] (d) a position selected from the group consisting of amino acids 45, 47, 61, 69, 123, 160, 162, 187, 203, 215, 258, 265, 278, 287, 326, 345, 377, 395, 420, 436, 440, 480, 516, 537, 590, 593, 597, 624, 627, 634, 652, 667, 677, 678, 699, 708, 734, and 782 of SEQ ID NO:7;

[0107] (e) a position selected from the group consisting of amino acids 20, 22, 36, 44, 98, 135, 137, 162, 178, 190, 233, 240, 253, 262, 301, 320, 352, 370, 395, 411, 415, 455, 491, 512, 565, 568, 572, 599, 602, 609, 627, 642, 652, 653, 674, 683, 709, and 757 of SEQ ID NO:8;

[0108] (f) a position selected from the group consisting of amino acids 20, 22, 36, 44, 99, 136, 138, 163, 179, 191, 234, 241, 254, 263, 302, 321, 353, 371, 396, 412, 416, 456, 492, 513, 566, 569, 573, 599, 602, 609, 627, 642, 653, 654, 675, 684, 710, and 759 of SEQ ID NO:9;

[0109] (g) a position selected from the group consisting of 9, 11, 13, 19, 23, 24, 40, 46, 47, 50, 51, 52, 63, 67, 70, 71, 77, 78, 83, 88, 98, 99, 111, 113, 115, 116, 119, 123, 129, 130, 147, 155, 161, 162, 166, 167, 171, 172, 174, 175, 182, 183, 184, 191, 203, 205, 206, 207, 219, 222, 224, 226, 229, 230, 231 1, 234, 236, 237, 238, 240, 242, 249, 250, 251, 263, 268, 275, 276, 279, 281, 294, 297, 298, 299, 300, 302, 304, 311, 316, 320, 321, 324, 328, 332, 333, 334, 346, 347, 348, 351, 355, 356, 358, 360, 362, 363, 369, 370, 373, 374, 382, ​​394, 395, 396, 400, 404, 406, 409, 413, 414, 417, 420, 421, 426, 430, 432, 437, 447, 453, 455, 463, 468, 477, 478, 482, 495, 496, 497, 498, 501, 504, 505, 509, 515, 520, 526, 528, 530, 532, 533, 536, 542, 550, 551, 552, 553, 554, 555, 556, 558, 559, 564, 567, 581, 585, 596, 597, 602, 608, 62 amino acids 5, 629, 633, 643, 650, 653, 660, 663, 664, 670, 674, 676, 680, 682, 686, 688, 690, 702, 707, 713, 715, 716, 721, 729, 730, 733, 734, 739, 744, 745, 746, 749, 751, 763, 768, 770, 773, 779, 780, 785, 787, 802, 804, 810, 811, 819, 821, 827, 829, 830, 834, and 835;

[0110] (h) a position selected from the group consisting of amino acids 12, 17, 20, 35, 37, 77, 118, 132, 158, 159, 178, 179, 188, 194, 195, 200, 212, 223, 280, 309, 314, 324, 343, 441, 485, 534, 558, 594, 599, 624, 654, 656, 657, 658, 659, 682, 683, 697, 700, 733, 761, 769, 774, 777, 798, and 821 of SEQ ID NO:6; or

[0111] (i) a position in SEQ ID NO:4 selected from the group consisting of 250, 259, 281, 285, 322, 337, 367, 368, 382, ​​383, 386, 393, 395, 409, 410, 415, 435, 444, 451, 455, 458, 472, 479, 481, 487, 491, 496, 513, 544, 545, 550, 569, 576, 583, 585, 597, 604, 609, 614, 627, 631, 632, 642, 659, 66 1, 673, 676, 683, 697, 727, 729, 746, 748, 749, 767, 777, 791, 794, 805, 822, 826, 829, 834, 850, 870, 904, 905, 906, 919, 920, 921, 923, 925, 927, 944, 948, 956, 960, 962, 970, 1002, 1006, 1007, 1008, 1011, 1020, 1027, 1040 and 1045 amino acids.

[0112] 12. The capping enzyme according to any one of items 6, 10-11, wherein the site-directed mutation is:

[0113] (a) performing a site-directed mutagenesis of SEQ ID NO: 1 selected from the group consisting of H21R / K, K23R / H, T37S / N / Q, P45C / G, A100V / I / L / M / F / Y / W, N137S / T / Q, K139R / H, T164S / N / Q, V180A / I / L / M / F / Y / W, P192C / G, R235H / K, Y242A / V / I / L / M / F / W, K255R / H, L264A / V / I / M / F / Y / W, Q303S / T / N, S322T / N / Q, N354S / T / Q, P372C / G, G3 97C / P, D413E, K417R / H, K457R / H, E493D, K514R / H, I567A / V / L / M / F / Y / W, Q570S / T / N, G574C / P, G600C / P, M603A / V / I / L / F / Y / W, N 610S / T / Q, S628T / N / Q, L643A / V / I / M / F / Y / W, M653A / V / I / L / F / Y / W, A654V / I / L / M / F / Y / W, E675D, G684C / P, S710T / N / Q and K759R / H;

[0114] (b) performing a site-directed mutagenesis of SEQ ID NO: 2 selected from the group consisting of Q20S / T / N, A22V / I / L / M / F / Y / W, T36S / N / Q, P44C / G, S98T / N / Q, N135S / T / Q, K137R / H, T162S / N / Q, A178V / I / L / M / F / Y / W, P190C / G, R233H / K, Y240A / V / I / L / M / F / W, D253E, L262A / V / I / M / F / Y / W, Q301S / T / N, S320T / N / Q, N352S / T / Q, S3 70T / N / Q, G395C / P, E411D, K415R / H, K455R / H, E491D, L512A / V / I / M / F / Y / W, K565R / H, Q568S / T / N, G572C / P, G598C / P, S601T / N / Q, N608S / T / Q, G626C / P, V641A / I / L / M / F / Y / W, F651A / V / I / L / M / Y / W, S652T / N / Q, N673S / T / Q, K682R / H, P708C / G and G757C / P;

[0115] (c) performing site-directed mutagenesis on SEQ ID NO: 3 selected from the group consisting of H21R / K, K23R / H, T37S / N / Q, S45T / N / Q, S99T / N / Q, N136S / T / Q, K138R / H, T163S / N / Q, T179S / N / Q, P191C / G, L234A / V / I / M / F / Y / W, Y241A / V / I / L / M / F / W, D254E, T263S / N / Q, A302V / I / L / M / F / Y / W, Q321S / T / N, H353R / K, P371C / G, G396C / P, K412R / H, E416D, K456R / H, E495D, L516A / V / I / M / F / Y / W, T569S / N / Q, R572H / K, G576C / P, K602R / H, M605A / V / I / L / F / Y / W, D 612E, N630S / T / Q, N645S / T / Q, F655A / V / I / L / M / Y / W, V656A / I / L / M / F / Y / W, E677D, G686C / P, A712V / I / L / M / F / Y / W and N761S / T / Q;

[0116] (d) performing a site-directed mutagenesis of SEQ ID NO:7 selected from the group consisting of H45R / K, K47R / H, T61S / N / Q, P69C / G, Q123S / T / N, N160S / T / Q, H162R / K, T187S / N / Q, V203A / I / L / M / F / Y / W, P215C / G, K258R / H, Y265A / V / I / L / M / F / W, K278R / H, V287A / I / L / M / F / Y / W, Q326S / T / N, Q345S / T / N, N377S / T / Q, P395C / G, G420C / P, E436D, K440R / H, K480R / H, E516D, K537R / H, V590A / I / L / M / F / Y / W, Q593S / T / N, G597C / P, G624C / P, L627A / V / I / M / F / Y / W, N63 4S / T / Q, N652S / T / Q, I667A / V / L / M / F / Y / W, A677V / I / L / M / F / Y / W, A678V / I / L / M / F / Y / W, E699D, G708C / P, S734T / N / Q and K782R / H;

[0117] (e) performing a site-directed mutagenesis of SEQ ID NO: 8 selected from the group consisting of H20R / K, K22R / H, A36V / I / L / M / F / Y / W, S44T / N / Q, G98C / P, G135C / P, Q137S / T / N, T162S / N / Q, V178A / I / L / M / F / Y / W, S190T / N / Q, Q233S / T / N, H240R / K, K253R / H, I262A / V / L / M / F / Y / W, Q301S / T / N, L320A / V / I / M / F / Y / W, N352S / T / Q, P370 C / G, E395D, E411D, K415R / H, K455R / H, S491T / N / Q, K512R / H, I565A / V / L / M / F / Y / W, Q568S / T / N, E572D, G599C / P, F602A / V / I / L / M / Y / W, N609S / T / Q, S627T / N / Q, I642A / V / L / M / F / Y / W, A652V / I / L / M / F / Y / W, S653T / N / Q, E674D, E683D, S709T / N / Q and K757R / H;

[0118] (f) performing a site-directed mutagenesis of SEQ ID NO:9 selected from the group consisting of Q20S / T / N, D22E, E36D, P44C / G, S99T / N / Q, D136E, V138A / I / L / M / F / Y / W, I163A / V / L / M / F / Y / W, V179A / I / L / M / F / Y / W, K191R / H, R234H / K, Q241S / T / N, K254R / H, L263A / V / I / M / F / Y / W, Q302S / T / N, K321R / H, H353R / K, S371T / N / Q, K396R / H, D412E, V416A / I / L / M / F / Y / W, K456R / H, K492R / H, K513R / H, I566A / V / L / M / F / Y / W, Q569S / T / N, T573S / N / Q, G599C / P, M602A / V / I / L / F / Y / W, N 609S / T / Q, K627R / H, I642A / V / L / M / F / Y / W, L653A / V / I / M / F / Y / W, S654T / N / Q, L675A / V / I / M / F / Y / W, G684C / P, S710T / N / Q and K759R / H;

[0119] (g) Perform site-directed mutagenesis on SEQ ID NO:12 selected from the following group: S9T / N / Q, A11V / I / L / M / F / Y / W, V13A / I / L / M / F / Y / W, V19A / I / L / M / F / Y / W, D23E, D24E, T40S / N / Q, F46A / V / I / L / M / Y / W, N47S / T / Q, V50A / I / L / M / F / Y / W, K51R / H, T52S / N / Q, A63V / I / L / M / F / Y / W, Q67S / T / N, E70D, E71D, R77H / K, C78G / P, D83E, V88A / I / L / M / F / Y / W, T98S / N / Q, N99S / T / Q, K111R / H, N113S / T / Q, E115D, E116D, L119A / V / I / M / F / Y / W, K123R / H, Y129A / V / I / L / M / F / W, N130S / T / Q, A147V / I / L / M / F / Y / W, I155A / V / L / M / F / Y / W, E161D, A162V / I / L / M / F / Y / W, Q166S / T / N, N167S / T / Q, K171R / H, K172R / H, K174R / H, E175D, P182C / G, I183A / V / L / M / F / Y / W, A184V / I / L / M / F / Y / W, F191A / V / I / L / M / Y / W, K194R / H, H203R / K, R205H / K, V206A / I / L / M / F / Y / W, I207A / V / L / M / F / Y / W, F219A / V / I / L / M / Y / W, F222A / V / I / L / M / Y / W, S224T / N / Q, R226H / K, S229T / N / Q, N230S / T / Q, G231C / P, L234A / V / I / M / F / Y / W, K236R / H, N237S / T / Q, T238S / N / Q, K240R / H, K242R / H, K249R / H, R250H / K, V251A / I / L / M / F / Y / W, M263A / V / I / L / F / Y / W, T268S / N / Q, L275A / V / I / M / F / Y / W, Y276A / V / I / L / M / F / W, K279R / H, I281A / V / L / M / F / Y / W, Y294A / V / I / L / M / F / W, R297H / K, Q298S / T / N, Y299A / V / I / L / M / F / W, S300T / N / Q, F302A / V / I / L / M / Y / W, F304A / V / I / L / M / Y / W, D311E, S316T / N / Q, K320R / H, E321D, F324A / V / I / L / M / Y / W,I328A / V / L / M / F / Y / W、K332R / H、D333E、N334S / T / Q、G346C / P、N347S / T / Q、V348A / I / L / M / F / Y / W、K351R / H、K355R / H、I356A / V / L / M / F / Y / W、E358D、Q360S / T / N、I362A / V / L / M / F / Y / W、N363S / T / Q、Y369A / V / I / L / M / F / W、K370R / H、A373V / I / L / M / F / Y / W、T374S / N / Q、V382A / I / L / M / F / Y / W、S394T / N / Q、A395V / I / L / M / F / Y / W、K396R / H、H400R / K、E404D、R406H / K、F409A / V / I / L / M / Y / W、L413A / V / I / M / F / Y / W、D414E、D417E、N420S / T / Q、V421A / I / L / M / F / Y / W、P426C / G、L430A / V / I / M / F / Y / W、S432T / N / Q、V437A / I / L / M / F / Y / W、P447C / G、S453T / N / Q、S455T / N / Q、A463V / I / L / M / F / Y / W、F468A / V / I / L / M / Y / W、L477A / V / I / M / F / Y / W、I478A / V / L / M / F / Y / W、K482R / H、K495R / H、D496E、E497D、P498C / G、G501C / P、N504S / T / Q、I505A / V / L / M / F / Y / W、T509S / N / Q、F515A / V / I / L / M / Y / W、A520V / I / L / M / F / Y / W、D526E、C528G / P、G530C / P、F532A / V / I / L / M / Y / W、G533C / P、I536A / V / L / M / F / Y / W、P542C / G、I550A / V / L / M / F / Y / W、L551A / V / I / M / F / Y / W、N552S / T / Q、G553C / P、554S / N / Q、D555E、K556R / H、Y558A / V / I / L / M / F / W、K559R / H、A564V / I / L / M / F / Y / W、T567S / N / Q、S581T / N / Q、Y585A / V / I / L / M / F / W、N596S / T / Q、N597S / T / Q、G602C / P、S608T / N / Q、S625T / N / Q、K629R / H、Y633A / V / I / L / M / F / W、E643D、H650R / K、I653A / V / L / M / F / Y / W、R660H / K、K663R / H、E664D、D670E、V674A / I / L / M / F / Y / W, R676H / K, E680D, E682D, N686S / T / Q, V688A / I / L / M / F / Y / W, S690T / N / Q, G702C / P, N707S / T / Q, D713E, K715R / H, V716A / I / L / M / F / Y / W, F721A / V / I / L / M / Y / W, N729S / T / Q, M730A / V / I / L / F / Y / W, D733E, V734A / I / L / M / F / Y / W, K739R / H, E744D, L745A / V / I / M / F / Y / W, F746A / V / I / L / M / Y / W, D749E, T751S / N / Q, Y763A / V / I / L / M / F / W, S768T / N / Q, V770A / I / L / M / F / Y / W, T773S / N / Q, N779S / T / Q, L780A / V / I / M / F / Y / W, Q785S / T / N, T787S / N / Q, F802A / V / I / L / M / Y / W, N804S / T / Q, I810A / V / L / M / F / Y / W, E811D, S819T / N / Q, Y821A / V / I / L / M / F / W, G827C / P, T829S / N / Q, M830A / V / I / L / F / Y / W, S834T / N / Q and E835D;

[0120] (h)Perform site-directed mutagenesis on SEQ ID NO:6 selected from the following group: N12S / T / Q, I17A / V / L / M / F / Y / W, S20T / N / Q, P35C / G, N37S / T / Q, N77S / T / Q, G118C / P, K132R / H, Q158S / T / N, R159H / K, A178V / I / L / M / F / Y / W, D179E, T188S / N / Q, T194S / N / Q, N195S / T / Q, V200A / I / L / M / F / Y / W, V212A / I / L / M / F / Y / W, F223A / V / I / L / M / Y / W, A280V / I / L / M / F / Y / W, H309R / K, A314V / I / L / M / F / Y / W, V324A / I / L / M / F / Y / W, Y343A / V / I / L / M / F / W, Y441A / V / I / L / M / F / W, Q485S / T / N, N534S / T / Q, H558R / K, G594C / P, D599E, H624R / K, G654C / P, R656H / K, N657S / T / Q, M658A / V / I / L / F / Y / W, H659R / K, E682D, N683S / T / Q, H697R / K, C700G / P, P733C / G, A761V / I / L / M / F / Y / W, V769A / I / L / M / F / Y / W, S774T / N / Q, P777C / G, E798D, M821A / V / I / L / F / Y / W; or

[0121] (i) performing site-directed mutagenesis on SEQ ID NO: 4 selected from the group consisting of A250V / I / L / M / F / Y / W, M259A / V / I / L / F / Y / W, G281C / P, E285D, Y322A / V / I / L / M / F / W, N337S / T / Q, P367C / G, S368T / N / Q, K382R / H, G383C / P, D386E, F393A / V / I / L / M / Y / W, E395D, V409A / I / L / M / F / Y / W, M 410A / V / I / L / F / Y / W, G415C / P, N435S / T / Q, F444A / V / I / L / M / Y / W, D451E, E455D, Y458A / V / I / L / M / F / W, A472V / I / L / M / F / Y / W, K479R / H, S481T / N / Q, K487R / H, D491E, Y496A / V / I / L / M / F / W, V513A / I / L / M / F / Y / W, K544R / H,D545E, L550A / V / I / M / F / Y / W, C569G / P, K576R / H, Q583S / T / N, T585S / N / Q, N597S / T / Q, E604D, D609E, N614S / T / Q, P627C / G, E631D, R632H / K, Y642A / V / I / L / M / F / W, I659A / V / L / M / F / Y / W, V661A / I / L / M / F / Y / W, I673A / V / L / M / F / Y / W, S676T / N / Q, K683R / H, K697R / H, A727V / I / L / M / F / Y / W, S729T / N / Q, C746G / P, P748C / G, M749A / V / I / L / F / Y / W, Q767S / T / N, V777A / I / L / M / F / Y / W, T791S / N / Q, I794A / V / L / M / F / Y / W, Q805S / T / N, G822C / P, N826 S / T / Q, D829E, L834A / V / I / M / F / Y / W, N850S / T / Q, L870A / V / I / M / F / Y / W, V904A / I / L / M / F / Y / W, E905D, L906A / V / I / M / F / Y / W, N919S / T / Q, D920E, K921R / H, D923E, K925R / H, L927A / V / I / M / F / Y / W, G944C / P, P948C / G, F956 A / V / I / L / M / Y / W, G960C / P, Y962A / V / I / L / M / F / W, K970R / H, F1002A / V / I / L / M / Y / W, A1006V / I / L / M / F / Y / W, Q1 007S / T / N, Y1008A / V / I / L / M / F / W, N1011S / T / Q, N1020S / T / Q, S1027T / N / Q, R1040H / K and Y1045A / V / I / L / M / F / W. ,

[0122] 13. A combination of capping enzymes, comprising at least two capping enzymes of different sequences according to any one of items 1 to 12.

[0123] 14. A nucleic acid sequence encoding the capping enzyme according to any one of items 1 to 12.

[0124] 15. A nucleic acid construct comprising the nucleic acid sequence described in item 14.

[0125] 16. The nucleic acid construct according to item 15, further comprising a resistance gene, wherein the resistance gene is selected from the group consisting of an ampicillin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, a spectinomycin resistance gene, a tetracycline resistance gene, a bleomycin resistance gene, a streptomycin resistance gene, a hygromycin resistance gene, a gentamicin resistance gene, an apramycin sulfate resistance gene, an erythromycin resistance gene, a nourseoinhibin resistance gene, a geneticin resistance gene, and a puromycin resistance gene.

[0126] 17. The nucleic acid construct according to item 15 or 16, which is obtained by introducing the nucleic acid sequence according to item 14 into a plasmid vector, wherein the plasmid vector is selected from the group consisting of a prokaryotic expression vector, a yeast expression vector, a plant expression vector, an insect expression vector and a mammalian expression vector.

[0127] 18. A recombinant host cell comprising the nucleic acid sequence of item 14 or the expression construct of any one of items 15-17.

[0128] 19. A recombinant host cell according to item 18, wherein the cell is prepared by introducing the nucleic acid sequence described in item 14 or the nucleic acid construct described in any one of items 14-16 into a host cell, preferably the host cell is Escherichia coli, a yeast cell or a mammalian cell, more preferably an Escherichia coli Top10 or DH5α cell, a Saccharomyces cerevisiae cell, a Chinese hamster ovary cell or a human embryonic kidney cell.

[0129] 20. A kit for capping RNA, comprising:

[0130] (i) the capping enzyme described in any one of items 1 to 12 or the combination described in item 13;

[0131] (ii) a storage buffer in which the capping enzyme or combination is located; and

[0132] (iii) a reaction buffer,

[0133] The storage buffer is the same as or different from the reaction buffer.

[0134] 21. The kit according to item 20, further comprising:

[0135] (iv) guanosine triphosphate (GTP) or modified GTP;

[0136] (v) a methyl donor; and

[0137] (vi) Optional target RNA to be capped.

[0138] 22. A kit according to item 20 or 21, wherein the storage buffer comprises or consists of: 20-100 mM Tris-HCl, 1-20 mM KCl, 0.5-5 mM MgCl2 and 0.5-10 mM dithiothreitol (DTT), preferably the storage buffer comprises or consists of: 50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2 and 1 mM DTT.

[0139] 23. A kit according to any one of items 20-22, wherein the reaction buffer comprises or consists of: 20-100 mM Tris-HCl, 1-20 mM KCl, 0.5-5 mM MgCl2 and 0.5-10 mM dithiothreitol (DTT), preferably the reaction buffer comprises or consists of: 50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2 and 1 mM DTT.

[0140] 24. A kit according to any one of items 21-23, wherein the modified GTP is selected from the group consisting of GTP modified on the base, GTP modified on the ribose and GTP modified on the α-phosphate group; preferably, the modified GTP is selected from the group consisting of GTP modified on the 2'-OH of ribose and GTP modified on the 3'-OH of ribose; more preferably, the modified GTP is selected from the group consisting of GTP modified by O-methylation on the 3'-OH of ribose, GTP modified by NH2 on the 3'-OH of ribose and dGTP modified by N3 on the 3'-OH of ribose.

[0141] 25. A kit according to any one of items 21-24, wherein the methyl donor is selected from the group consisting of choline, trimethylglycine, methionine, S-adenosylmethionine (SAM), methylfolate and methylcobalamin, preferably the methyl donor is SAM.

[0142] 26. A method for capping RNA in vitro, comprising:

[0143] (i) providing an uncapped RNA sample;

[0144] (ii) adding the capping enzyme of any one of items 1 to 12 or the combination of item 13, guanosine triphosphate (GTP) or modified GTP and a methyl donor to a reaction buffer to provide a capping reaction mixture;

[0145] (iii) reacting the RNA sample with the mixture at a temperature of 30° C. to 60° C. for 10 to 200 minutes to form a reaction product, i.e., capped RNA.

[0146] 27. A method according to item 26, wherein in step (i) the uncapped RNA sample is incubated at 50-80°C for 3-15 minutes, preferably at 55-70°C for 5-10 minutes, and then ice-bathed, preferably the ice-bath is carried out for 5-10 minutes.

[0147] 28. The method according to item 26 or 27, wherein in step (ii) the capping enzyme or combination is added to the reaction buffer in an amount of 0.2-2.0, preferably 0.5-1.0 U / μL.

[0148] 29. A method according to any one of items 26-28, wherein in step (ii) the GTP or modified GTP is added to the reaction buffer in an amount of 0.2-5 mM, preferably 0.5-1 mM.

[0149] 30. A method according to any one of items 26-29, wherein in step (ii) the methyl donor is added to the reaction buffer in an amount of 0.1-0.8 mM.

[0150] 31. A method according to any one of items 26-30, wherein in step (ii) the buffer comprises or consists of: 20-100 mM Tris-HCl, 1-20 mM KCl, 0.5-5 mM MgCl2 and 0.5-10 mM dithiothreitol (DTT), preferably the buffer comprises or consists of: 50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2 and 1 mM DTT.

[0151] 32. The method according to any one of items 26 to 31, wherein step (iii) is reacted at 30-42°C for 45-90 minutes.

[0152] 33. The method according to any one of items 26-32, further comprising:

[0153] (iv) purifying the reaction product and / or storing the reaction product or the purified reaction product at -80°C.

[0154] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below in conjunction with specific implementation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0155] Figure 1 is the relative activity of the capping enzyme of the present invention (293T cells);

[0156] Figure 2 is the relative activity of the capping enzyme of the present invention (CHO cells);

[0157] Figure 3is the relative activity of the capping enzyme of the present invention (yeast cells);

[0158] Figure 4A -B is a comparison of the relative activity of the capping enzyme of the present invention in different species, wherein Figure 4A For the comparison of the activity in yeast and 293T cells, Figure 4B Comparison of the activities in yeast and CHO cells. The letters in the legend represent the same type of capping enzymes of the present invention, for example, MRV represents MRV_1-6, FTV represents FTV_1-2, PTV represents PTV_1 / 4 / 6-7, MMV represents MMV_3-4, REV represents REV_3-4 / 6 / 8-14 / 16, ASFV represents ASFV_1, PCV represents PCV_1 / 3, in addition, NP868R represents the positive control (the most active capping enzyme in the prior art), and NO_CE represents the negative control without capping enzyme;

[0159] Figure 5 is the relative abundance of the cap at position 0 (Cap0) after the capping enzyme reaction of the present invention (the two sequences of the present invention vs. the commercially available capping enzyme); and

[0160] Figure 6 is the relative abundance of Cap0 after the capping enzyme reaction of the present invention (five sequences of the present invention vs. commercially available capping enzyme). DETAILED DESCRIPTION OF THE INVENTION

[0162] 1. Definition

[0163] Capping enzyme: mRNA in eukaryotic organisms is modified after transcription to form a special structure at the 5' end, namely the cap structure, which plays an important role in the stability, transport and translation of mRNA, and can reduce its immunogenicity in cells. Capping enzyme is an effective enzyme that catalyzes the formation of the cap structure. It has the activities of RNA 5' triphosphatase (TPase / RT), guanylate transferase (GTase / GT) and RNA guanine-N7 methyltransferase (MTase / RNMT), and can connect the 7-methylguanine cap structure (m7Gppp) to the 5' end of RNA.

[0164] Isolated or purified: The terms "isolated" or "purified" mean a polypeptide or polynucleotide that is removed from at least one component with which it is naturally associated. For example, a polypeptide can be at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure, or at least 95% pure, as determined by SDS-PAGE, while a polynucleotide can be at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure, or at least 95% pure, as determined by agarose electrophoresis.

[0165] Sequence identity: The parameter "sequence identity" describes the relatedness between two amino acid sequences or between two nucleotide sequences.

[0166] For the purposes of the present invention, the degree of sequence identity between two amino acid sequences can be determined using the Clustal Omega algorithm (Sievers F, et al. (2011). Molecular Systems Biology 7:539) as implemented in the online align tool (https: / / www.uniprot.org / align) of the UniProt website, preferably version 1.2.4 or higher. The optional parameter used is iteration 0. The percent identity is obtained using the Percent Identity Matrix output by the program, which is calculated as follows:

[0167] (same residues x 100) / (alignment length - total number of gaps in the alignment)

[0168] For the present invention, the degree of sequence identity between two nucleotide sequences can be determined using the Clustal Omega algorithm (Sievers F, et al. (2011). Molecular Systems Biology 7:539) as implemented in the online align tool (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) of the EBI website, preferably version 1.2.4 or higher. Default parameters are used. The percent identity is obtained using the Percent Identity Matrix output by the program, which is calculated as follows:

[0169] (same deoxyribonucleotides x 100) / (alignment length - total number of gaps in the alignment)

[0170] For the present invention, the sequence identity degree between two amino acid sequences can be measured using the Needle program as in the EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) performed in version 3.0.0 or higher. The optional parameters used are gap penalty (gap penalty) 10, gap extension penalty (gap extension penalty) 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. Use the output result (obtained using the -nobrief option) marked as "highest identity (longest identity)" using Needle as the identity percentage, and calculate as follows:

[0171] (same residues x 100) / (alignment length - total number of gaps in the alignment)

[0172] For the present invention, the sequence identity degree between two nucleotide sequences can be measured using the Needle program as in the EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite, Rice etc., 2000, see above), preferably the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, see above) performed in version 3.0.0 or higher. The optional parameters used are gap penalty 10, gap extension penalty 0.5 and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. Use the output result (obtained using the -nobrief option) marked as "highest identity" using Needle as identity percentage, and calculate as follows:

[0173] (same deoxyribonucleotides x 100) / (alignment length - total number of gaps in the alignment)

[0174] Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are usually determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG, and ends with a stop codon such as TAA, TAG and TGA. The coding sequence can be a DNA, cDNA, synthetic or recombinant polynucleotide.

[0175] Nucleic acid construct: The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or that is modified in a manner that does not otherwise exist in nature to contain a segment of nucleic acid, or that is synthetic. When the nucleic acid construct contains the regulatory sequences required for expression of the coding sequence of the present invention, the term nucleic acid construct is synonymous with the term "expression cassette".

[0176] Control sequence: The term "control sequence" is meant to include all components that are necessary or advantageous for the expression of a polynucleotide encoding a polypeptide. Each control sequence may be native or foreign to the polynucleotide encoding the polypeptide, or each control sequence may be native or foreign to each other. These control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, a control sequence includes a promoter and termination signals for transcription and translation. The control sequence may be provided together with a linker for introducing specific restriction sites that facilitate the connection of the control sequence to the coding region of the polynucleotide encoding the polypeptide.

[0177] Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

[0178] Expression: The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0179] Expression vector: The term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide operably linked to additional nucleotides that provide for its expression.

[0180] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. using a nucleic acid construct comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0181] 2. Capping enzymes of the present invention

[0182] In one aspect, the present invention provides a capping enzyme, which: (i) comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of SEQ ID NOs: 1-28, or consists of said amino acid sequence; or (2) comprises an amino acid sequence encoded by a nucleotide sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of SEQ ID NOs: 29-56, or consists of said amino acid sequence.

[0183] In one embodiment, the capping enzyme of the present invention can be expressed in vitro. In one embodiment, the capping enzyme of the present invention can be expressed in human cells. In one embodiment, the capping enzyme of the present invention can be expressed in mouse cells. In one embodiment, the capping enzyme of the present invention can be expressed in yeast cells. In one embodiment, the capping enzyme of the present invention has three activities of RNA 5' triphosphatase (RT), guanylate transferase (GT) and RNA guanine-N7 methyltransferase (RNMT) at the same time. In one embodiment, the capping enzyme of the present invention can cap RNA (preferably mRNA). In one embodiment, the capping enzyme of the present invention can cap RNA (preferably mRNA) at the 5' end. In one embodiment, the capping enzyme of the present invention can connect a 7-methylguanine cap structure (m7Gppp) to the 5' end of RNA (preferably mRNA).

[0184] In one embodiment, the capping enzyme of the present invention comprises amino acid substitution, addition and / or deletion of any of SEQ ID NOs: 1-28. In one embodiment, the capping enzyme of the present invention is a mutant obtained by amino acid substitution, addition and / or deletion of any of SEQ ID NOs: 1-28. In one embodiment, the capping enzyme of the present invention is a mutant obtained by amino acid substitution and / or deletion of any of SEQ ID NOs: 1-28.

[0185] In one embodiment, the capping enzyme of the present invention comprises a deletion of any of SEQ ID NOs: 1-28. In one embodiment, the capping enzyme of the present invention is a mutant obtained by deleting any of SEQ ID NOs: 1-28. In one embodiment, the deletion is an N-terminal truncation and / or a C-terminal truncation. In one embodiment, the deletion is a truncation of the N-terminus of SEQ ID NO: 4, preferably a deletion of residues 1-6 of SEQ ID NO: 4. In one embodiment, the deletion is a truncation of the N-terminus of SEQ ID NO: 7, preferably a deletion of residues 1-24 of SEQ ID NO: 7. In one embodiment, the deletion is a truncation of the C-terminus of SEQ ID NO: 5, preferably a deletion of residues 813-1003 of SEQ ID NO: 5.

[0186] In one embodiment, the capping enzyme of the present invention comprises a substitution of any sequence in SEQ ID NO: 1-28. In one embodiment, the capping enzyme of the present invention is a mutant obtained by substituting any sequence in SEQ ID NO: 1-28. In one embodiment, the substitution is selected from the group consisting of motif shuffling and site-directed mutagenesis.

[0187] In one embodiment, the motif rearrangement is to rearrange the motifs in SEQ ID NO:1-28. In one embodiment, the motif rearrangement is to rearrange the motifs in SEQ ID NO:1-3, 7-9, 12-13, 16 and 21. In one embodiment, the motif rearrangement is to rearrange the following motifs: (a) a motif selected from the group consisting of amino acids 37-41, 108-110, 355-361, 443-445, 495-497, 532-541, 649-653 and 677-684 in SEQ ID NO:2; (b) a motif selected from the group consisting of amino acids 37-41, 108-110, 355-361, 443-445, 495-497, 532-541, 650-654 and 678-685 in SEQ ID NO:8; (c) a motif selected from the group consisting of amino acids 37-41, 108-110, 355-361, 443-445, 495-497, 532-541, 650-654 and 678-685 in SEQ ID NO: (d) a motif selected from the group consisting of amino acids 37-41, 109-111, 356-362, 444-446, 496-498, 533-542, 651-655, and 679-686 in SEQ ID NO:7; (e) a motif selected from the group consisting of amino acids 38-42, 110-112, 357-363, 445-447, 497-499, 534-543, 651-655, and 679-686 in SEQ ID NO:9; (g) a motif selected from the group consisting of amino acids 63-71, 263-269, 452-459, and 759-763 in SEQ ID NO:16; (h) a motif selected from the group consisting of amino acids 63-71, 263-269, 452-459, and 759-763 in SEQ ID NO:12; (i) a motif selected from the group consisting of amino acids 63-71, 263-269, 452-459, and 759-763 in SEQ ID NO:13; or (j) a motif selected from the group consisting of amino acids 63-71, 263-269, 452-459, and 759-763 in SEQ ID NO:13. A motif in NO:21 selected from the group consisting of amino acids 63-71, 263-269, 452-459 and 759-763.

[0188] In one embodiment, the motif rearrangement is: (a) performing a motif rearrangement on SEQ ID NO: 2 selected from the group consisting of: replacing amino acids 37-41 with DRHQR, ERHQN, GKHKS or ERQQV, replacing amino acids 108-110 with ERS, ERN or DRN, replacing amino acids 355-361 with QEAKVIA, QSAHIIS or EELGIVS, replacing amino acids 443-445 with AHQ, AEQ, ENL or AEL, and replacing amino acids 495-497 with ENS or EHQ , amino acids 532-541 are replaced with IKTVLSVWGD, VKTVLSVWAN, STTVVSVWND or MTTVLSVWGN, amino acids 649-653 are replaced with VDMAS, VDFVS, MDAAS, VDAST or VDLSS, and amino acids 677-684 are replaced with SLSRVGAL, SLSKNGAM, ALLETGSM, LLLIKEAV or SLSKQGSL; (b) the amino acids 532-541 are replaced with IKTVLSVWGD, VKTVLSVWAN, STTVVSVWND or MTTVLSVWGN, amino acids 649-653 are replaced with VDMAS, VDFVS, MDAAS, VDAST or VDLSS, and amino acids 677-684 are replaced with SLSRVGAL, SLSKNGAM, ALLETGSM, LLLIKEAV or SLSKQGSL; (c) the amino acids 532-541 are replaced with IKTVLSVWGD, VKTVLSVWAN, STTVVSVWND or MTTVLSVWGN, amino acids 649-653 are replaced with VDMAS, VDFVS, MDAAS, VDAST or VDLSS, and amino acids 677-684 are replaced with SLSRVGAL, SLSKNGAM, ALLETGSM, LLLIKEAV or SLSKQGSL; NO:8 performs a motif rearrangement selected from the following group: replacing amino acids 37-41 with DRHQR, DRHQK, ERHQN or ERQQV, replacing amino acids 108-110 with ERS, DRS or ERN, replacing amino acids 355-361 with QEAKVIA, QEARVIA or EELGIVS, replacing amino acids 443-445 with AHQ, ADQ, AEQ or AEL, replacing amino acids 495-497 with SHP or EHQ, amino acids 532-541 are substituted with IKTVLSVWGD, VKTVLSVWGN, VKTVLSVWAN or MTTVLSVWGN, amino acids 650-654 are substituted with VDMAS, VDFSS, VDFVS, MDAAS or VDLSS, and amino acids 678-685 are substituted with SLSRVGAL, SMSSDKAM, SLSKNGAM, ALLETGSM or SLSKQGSL;(c) rearranging SEQ ID NO:9 by a motif selected from the group consisting of: replacing amino acids 37-41 with DRHQR, DRHQK, ERHQN or GKHKS, replacing amino acids 109-111 with ERS, DRS or DRN, replacing amino acids 356-362 with QEAKVIA, QEARVIA or QSAHIIS, replacing amino acids 444-446 with AHQ, ADQ, AEQ or ENL, and replacing amino acids 496-498 with SHP or ENS , amino acids 533-542 are replaced with IKTVLSVWGD, VKTVLSVWGN, VKTVLSVWAN or STTVVSVWND, amino acids 651-655 are replaced with VDMAS, VDFSS, VDFVS, MDAAS or VDAST, and amino acids 679-686 are replaced with SLSRVGAL, SMSSDKAM, SLSKNGAM, ALLETGSM or LLLEKEAV; (d) amino acids 533-542 are replaced with IKTVLSVWGD, VKTVLSVWGN, VKTVLSVWAN or STTVVSVWND, amino acids 651-655 are replaced with VDMAS, VDFSS, VDFVS, MDAAS or VDAST, and amino acids 679-686 are replaced with SLSRVGAL, SMSSDKAM, SLSKNGAM, ALLETGSM or LLLEKEAV; NO:7 performs a motif rearrangement selected from the following group: replacing amino acids 62-66 with DRHQR, DRHQK, ERHQN or ERQQV, replacing amino acids 133-135 with ERS, DRS or ERN, replacing amino acids 380-386 with QEAKVIA, QEARVIA or EELGIVS, replacing amino acids 468-470 with AHQ, ADQ, AEQ or AEL, replacing amino acids 520-522 with SHP or EHQ, amino acids 557-566 are substituted with IKTVLSVWGD, VKTVLSVWGN, VKTVLSVWAN, or MTTVLSVWGN, amino acids 675-679 are substituted with VDMAS, VDFSS, VDFVS, VDAST, or VDLSS, and amino acids 703-710 are substituted with SLSRVGAL, SMSSDKAM, SLSKNGAM, LLLEKEAV, or SLSKQGSL;(e) rearranging SEQ ID NO:1 by a motif selected from the group consisting of: replacing amino acids at positions 38-42 with DRHQK, ERHQN, GKHKS or ERQQV, replacing amino acids at positions 110-112 with DRS, ERN or DRN, replacing amino acids at positions 357-363 with QEARVIA, QSAHIIS or EELGIVS, replacing amino acids at positions 445-447 with ADQ, AEQ, ENL or AEL, and replacing amino acids at positions 497-499 with ENS or EHQ , replacing amino acids at positions 534-543 with VKTVLSVWGN, VKTVLSVWAN, STTVVSVWND or MTTVLSVWGN, replacing amino acids at positions 651-655 with VDFSS, VDFVS, MDAAS, VDAST or VDLSS, and replacing amino acids at positions 679-686 with SMSSDKAM, SLSKNGAM, ALLETGSM, LLLEKEAV or SLSKQGSL; (f) modifying SEQ ID NO:3 performs a motif rearrangement selected from the following group: replacing amino acids 38-42 with DRHQR, DRHQK, GKHKS or ERQQV, replacing amino acids 109-111 with ERS, DRS or DRN, replacing amino acids 356-362 with QEAKVIA, QSAHIIS or EELGIVS, replacing amino acids 444-446 with AHQ, ADQ, ENL or AEL, replacing amino acids 499-501 with ENS or EHQ, Amino acids 536-545 are replaced with IKTVLSVWGD, VKTVLSVWGN, STTVVSVWND or MTTVLSVWGN, amino acids 653-657 are replaced with VDMAS, VDFSS, MDAAS, VDAST or VDLSS, and amino acids 681-688 are replaced with SLSRVGAL, SMSSDKAM, ALLETGSM, LLLEKEAV or SLSKQGSL; (g) SEQ ID NO:16 performs a motif rearrangement selected from the following group: replacing amino acids 63-71 with AIEIQSLEE, AIALQSLEE, AIEIQKIDE, AVEIQSPDE, AVEVQGIEE, AVQVDNDAE or SLIAINKDK, replacing amino acids 263-269 with MNVITTI, LNVLTAI, LNVVTTI, LNVITTI, LNVITTV or CNVLSTI, replacing amino acids 452-459 with TSMSDYIV, VLNDSYIV, TLKNEYVI, TLPNDYIV, KMKNEYIV, PVNNSYIV or PTN-KFVV, and replacing amino acids 759-763 with SDGIY, SDEVY, SDGIH, TDDVY or TDSIY;(h) rearranging the sequence of SEQ ID NO:12 by replacing amino acids 63-71 with AIALQSLEE, AVEIQEMDE, AIEIQKIDE, AVEIQSPDE, AVEVQGIEE, AVQVDNDAE or SLIAINKDK, replacing amino acids 263-269 with LNVLTAI, LNVLTTL, LNVVTTI, LNVITTI, LNVITTV or CNVLSTI, replacing amino acids 452-459 with VLNDSYIV, TLRNEYVV, TLKNEYVI, TLPNDYIV, KMKNEYIV, PVNNSYIV or PTN-KFVV, and replacing amino acids 759-763 with SDEIY, SDEVY, SDGIH, TDDVY or TDSIY; (i) rearranging the sequence of SEQ ID NO:12 by replacing amino acids 63-71 with AIALQSLEE, AVEIQEMDE, AIEIQKIDE, AVEIQSPDE, AVEVQGIEE, AVQVDNDAE or SLIAINKDK, replacing amino acids 263-269 with LNVLTAI, LNVLTTL, LNVVTTI, LNVITTI, LNVITTV or CNVLSTI, replacing amino acids 452-459 with VLNDSYIV, TLRNEYVV, TLKNEYVI, TLPNDYIV, KMKNEYIV, PVNNSYIV or PTN-KFVV NO:13 is selected from the group consisting of: replacing amino acids 63-71 with AIEIQSLEE, AIALQSLEE, AVEIQEMDE, AIEIQKIDE, AVEVQGIEE, AVQVDNDAE or SLIAINKDK, replacing amino acids 263-269 with MNVITTI, LNVLTAI, LNVLTTL, LNVITTI, LNVITTV or CNVLSTI, replacing amino acids 452-459 with TSMSDYIV, VLNDSYIV, TLRNEYVV, TLKNEYVI, KMKNEYIV, PVNNSYIV or PTN-KFVV, replacing amino acids 759-763 with SDGIY, SDEIY, SDEVY, TDDVY or TDSIY; or (j) replacing amino acids 759-763 with SDGIY, SDEIY, SDEVY, TDDVY or TDSIY; NO:21 performs a motif rearrangement selected from the following group: replacing amino acids 63-71 with AIEIQSLEE, AIALQSLEE, AVEIQEMDE, AVEIQSPDE, AVEVQGIEE, AVQVDNDAE or SLIAINKDK, replacing amino acids 263-269 with MNVITTI, LNVLTAI, LNVVTTI, LNVITTI, LNVITTV or CNVLSTI, replacing amino acids 452-459 with TSMSDYIV, VLNDSYIV, TLRNEYVV, TLPNDYIV, KMKNEYIV, PVNNSYIV or PTN-KFVV, and replacing amino acids 759-763 with SDGIY, SDEIY, SDGIH, TDDVY or TDSIY. ;

[0189] In one embodiment, the site-directed mutagenesis is performed on the sequences of SEQ ID NOs: 1-28. In one embodiment, the site-directed mutagenesis is performed on the sequences of SEQ ID NOs: 1-4, 6-9 and 12. In one embodiment, the site-directed mutagenesis is performed on the following sites: (a) a site selected from the group consisting of amino acids 21, 23, 37, 45, 100, 137, 139, 164, 180, 192, 235, 242, 255, 264, 303, 322, 354, 372, 397, 413, 417, 457, 493, 514, 567, 570, 574, 600, 603, 610, 628, 643, 653, 654, 675, 684, 710 and 759 in SEQ ID NO: 1; (b) a site selected from the group consisting of amino acids 21, 23, 37, 45, 100, 137, 139, 164, 180, 192, 235, 242, 255, 264, 303, 322, 354, 372, 397, 413, 417, 457, 493, 514, 567, 570, 574, 600, 603, 610, 628, 643, 653, 654, 675, 684, 710 and 759 in SEQ ID NO: 1; NO:2 is selected from the group consisting of amino acids 20, 22, 36, 44, 98, 135, 137, 162, 178, 190, 233, 240, 253, 262, 301, 320, 352, 370, 395, 411, 415, 455, 491, 512, 565, 568, 572, 598, 601, 608, 626, 641, 651, 652, 673, 682, 708, and 757; (c) SEQ ID NO:3 is selected from the group consisting of amino acids 21, 23, 37, 45, 99, 136, 138, 163, 179, 191, 234, 241, 254, 263, 302, 321, 353, 371, 396, 412, 416, 456, 495, 516, 569, 572, 576, 602, 605, 612, 630, 645, 655, 656, 677, 686, 712, and 761; (d) SEQ ID NO:7 is selected from the group consisting of amino acids 45, 47, 61, 69, 123, 160, 162, 187, 203, 215, 258, 265, 278, 287, 326, 345, 377, 395, 420, 436, 440, 480, 516, 537, 590, 593, 597, 624, 627, 634, 652, 667, 677, 678, 699, 708, 734, and 782; (e) SEQ ID NO:8 is selected from the group consisting of amino acids 20, 22, 36, 44, 98, 135, 137, 162, 178, 190, 233, 240, 253, 262, 301, 320, 352, 370, 395, 411, 415, 455, 491, 512, 565, 568, 572, 599, 602, 609, 627, 642, 652, 653, 674, 683, 709, and 757;(f) a position selected from the group consisting of amino acids 20, 22, 36, 44, 99, 136, 138, 163, 179, 191, 234, 241, 254, 263, 302, 321, 353, 371, 396, 412, 416, 456, 492, 513, 566, 569, 573, 599, 602, 609, 627, 642, 653, 654, 675, 684, 710, and 759 of SEQ ID NO:9;(g) a position selected from the group consisting of 9, 11, 13, 19, 23, 24, 40, 46, 47, 50, 51, 52, 63, 67, 70, 71, 77, 78, 83, 88, 98, 99, 111, 113, 115, 116, 119, 123, 129, 130, 147, 155, 161, 162, 166, 167, 171, 172, 174, 175, 182, 183, 184, 191, 203, 205, 206, 207, 219, 222, 224, 226, 229, 230, 231 1, 234, 236, 237, 238, 240, 242, 249, 250, 251, 263, 268, 275, 276, 279, 281, 294, 297, 298, 299, 300, 302, 304, 311, 316, 320, 321, 324, 328, 332, 333, 334, 346, 347, 348, 351, 355, 356, 358, 360, 362, 363, 369, 370, 373, 374, 382, ​​394, 395, 396, 400, 404, 406, 409, 413, 414, 417, 420, 421, 426, 430, 432, 437, 447, 453, 455, 463, 468, 477, 478, 482, 495, 496, 497, 498, 501, 504, 505, 509, 515, 520, 526, 528, 530, 532, 533, 536, 542, 550, 551, 552, 553, 554, 555, 556, 558, 559, 564, 567, 581, 585, 596, 597, 602, 608, 62 amino acids 5, 629, 633, 643, 650, 653, 660, 663, 664, 670, 674, 676, 680, 682, 686, 688, 690, 702, 707, 713, 715, 716, 721, 729, 730, 733, 734, 739, 744, 745, 746, 749, 751, 763, 768, 770, 773, 779, 780, 785, 787, 802, 804, 810, 811, 819, 821, 827, 829, 830, 834, and 835;(h) a position selected from the group consisting of amino acids 12, 17, 20, 35, 37, 77, 118, 132, 158, 159, 178, 179, 188, 194, 195, 200, 212, 223, 280, 309, 314, 324, 343, 441, 485, 534, 558, 594, 599, 624, 654, 656, 657, 658, 659, 682, 683, 697, 700, 733, 761, 769, 774, 777, 798, and 821 of SEQ ID NO:6; or (i) an amino acid selected from the group consisting of amino acids 12, 17, 20, 35, 37, 77, 118, 132, 158, 159, 178, 179, 188, 194, 195, 200, 212, 223, 280, 309, 314, 324, 343, 441, NO: 4 is selected from the following positions: 250, 259, 281, 285, 322, 337, 367, 368, 382, ​​383, 386, 393, 395, 409, 410, 415, 435, 444, 451, 455, 458, 472, 479, 481, 487, 491, 496, 513, 544, 545, 550, 569, 576, 583, 585, 597, 604, 609, 614, 627, 631, 632, 642, 659, 66 1, 673, 676, 683, 697, 727, 729, 746, 748, 749, 767, 777, 791, 794, 805, 822, 826, 829, 834, 850, 870, 904, 905, 906, 919, 920, 921, 923, 925, 927, 944, 948, 956, 960, 962, 970, 1002, 1006, 1007, 1008, 1011, 1020, 1027, 1040 and 1045 amino acids. ;

[0190] In one embodiment, the site-directed mutagenesis is: (a) performing a site-directed mutagenesis on SEQ ID NO: 1 selected from the group consisting of H21R / K, K23R / H, T37S / N / Q, P45C / G, A100V / I / L / M / F / Y / W, N137S / T / Q, K139R / H, T164S / N / Q, V180A / I / L / M / F / Y / W, P192C / G, R235H / K, Y242A / V / I / L / M / F / W, K255R / H, L264A / V / I / M / F / Y / W, Q303S / T / N, S322T / N / Q, N354S / T / Q, P372C / G, G397C / G / P, D413E, K417R / H, K457R / H, E493D, K514R / H, I567A / V / L / M / F / Y / W, Q570S / T / N, G574C / P, G600C / P, M603A / V / I / L / F / Y / W, N610S / T / Q, S628T / N / Q, L643A / V / I / M / F / Y / W, M653A / V / I / L / F / Y / W, A654V / I / L / M / F / Y / W, E675D, G684C / P, S710T / N / Q and K759R / H; (b) for SEQ ID NO:2 site-directed mutagenesis selected from the group consisting of Q20S / T / N, A22V / I / L / M / F / Y / W, T36S / N / Q, P44C / G, S98T / N / Q, N135S / T / Q, K137R / H, T162S / N / Q, A178V / I / L / M / F / Y / W, P190C / G, R233H / K, Y240A / V / I / L / M / F / W, D253E, L262A / V / I / M / F / Y / W, Q301S / T / N, S320T / N / Q, N352S / T / Q, S370T / N / Q, G395C / P, E411D, K415R / H, K455R / H, E491D, L512A / V / I / M / F / Y / W, K565R / H, Q568S / T / N, G572C / P, G598C / P, S601T / N / Q, N 608S / T / Q, G626C / P, V641A / I / L / M / F / Y / W, F651A / V / I / L / M / Y / W, S652T / N / Q, N673S / T / Q, K682R / H, P708C / G and G757C / P; (c) For SEQ ID NO:3 site-directed mutagenesis selected from the group consisting of H21R / K, K23R / H, T37S / N / Q, S45T / N / Q, S99T / N / Q, N136S / T / Q, K138R / H, T163S / N / Q, T179S / N / Q, P191C / G, L234A / V / I / M / F / Y / W,Y241A / V / I / L / M / F / W, D254E, T263S / N / Q, A302V / I / L / M / F / Y / W, Q321S / T / N, H353R / K, P37 1C / G, G396C / P, K412R / H, E416D, K456R / H, E495D, L516A / V / I / M / F / Y / W, T569S / N / Q, R572H / K, G576C / P, K602R / H, M605A / V / I / L / F / Y / W, D612E, N630S / T / Q, N645S / T / Q, F655A / V / I / L / M / Y / W, V656A / I / L / M / F / Y / W, E677D, G686C / P, A712V / I / L / M / F / Y / W and N761S / T / Q; (d) for SEQ ID NO:7 site-directed mutagenesis selected from the group consisting of H45R / K, K47R / H, T61S / N / Q, P69C / G, Q123S / T / N, N160S / T / Q, H162R / K, T187S / N / Q, V203A / I / L / M / F / Y / W, P215C / G, K258R / H, Y265A / V / I / L / M / F / W, K278R / H, V287A / I / L / M / F / Y / W, Q326S / T / N, Q345S / T / N, N377S / T / Q, P395C / G, G420C / P, E 436D, K440R / H, K480R / H, E516D, K537R / H, V590A / I / L / M / F / Y / W, Q593S / T / N, G597C / P, G624C / P, L627A / V / I / M / F / Y / W, N634S / T / Q, N652S / T / Q, I667A / V / L / M / F / Y / W, A677V / I / L / M / F / Y / W, A678V / I / L / M / F / Y / W, E699D, G708C / P, S734T / N / Q and K782R / H; (e) For SEQ ID NO:8 site-directed mutagenesis selected from the group consisting of H20R / K, K22R / H, A36V / I / L / M / F / Y / W, S44T / N / Q, G98C / P, G135C / P, Q137S / T / N, T162S / N / Q, V178A / I / L / M / F / Y / W, S190T / N / Q, Q233S / T / N, H240R / K , K253R / H, I262A / V / L / M / F / Y / W, Q301S / T / N, L320A / V / I / M / F / Y / W, N352S / T / Q, P37 0C / G, E395D, E411D, K415R / H, K455R / H, S491T / N / Q, K512R / H, I565A / V / L / M / F / Y / W,Q568S / T / N, E572D, G599C / P, F602A / V / I / L / M / Y / W, N609S / T / Q, S627T / N / Q, I642A / V / L / M / F / Y / W, A652V / I / L / M / F / Y / W, S653T / N / Q, E674D, E683D, S709T / N / Q and K757R / H; (f) site-directed mutagenesis of SEQ ID NO:9 selected from the group consisting of: Q20S / T / N, D22E, E36D, P44C / G, S99T / N / Q, D136E, V138A / I / L / M / F / Y / W, I163A / V / L / M / F / Y / W, V179A / I / L / M / F / Y / W, K191R / H, R234H / K, Q241S / T / N, K254R / H, L263A / V / I / M / F / Y / W, Q302S / T / N, K321R / H, H353R / K, S371T / N / Q, K396R / H, D412E, V416A / I / L / M / F / Y / W, K456R / H, K492R / H, K513R / H, I566A / V / L / M / F / Y / W, Q569S / T / N, T573S / N / Q, G599C / P, M602A / V / I / L / F / Y / W, N609S / T / Q, K627R / H, I642A / V / L / M / F / Y / W, L653A / V / I / M / F / Y / W, S654T / N / Q, L675A / V / I / M / F / Y / W, G684C / P, S710T / N / Q and K759R / H; (g) site-directed mutagenesis of SEQ ID NO:12 selected from the group consisting of: S9T / N / Q, A11V / I / L / M / F / Y / W, V13A / I / L / M / F / Y / W, V19A / I / L / M / F / Y / W, D23E, D24E, T40S / N / Q, F46A / V / I / L / M / Y / W, N47S / T / Q, V50A / I / L / M / F / Y / W, K51R / H, T52S / N / Q, A63V / I / L / M / F / Y / W, Q67S / T / N, E70D, E71D, R77H / K, C78G / P, D83E, V88A / I / L / M / F / Y / W, T98S / N / Q, N99S / T / Q, K111R / H, N113S / T / Q, E115D, E116D, L119A / V / I / M / F / Y / W, K123R / H, Y129A / V / I / L / M / F / W, N130S / T / Q, A147V / I / L / M / F / Y / W, I155A / V / L / M / F / Y / W, E161D, A162V / I / L / M / F / Y / W, Q166S / T / N, N167S / T / Q,K171R / H、K172R / H、K174R / H、E175D、P182C / G、I183A / V / L / M / F / Y / W、A184V / I / L / M / F / Y / W、F191A / V / I / L / M / Y / W、K194R / H、H203R / K、R205H / K、V206A / I / L / M / F / Y / W、I207A / V / L / M / F / Y / W、F219A / V / I / L / M / Y / W、F222A / V / I / L / M / Y / W、S224T / N / Q、R226H / K、S229T / N / Q、N230S / T / Q、G231C / P、L234A / V / I / M / F / Y / W、K236R / H、N237S / T / Q、T238S / N / Q、K240R / H、K242R / H、K249R / H、R250H / K、V251A / I / L / M / F / Y / W、M263A / V / I / L / F / Y / W、T268S / N / Q、L275A / V / I / M / F / Y / W、Y276A / V / I / L / M / F / W、K279R / H、I281A / V / L / M / F / Y / W、Y294A / V / I / L / M / F / W、R297H / K、Q298S / T / N、Y299A / V / I / L / M / F / W、S300T / N / Q、F302A / V / I / L / M / Y / W、F304A / V / I / L / M / Y / W、D311E、S316T / N / Q、K320R / H、E321D、F324A / V / I / L / M / Y / W、I328A / V / L / M / F / Y / W、K332R / H、D333E、N334S / T / Q、G346C / P、N347S / T / Q、V348A / I / L / M / F / Y / W、K351R / H、K355R / H、I356A / V / L / M / F / Y / W、E358D、Q360S / T / N、I362A / V / L / M / F / Y / W、N363S / T / Q、Y369A / V / I / L / M / F / W、K370R / H、A373V / I / L / M / F / Y / W、T374S / N / Q、V382A / I / L / M / F / Y / W、S394T / N / Q、A395V / I / L / M / F / Y / W、K396R / H、H400R / K、E404D、R406H / K、F409A / V / I / L / M / Y / W、L413A / V / I / M / F / Y / W、D414E、D417E、N420S / T / Q、V421A / I / L / M / F / Y / W、P426C / G、L430A / V / I / M / F / Y / W、S432T / N / Q、V437A / I / L / M / F / Y / W、P447C / G、S453T / N / Q、S455T / N / Q、A463V / I / L / M / F / Y / W、F468A / V / I / L / M / Y / W、L477A / V / I / M / F / Y / W、I478A / V / L / M / F / Y / W、K482R / H、K495R / H、D496E、E497D、P498C / G、G501C / P、N504S / T / Q、I505A / V / L / M / F / Y / W、T509S / N / Q、F515A / V / I / L / M / Y / W、A520V / I / L / M / F / Y / W、D526E、C528G / P、G530C / P、F532A / V / I / L / M / Y / W、G533C / P、I536A / V / L / M / F / Y / W、P542C / G、I550A / V / L / M / F / Y / W、L551A / V / I / M / F / Y / W、N552S / T / Q、G553C / P、554S / N / Q、D555E、K556R / H、Y558A / V / I / L / M / F / W、K559R / H、A564V / I / L / M / F / Y / W、T567S / N / Q、S581T / N / Q、Y585A / V / I / L / M / F / W、N596S / T / Q、N597S / T / Q、G602C / P、S608T / N / Q、S625T / N / Q、K629R / H、Y633A / V / I / L / M / F / W、E643D、H650R / K、I653A / V / L / M / F / Y / W、R660H / K、K663R / H、E664D、D670E、V674A / I / L / M / F / Y / W、R676H / K、E680D、E682D、N686S / T / Q、V688A / I / L / M / F / Y / W、S690T / N / Q、G702C / P、N707S / T / Q、D713E、K715R / H、V716A / I / L / M / F / Y / W、F721A / V / I / L / M / Y / W、N729S / T / Q、M730A / V / I / L / F / Y / W、D733E、V734A / I / L / M / F / Y / W、K739R / H、E744D、L745A / V / I / M / F / Y / W、F746A / V / I / L / M / Y / W、D749E、T751S / N / Q、Y763A / V / I / L / M / F / W、S768T / N / Q、V770A / I / L / M / F / Y / W、T773S / N / Q、N779S / T / Q、L780A / V / I / M / F / Y / W、Q785S / T / N、T787S / N / Q、F802A / V / I / L / M / Y / W、N804S / T / Q、I810A / V / L / M / F / Y / W、E811D、(h) subjecting SEQ ID NO:6 to a site-directed mutagenesis selected from the group consisting of N12S / T / Q, I17A / V / L / M / F / Y / W, S20T / N / Q, P35C / G, N37S / T / Q, N77S / T / Q, G118C / P, K132R / H, Q158S / T / N, R159H / K, A178V / I / L / M / F / Y / W, D179E, T1 88S / N / Q, T194S / N / Q, N195S / T / Q, V200A / I / L / M / F / Y / W, V212A / I / L / M / F / Y / W, F223 A / V / I / L / M / Y / W, A280V / I / L / M / F / Y / W, H309R / K, A314V / I / L / M / F / Y / W, V324A / I / L / M / F / Y / W, Y343A / V / I / L / M / F / W, Y441A / V / I / L / M / F / W, Q485S / T / N, N534S / T / Q, H558 R / K, G594C / P, D599E, H624R / K, G654C / P, R656H / K, N657S / T / Q, M658A / V / I / L / F / Y / W, H659R / K, E682D, N683S / T / Q, H697R / K, C700G / P, P733C / G, A761V / I / L / M / F / Y / W, V769A / I / L / M / F / Y / W, S774T / N / Q, P777C / G, E798D, M821A / V / I / L / F / Y / W; or (i) to SEQ ID NO:4 site-directed mutagenesis selected from the group consisting of A250V / I / L / M / F / Y / W, M259A / V / I / L / F / Y / W, G281C / P, E285D, Y322A / V / I / L / M / F / W, N337S / T / Q, P367C / G, S368T / N / Q, K382R / H, G383C / P, D386E, F393A / V / I / L / M / Y / W, E395D, V409A / I / L / M / F / Y / W, M410 A / V / I / L / F / Y / W, G415C / P, N435S / T / Q, F444A / V / I / L / M / Y / W, D451E, E455D, Y458A / V / I / L / M / F / W, A472V / I / L / M / F / Y / W, K479R / H, S481T / N / Q, K487R / H, D491E, Y496A / V / I / L / M / F / W, V513A / I / L / M / F / Y / W, K544R / H, D545E,L550A / V / I / M / F / Y / W, C569G / P, K576R / H, Q583S / T / N, T585S / N / Q, N597S / T / Q, E604D, D609E, N614S / T / Q, P627C / G, E631D, R632H / K, Y642A / V / I / L / M / F / W, I659A / V / L / M / F / Y / W, V661A / I / L / M / F / Y / W, I673A / V / L / M / F / Y / W, S676T / N / Q, K683R / H, K697R / H, A727V / I / L / M / F / Y / W, S729T / N / Q, C746G / P, P748C / G, M749A / V / I / L / F / Y / W, Q767S / T / N, V777A / I / L / M / F / Y / W, T791S / N / Q, I794A / V / L / M / F / Y / W, Q805S / T / N, G822C / P, N826S / T / Q, D829E, L834A / V / I / M / F / Y / W, N850S / T / Q, L870A / V / I / M / F / Y / W, V904A / I / L / M / F / Y / W, E905D, L906A / V / I / M / F / Y / W, N919S / T / Q, D920E, K921R / H, D923E, K925R / H, L927A / V / I / M / F / Y / W, G944C / P, P948C / G, F956A / V / I / L / M / Y / W, G960C / P, Y962A / V / I / L / M / F / W, K970R / H, F1002A / V / I / L / M / Y / W, A1006V / I / L / M / F / Y / W, Q1007S / T / N, Y1008A / V / I / L / M / F / W, N1011S / T / Q, N1020S / T / Q, S1027T / N / Q, R1040H / K, and Y1045A / V / I / L / M / F / W.

[0191] In one embodiment, the capping enzyme of the present invention is from a source selected from the group consisting of Marseillevirus, Pithovirus, Catovirus, Rotavirus, Orbivirus, Moumouvirus, African swine fever virus, and Seadornavirus. In one embodiment, the capping enzyme of the present invention is from Marseillevirus. In one embodiment, the capping enzyme of the present invention is from the family Marseilleviridae. In one embodiment, the capping enzyme of the present invention is from the genus Marseillevirus.

[0192] The capping enzyme of the present invention can be in any form suitable for the process described herein, such as crude fermentation broth with or without cells removed, dry powder or granules, dust-free granules, liquid, stabilized liquid or stabilized protected enzyme. Liquid enzyme preparations can be stabilized according to established processes, for example by adding stabilizers such as sugars, sugar alcohols or other polyols, and / or lactic acid or other organic acids.

[0193] In one embodiment, the capping efficiency of the capping enzyme of the present invention is 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times or 2 times higher than that of the commercial capping enzyme. In one embodiment, the capping efficiency is characterized by the percentage of capped RNA to total RNA.

[0194] 3. Nucleic acid sequences, nucleic acid constructs, expression vectors and host cells of the present invention

[0195] In one aspect, the present invention provides a nucleic acid sequence encoding a capping enzyme of the present invention. In one embodiment, the nucleic acid sequence comprises or consists of: (a) a polynucleotide sequence encoding any one of the amino acid sequences in SEQ ID NO: 1-28, or (b) a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the polynucleotide sequence. In one embodiment, the nucleic acid sequence comprises or consists of: (a) any one of the polynucleotide sequences in SEQ ID NO: 29-56, or (b) a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the polynucleotide sequence.

[0196] In one aspect, the present invention provides a nucleic acid construct comprising the nucleic acid sequence of the present invention. In one embodiment, the nucleic acid construct of the present invention may further comprise elements or regulatory sequences required for expressing the nucleic acid sequence.

[0197] In one embodiment, nucleic acid construct of the present invention can be constructed as follows: the nucleotide sequence is operably connected to one or more (several) regulating and controlling sequences, and the regulating and controlling sequences instruct the expression of the nucleotide sequence under the conditions compatible with the regulating and controlling sequences in a suitable host cell. Depending on the type of nucleic acid construct, it may be desirable or necessary to operate the nucleotide sequence before the nucleotide sequence is inserted into a plasmid or other vector. The technology of using recombinant DNA methods to modify the nucleotide sequence is known in the art.

[0198] In one embodiment, the regulatory sequence may be a suitable promoter sequence, which is a nucleotide sequence recognized by a host cell for expressing a polynucleotide encoding a capping enzyme. The promoter sequence contains a transcriptional regulatory sequence that mediates the expression of the capping enzyme. The promoter may be any nucleotide sequence that shows transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular capping enzyme that is homologous or heterologous to the host cell.

[0199] Suitable promoters for directing transcription of the nucleic acid constructs of the invention in bacterial host cells can be obtained from the genes for the following enzymes: the Escherichia coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes and prokaryotic β-lactamase genes (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75:3727-3731), and the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80:21-25). Additional promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; and in Sambrook et al., 1989, supra.

[0200] Suitable promoters for directing transcription of the nucleic acid constructs of the invention in a filamentous fungal host cell can be obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium venenatum amyloglucosidase (WO 00 / 56900), Fusarium venenatum Daria (WO 00 / 56900), Fusarium venenatum Quinn (WO 00 / 56900), Rhizomucor manhei (WO 00 / 56900), miehei) lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei β-xylosidase, and NA2-tpi promoter (a modified promoter from the gene encoding a neutral α-amylase in penicillin, in which its untranslated leader sequence is replaced by the untranslated leader sequence from the gene encoding a triose phosphate isomerase in Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated and hybrid promoters thereof.

[0201] Suitable promoters for directing transcription of the nucleic acid construct of the present invention in a yeast host can be obtained from genes for the following enzymes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1) and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells include, for example, a xylose-inducible promoter (regulated by the XylR transcription factor derived from Bacillus licheniformis) and promoters described by Romanos et al., 1992, Yeast 8:423-488 and Chen et al., 2020, Nature Microbiology 5:1349-1360.

[0202] The promoters used to direct the expression of the nucleic acid construct of the present invention in a mammalian host include, but are not limited to, a strong mammalian expression promoter derived from human cytomegalovirus (CMV), a strong mammalian expression promoter derived from human elongation factor 1α (EF1α), a mammalian expression promoter derived from simian vacuolating virus 40 (SV40), a mammalian promoter derived from phosphoglycerate kinase gene (PGK1), and the like.

[0203] In one embodiment, the regulatory sequence may be a suitable transcription terminator sequence, which is a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably connected to the 3' end of the nucleotide sequence encoding the capping enzyme. Any terminator that is functional in the selected host cell can be used in the present invention. For filamentous fungal host cells, preferred terminators can be obtained from the genes of the following enzymes: Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase and Fusarium oxysporum trypsin-like protease. For yeast host cells, preferred terminators can be obtained from the genes of the following enzymes: Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1) and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. For yeast host cells, other useful terminators are described by Romanos et al., 1992 and Chen et al., 2020.

[0204] In one embodiment, the regulatory sequence may be a suitable leader sequence, which is an mRNA non-translated region important for the translation of the host cell. The leader sequence may be operably linked to the 5'-end of the nucleotide sequence encoding the capping enzyme. Any leader sequence that is functional in the selected host cell may be used in the present invention. Preferred leaders for filamentous fungal host cells are obtained from the genes of the following enzymes: Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes of the following enzymes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0205] In one embodiment, the regulatory sequence may be a polyadenylation sequence, which is a sequence operably connected to the 3' end of the nucleotide sequence, and when transcribed, the host cell recognizes it as a signal for adding polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the selected host cell can be used in the present invention. For filamentous fungal host cells, preferred polyadenylation sequences are obtained from the genes of the following enzymes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease and Aspergillus niger α-glucosidase. For yeast host cells, useful polyadenylation sequences are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990. For mammalian host cells, useful polyadenylation sequences are described by Qin et al., 2023, Nature Communications 14: 1500.

[0206] In one embodiment, the regulatory sequence may be a signal peptide coding region, which encodes a signal peptide connected to the N-terminus of the capping enzyme and directs the capping enzyme to enter the cell secretory pathway. The 5' end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence, which is naturally connected in the translation reading frame together with the coding sequence segment encoding the capping enzyme. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is exogenous to the coding sequence. An exogenous signal peptide coding sequence may be necessary when the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, an exogenous signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance the secretion of the capping enzyme. However, any signal peptide coding sequence that directs the expressed capping enzyme to enter the secretory pathway of the selected host cell may be used. Signal peptide coding sequences useful for bacterial host cells are those obtained from the genes for Bacillus sp. NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral proteinase (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137. Signal peptide coding sequences useful for filamentous fungal host cells are those obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor manhei aspartic proteinase. Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

[0207] In one embodiment, the regulatory sequence may be a propeptide coding sequence encoding a propolypeptide located at the N-terminus of the capping enzyme. The resulting polypeptide is referred to as a proenzyme or propolypeptide (or in some cases a zymogen). The propolypeptide is generally inactive and can be converted from the propolypeptide to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide. The propeptide coding sequence can be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor manhei aspartic protease, and Saccharomyces cerevisiae alpha factor.

[0208] In one embodiment, when both the signal peptide and propeptide sequences are present at the N-terminus of the capping enzyme, the propeptide sequence is placed next to the capping enzyme N-terminus and the signal peptide sequence is placed next to the amino terminus of the propeptide sequence.

[0209] In one embodiment, it is also desirable to add a regulatory sequence that allows the expression of the capping enzyme to be regulated relative to the growth of the host cell. Examples of regulatory systems are those systems that cause gene expression to respond to chemical or physical stimuli, including those systems that are turned on or off by the presence of regulatory compounds. Regulatory systems in prokaryotic systems include lac, tac, and trp operator systems. In yeast, the ADH2 system or the GAL1 system and the XYL system can be used. In filamentous fungi, Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA α-amylase promoter, and Aspergillus oryzae glucoamylase promoter can be used as regulatory sequences. In mammalian cells, constitutive promoters including EF1α, CMV, and SV40 and other induction systems can be used for regulation. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate, and metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the capping enzyme is operably connected to the regulatory sequence.

[0210] In one embodiment, the nucleic acid construct of the present invention may further comprise one or more (several) convenient restriction sites to allow insertion or substitution of the isolated polynucleotide encoding the capping enzyme at these sites. In one embodiment, the nucleic acid sequence may be expressed by inserting a nucleotide sequence or nucleic acid construct comprising the sequence into an appropriate vector for expression. In the process of preparing the nucleic acid construct, the coding sequence is placed in the vector so that the coding sequence is operably linked to an appropriate regulatory sequence for expression.

[0211] In one embodiment, the vector for expression can be any vector (for example, plasmid or virus), which can easily carry out the recombinant DNA step, and can produce the expression of the nucleic acid sequence. The selection of the vector will usually depend on the compatibility of the vector with the host cell into which the vector will be introduced. The vector can be a linear or closed circular plasmid. In one embodiment, the vector can be an autonomously replicating vector, that is, a vector existing as an extrachromosomal entity (entity), and its replication is independent of chromosomal replication, for example, a plasmid, an extrachromosomal element, a minichromosome (minichromosome) or an artificial chromosome. In one embodiment, the vector can contain any feature for ensuring self-replication. In one embodiment, the vector can be a vector that is integrated into the genome and replicated together with the chromosome into which the vector is integrated when introduced into the host cell. In one embodiment, a single vector or plasmid or two or more vectors or plasmids can be used, which contain the complete DNA (total DNA) to be introduced into the host cell genome together, or a transposon (transposon) can be used.

[0212] In one embodiment, the vector preferably contains one or more (several) selective markers, which allow simple selection of cells transformed, transfected, transduced, etc. Selectable markers are genes whose products provide biocide or virus resistance, resistance to heavy metals, prototrophy to auxotrophs, etc. In one embodiment, examples of bacterial selective markers are dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance. In one embodiment, the resistance gene is selected from the group consisting of ampicillin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene, spectinomycin resistance gene, tetracycline resistance gene, bleomycin resistance gene, streptomycin resistance gene, hygromycin resistance gene, gentamicin resistance gene, apramycin sulfate resistance gene, erythromycin resistance gene, nourseoinhibitor resistance gene, geneticin resistance gene, and puromycin resistance gene. In one embodiment, the resistance gene is selected from the group consisting of an ampicillin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, or a tetracycline resistance gene. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenylyltransferase), and trpC (anthranilate synthase), and their equivalents. Fluorescent markers such as mCherry are included for mammalian cells. Preferred for use in Aspergillus cells are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.

[0213] In one embodiment, the vector preferably contains elements that allow the vector to be integrated into the host cell genome or the vector to replicate autonomously in the cell independently of the genome. In order to be integrated into the host cell genome, the vector can rely on the sequence of the polynucleotide encoding the capping enzyme or any other vector element for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional nucleotide sequences for guiding the precise position of integration into the host cell genome chromosome by homologous recombination. In order to increase the possibility of integration at a precise position, the integration element should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a high degree of identity with the corresponding target sequence to enhance the probability of homologous recombination. The integration element can be any sequence, which is homologous to the target sequence in the host cell genome. In addition, the integration element can be a non-coding or encoding nucleotide sequence. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

[0214] In one embodiment, for autonomous replication, the vector may further comprise an origin of replication that enables the vector to replicate autonomously in the host cell. The origin of replication may be any plasmid replicator that mediates autonomous replication, which functions in the cell. The term "origin of replication" or "plasmid replicator" is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo. Examples of origins of replication used in bacterial host cells are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 that allow replication in Escherichia coli, and the origins of replication of plasmids pUB110, pE194, pTA1060, and pAMβ1 that allow replication in Bacillus. Examples of origins of replication used in yeast host cells are the 2 micron origin of replication, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6. Examples of origins of replication for use in filamentous fungal host cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Research 15:9163-9175; WO 00 / 24883). Isolation of the AMA1 gene and construction of a plasmid or vector comprising the gene can be accomplished according to the methods disclosed in WO 00 / 24883.

[0215] In one embodiment, more than one copy of the nucleic acid can be inserted into the host cell to increase the production of the expression product. The increase in nucleic acid copy number can be obtained by the following method: the sequence of at least one additional copy is integrated into the host cell genome, or an amplifiable selective marker gene is included in a polynucleotide, wherein the amplified copy containing the selective marker gene can be selected by culturing cells in the presence of a suitable selection agent (selectable agent), thereby also containing cells of additional copies of the polynucleotide. The method for connecting the above-mentioned elements to construct a nucleic acid construct is well known to those skilled in the art (see, for example, Sambrook et al., 1989, see above).

[0216] In one embodiment, the vector is a plasmid vector. In one embodiment, the plasmid vector is selected from the group consisting of a prokaryotic expression vector, a yeast expression vector, a plant expression vector, an insect expression vector, and a mammalian expression vector.

[0217] In one aspect, the present invention provides a recombinant host cell comprising a nucleic acid sequence of the present invention or a nucleic acid construct of the present invention. In one embodiment, the recombinant host cell is prepared by introducing the nucleic acid sequence of the present invention or a nucleic acid construct into a host cell. In one embodiment, the host cell is an Escherichia coli cell, a yeast cell or a mammalian cell, more preferably an Escherichia coli Top10 or DH5α cell, a Saccharomyces cerevisiae cell (such as a Saccharomyces cerevisiae BY4741 cell), a Chinese hamster ovary (CHO) cell or a human embryonic kidney cell (such as a HEK293T cell).

[0218] In one embodiment, the recombinant host cell comprises one or more isolated polynucleotides encoding a capping enzyme, and the recombinant host cell is advantageously used for the recombinant production of the capping enzyme. The vector is introduced into the host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector, as described above. The term "host cell" includes any progeny of a parent cell that is different from the parent cell because of mutations that occur during replication. The choice of host cells will largely depend on the gene encoding the capping enzyme and its source.

[0219] In one embodiment, the host cell can be any cell useful in the recombinant production of the capping enzyme of the invention, eg, a prokaryotic or eukaryotic cell.

[0220] In one embodiment, the prokaryotic host cell can be any gram-positive bacteria or gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus and Ocean Bacillus. Gram-negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Silvae, Neisseria and Ureaplasma.

[0221] In one embodiment, the bacterial host cell can be any Bacillus cell. Useful Bacillus cells in the implementation of the present invention include, but are not limited to, alkaliphilic Bacillus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus radiata, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus stearothermophilus, Bacillus subtilis and Bacillus thuringiensis cells. In one embodiment, the bacterial host cell is Bacillus amyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cells. In one embodiment, the bacterial host cell is Bacillus amyloliquefaciens cells. In one embodiment, the bacterial host cell is Bacillus clausii cells. In one embodiment, the bacterial host cell is Bacillus licheniformis cells. In one embodiment, the bacterial host cell is Bacillus subtilis cells.

[0222] In one embodiment, the bacterial host cell can be any Streptococcus cell. Streptococcus cells useful in the practice of the present invention include, but are not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells. In one embodiment, the bacterial host cell is a Streptococcus equisimilis cell. In one embodiment, the bacterial host cell is a Streptococcus pyogenes cell. In one embodiment, the bacterial host cell is a Streptococcus uberis cell. In one embodiment, the bacterial host cell is a Streptococcus equi subsp. Zooepidemicus cell.

[0223] In one embodiment, the bacterial host cell can be any Streptomyces cell. Useful Streptomyces cells in the practice of the present invention include, but are not limited to, achromogenic Streptomyces, avermitilis Streptomyces, coelicolor Streptomyces, griseus Streptomyces, and lividans Streptomyces cells. In one embodiment, the bacterial host cell is achromogenic Streptomyces cell. In one embodiment, the bacterial host cell is a avermitilis Streptomyces cell. In one embodiment, the bacterial host cell is a coelicolor Streptomyces cell. In one embodiment, the bacterial host cell is a griseus Streptomyces cell. In one embodiment, the bacterial host cell is a lividans Streptomyces cell.

[0224] Introduction of DNA into Bacillus cells can be achieved by methods such as protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), use of competent cells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829 or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5271-5278). Introduction of DNA into E. coli cells can be achieved by methods such as protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166:557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). Introduction of DNA into Streptomyces cells can be achieved by methods such as protoplast transformation and electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294). Introduction of DNA into Pseudomonas cells can be achieved by methods such as electroporation (see, eg, Choi et al., 2006, J. Microbiol. Methods 64:391-397) or conjugation (see, eg, Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71:51-57).Introduction of DNA into Streptococcus cells can be achieved by methods such as natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios. 68: 189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0225] In one embodiment, the host cell can be a eukaryotic organism, such as a mammalian, insect, plant or fungal cell.

[0226] In one embodiment, the host cell is a fungal cell. "Fungus" as used herein includes the following phyla: Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., in Ainsworth and Bisby's Dictionary of The Fungi, 8th ed., 1995, CAB International, University Press, Cambridge, UK) and Oomycota (as cited in Hawksworth et al., 1995, supra, p. 171), and all mitosporic fungi (Hawksworth et al., 1995, supra).

[0227] In one embodiment, the fungal host cell is a yeast cell. "Yeast" is used herein to include ascosporogenous yeast (Endomycetales), basidiosporogenous yeast and yeast belonging to Fungi Imperfecti (Blastomycetes). Because the classification of yeast may change in the future, for the present invention, yeast is defined as described in Biology and Activities of Yeast (Skinner, FA, Passmore, SM, and Davenport, RR, Soc. App. Bacteriol. Symposium Series No. 9, 1980). In one embodiment, the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia cell. In one embodiment, the yeast host cell is Karlsaccharomyces, Saccharomyces cerevisiae, Saccharomyces diastaticus, Douglas yeast, Kluyveromyces, Nordy yeast or oval yeast cell. In another most preferred aspect, the yeast host cell is a Kluyveromyces lactis cell.In another most preferred aspect, the yeast host cell is a Yarrowia lipolytica cell.

[0228] In one embodiment, the fungal host cell is a filamentous fungal cell." filamentous fungi " include all filamentous forms of the subphylum of Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by the mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Nutritional growth is carried out by hyphae extension, and carbon catabolism is an obligate aerobic. On the contrary, the nutritional growth of yeast such as saccharomyces cerevisiae is carried out by budding of unicellular thallus, and carbon catabolism can be fermented.

[0229] In one embodiment, the filamentous fungal host cell is a Acremonium, Aspergillus, Pullulan, Bjerkandera, Ceroplasts, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Pyrospora, Mucor, Myceliophthora, Neocomas, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Chytridiomyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Toxicodendron, Trametes, or Trichoderma cell.

[0230] In one embodiment, the filamentous fungal host cell is Aspergillus awamori, Aspergillus fumigatus, Aspergillus odoratus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae. In another most preferred aspect, the filamentous fungal host cell is a rod-shaped fusarium, a graminearum fusarium, a Kuwei fusarium, a broadsword fusarium, a gramineous fusarium, a gramineous fusarium, a heterosporous fusarium, albizzia fusarium, a sharp fusarium, a multi-branched fusarium, a pink fusarium, an elder fusarium, a skin color fusarium, a pseudo-branched fusarium, a sulfur-colored fusarium, a round fusarium, a pseudo-spored fusarium or a veneer fusarium cell. In another most preferred aspect, the filamentous fungal host cell is Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicalis, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium queenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cells.

[0231] Fungal cells can be transformed in a manner known per se by methods involving protoplast formation, protoplast transformation, and cell wall reconstruction. Suitable methods for transforming Aspergillus and Trichoderma host cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156 and WO 96 / 00787. Yeast can be transformed using the methods described by Becker and Guarente, in Abelson, JN and Simon, MI, eds., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153:163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75:1920.

[0232] 4. Combinations of capping enzymes of the present invention

[0233] In one aspect, the invention provides a combination of capping enzymes comprising or consisting of capping enzymes of the invention derived from at least two, at least three, at least four or more sources.

[0234] In one embodiment, the combination of capping enzymes comprises or consists of at least two, at least three, at least four or more sequences of capping enzymes of the present invention. In one embodiment, the capping enzymes of at least two, at least three, at least four or more sequences are from the same source or from different sources. In one embodiment, the capping enzymes of at least two, at least three, at least four or more sequences are from the family Marseilleviridae, preferably the genus Marseillevirus.

[0235] 5. Kit or composition of the present invention

[0236] In one aspect, the present invention provides a kit or composition for capping RNA, comprising: (i) a capping enzyme of the present invention or a combination thereof; (ii) a storage buffer, in which the capping enzyme of the present invention or a combination thereof is located; and (iii) a reaction buffer. In one embodiment, the kit further comprises: (iv) guanosine triphosphate (GTP) or modified GTP; (v) a methyl donor; and (vi) an optional target RNA to be capped. In one embodiment, the storage buffer comprises or consists of: 20-100 mM Tris-HCl, 1-20 mM KCl, 0.5-5 mM MgCl2, and 0.5-10 mM dithiothreitol (DTT). In one embodiment, the storage buffer comprises or consists of: 50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2, and 1 mM DTT. In one embodiment, the reaction buffer comprises or consists of: 20-100mM Tris-HCl, 1-20mM KCl, 0.5-5mM MgCl2 and 0.5-10mM dithiothreitol (DTT). In one embodiment, the reaction buffer comprises or consists of: 50mM Tris-HCl, 5mM KCl, 1mM MgCl2 and 1mM DTT. In one embodiment, the modified GTP is selected from the group consisting of: GTP modified on the base, GTP modified on the ribose, and GTP modified on the alpha phosphate group. In one embodiment, the modified GTP is selected from the group consisting of GTP modified on ribose 2'-OH and GTP modified on ribose 3'-OH. In one embodiment, the modified GTP is selected from the group consisting of GTP modified by O methylation on ribose 3'-OH, GTP modified by NH2 on ribose 3'-OH, and dGTP modified by N3 on ribose 3'-OH. In one embodiment, the methyl donor is selected from the group consisting of choline, trimethylglycine, methionine, S-adenosylmethionine (SAM), methylfolate, and methylcobalamin. In one embodiment, the methyl donor is SAM.

[0237] 6. Capping method of the present invention

[0238] In one aspect, the present invention provides a method for capping RNA in vitro, comprising: (i) providing an uncapped RNA sample; (ii) adding the capping enzyme of the present invention or a combination thereof, guanosine triphosphate (GTP) or modified GTP and a methyl donor to a reaction buffer to provide a capping reaction mixture; (iii) reacting the RNA sample with the mixture at a temperature of 30°C-60°C for 10-200 minutes to form a reaction product, i.e., capped RNA.

[0239] In one embodiment, the uncapped RNA sample is incubated at 50-80°C for 3-15 minutes in step (i). In one embodiment, the uncapped RNA sample is incubated at 55-70°C for 5-10 minutes in step (i). In one embodiment, an ice bath is performed after incubation. In one embodiment, the ice bath is performed for 5-10 minutes. In one embodiment, the capping enzyme or combination is added to the reaction buffer in an amount of 0.2-2.0U / μL in step (ii). In one embodiment, the capping enzyme or combination is added to the reaction buffer in an amount of 0.5-1.0U / μL in step (ii). In one embodiment, the GTP or modified GTP is added to the reaction buffer in an amount of 0.2-5mM in step (ii). In one embodiment, the GTP or modified GTP is added to the reaction buffer in an amount of 0.5-1mM in step (ii). In one embodiment, in step (ii), the methyl donor is added to the reaction buffer in an amount of 0.1-0.8mM. In one embodiment, in step (ii), the buffer comprises the following and is composed of: 20-100mM Tris-HCl, 1-20mM KCl, 0.5-5mM MgCl2 and 0.5-10mM dithiothreitol (DTT). In one embodiment, in step (ii), the buffer comprises the following and is composed of: 50mM Tris-HCl, 5mM KCl, 1mM MgCl2 and 1mM DTT. In one embodiment, step (iii) is reacted at 30-42°C for 45-90 minutes. In one embodiment, the method of the present invention further comprises: (iv) purifying the reaction product and / or storing the reaction product or the purified reaction product at -80°C.

[0240] 7. Advantages of the present invention

[0241] The advantages of the present invention are at least embodied in the following aspects:

[0242] 1. The present invention tests the overall Cap0 capping activity, not the individual activities of the three steps of dephosphorylation, guanosine transfer, and methylation. The overall capping efficiency is better than or comparable to that of existing commercial poxvirus capping enzymes;

[0243] 2. The capping enzyme of the present invention has a sequence identity of less than 20% with the existing poxvirus capping enzyme, and is a brand-new capping enzyme with commercial potential;

[0244] 3. In the production and purification process of the capping enzyme, the present invention selects Pichia pastoris as the capping enzyme production and purification system, which is different from the conventional E. coli system. Compared with the prokaryotic expression system, this eukaryotic expression system has the function of post-translational protein processing and modification, and the purified protein is closer to mammalian cells in terms of physiological properties or functions;

[0245] 4. The capping enzyme derived from Marseille virus of the present invention has better activity in different species and has better versatility in cross-species expression than the positive control NP868R;

[0246] 5. The capping enzyme derived from orbivirus, Marseille virus or rotavirus of the present invention has a high activity and a sequence length of less than 800aa, and the shortest active sequence is 622aa, which is significantly shorter than the control capping enzyme sequence of 868aa. It is well known in the art that when the activity is basically equivalent, the delivery vector of the in vivo system has a payload limitation, and the shorter the sequence, the easier it is to be suitable for the delivery of the vector. In addition, the shorter the sequence of the enzyme, the more stable the conformation, and the easier it is to synthesize in vitro;

[0247] 6. The commercial capping enzyme derived from poxvirus needs to express two subunits at the same time to complete Cap0 capping, and the process is cumbersome and requires multiple purifications. The capping enzyme of the present invention can complete Cap0 capping with a single subunit, which greatly simplifies the production steps and reduces the production cost. DETAILED DESCRIPTION

[0248] The present invention will be further described below in conjunction with specific embodiments, but these specific embodiments cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art may make various changes or modifications to these specific embodiments without departing from the scope of the technical solution of the present invention, and the changed and modified embodiments still fall within the scope of protection of the present invention.

[0249] All chemicals or products used in the examples are commercially available.

[0250] Example 1. In vivo experiments of the capping enzyme of the present invention

[0251] The present invention constructs an intracellular expression system to test the activity of capping enzymes in vivo. The system fuses the candidate capping enzyme to the N' terminus of RNA polymerase T7 to drive reporter gene expression, and the positive control uses the NP868R capping enzyme, a single-chain protein with the highest in vivo activity in previous literature.

[0252] 1.1 Mammalian cell part

[0253] Cell culture: CHO and HEK 293T cells were cultured in DMEM medium (High Glucose Dulbecco's modified Eagle's media) supplemented with 10% FBS (Fetal bovine serum, GIBCO) and 1% penicillin-streptomycin (Hyclone). The cells were maintained at 5% CO2 and 37°C.

[0254] Plasmid construction: The sequence of the present invention was synthesized by GenScript Biotech Co., Ltd. onto a vector constructed in the laboratory in advance. The obtained plasmid was transformed into Top10 and DH5α E. coli competent cells, and single clones were screened by LB medium with ampicillin resistance or chloramphenicol resistance, and the plasmid was extracted (QIAprep Spin Miniprep) and sequenced (Beijing Ruibo Xingke Biotechnology Co., Ltd.).

[0255] Cell transfection: The required cells were plated into a 24-well cell culture plate at 10^5 / well and transfected when the cells grew to 70% confluence. The amount of transfected plasmid was 1 μg / well, and the transfection was performed using Lipofectamine 3000 (Invitrogen) reagent according to the instructions.

[0256] Flow cytometry: Samples were collected 2 days after transfection, the culture medium was removed, and the cells were collected in a 96-well plate with 200 μL 4% paraformaldehyde. BD Fortessa SORP machine was used for analysis, 10^5 cells were collected per well, and data analysis was performed using FlowJo (V10) software.

[0257] 1.2 Yeast

[0258] Yeast cell culture and transformation: The yeast strain used in the experiment was CYE72 (S.cerevisiaeBY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 derived from S288C). The culture medium used for the CYE72 strain was YPD, and the related plasmid was chloramphenicol resistant. The plasmid was linearized with BsaI enzyme and then transformed into yeast CYE72 competent cells. Yeast transformation was performed using the Frozen-EZ Yeast Transformation II Kit TM After transformation, the yeast strains were cultured in SD-U and SD-H solid medium for 2-4 days, and single clones were picked for verification and testing.

[0259] Yeast flow cytometry: Pick a single colony into 500 μL SD medium (with corresponding amino acid deficiency), place it in a 2 mL 96 deep well plate (covered with a breathable membrane), and incubate at 30 ° C, 900 rpm for 24 hours. Dilute the above culture solution 200 times, and grow it for another 16 hours overnight under the same conditions (add the required inducer). Dilute it 20 times with 1xPBS (Vyeast: VPBS = 1:10), and the final volume of each sample is 200 μL. Set up blank, negative and positive controls, mark them well, and use the cell flow high-throughput mode for detection.

[0260] Yeast flow cytometry: The number of cells measured for each sample was >50,000, and the machine used was a LSRII Fortessa flowcytometer (BD Biosciences).

[0261] Example 2. In vitro experiments of the capping enzyme of the present invention

[0262] 2.1 Yeast expression screening and protein purification methods

[0263] After the protein expression gene is synthesized, it is connected to the Pichia pastoris expression vector pPIC9K for intracellular expression. The constructed vector is linearized with Sal I and then electroporated into Pichia pastoris cells, and the primary screening is performed using a histidine-deficient MD culture plate, followed by high-copy screening using a YPD plate containing different G418 concentrations. After the clones grow out, the clones are picked into BMGY medium for culture, and then the cells are collected by centrifugation and suspended in BMMY containing 0.5% methanol for continued culture and induced expression for 72 hours (1% methanol is supplemented daily during this period). After the expression is completed, the cells are collected by centrifugation, suspended in 20mM Tris, 150mM NaCl, 5mM Imidazole, 5% Glycerol, pH8.0 for ultrasonic disruption, and then centrifuged at 12000g, 4 degrees for 10 minutes to collect the supernatant, which is purified by nickel affinity filler and washed with washing buffer (20mM Tris, 300mM NaCl, 20mMImidazole, 5% Glycerol, pH8.0), and finally eluted with elution buffer (20mM Tris, 300mM NaCl, 250mM Imidazole, 5% Glycerol, pH8.0). The protein expression of different cloned cells is tested by SDS. The corresponding high-expressing cells can be amplified and purified according to the above expression method. If necessary, the protein can be purified with molecular sieves, and the final protein concentration is 0.5-1 mg / ml and stored in storage buffer (20mM Tris, 300mM NaCl, 1mM EDTA, 0.1mM TCEP, 20% glycerol, pH7.4). The protein can be stored at -80 degrees for long-term storage.

[0264] 2.2 In vitro synthesis of IVT mRNA

[0265] The RNA required for the capping reaction was prepared by in vitro transcription using T7 RNA polymerase. The mRNA product is a GA-rich sequence of ~30 nt (5'-GGUAGGAGAGGAAGGAAGGGAAGAAAGAAG-3'). The mRNA in vitro transcription reaction system is 30 μL, including DNA template, 4mM / NTP, 40mM Tris-HCl, 30mM MgCl2, 1mM DTT, 1unit / μl RNA inhibitor, 8unit / ul T7 RNA polymerase (NEB, New England Biolabs), 0.0025U / ul pyrophosphatase (NEB, New England Biolabs), pH 7.9, 37°C for 4h, and the mRNA product is reacted by Purified using RNA Cleanup Kit (NEB, New England Biolabs).

[0266] 2.3 Preparation of capped mRNA

[0267] Incubate IVT mRNA at 65°C for 5 min, then place on ice for 5 min. Prepare mRNA capping reaction buffer, including 50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2, 1 mM DTT, 1 mM GTP, 0.8 mM SAM, 1 unit / ul Vaccinia Capping enzyme, pH 8. Place denatured mRNA in reaction buffer, incubate at 37°C for 0.5 h, and use The mRNA was purified using RNA Cleanup Kit (NEB, New England Biolabs). The capped mRNA was stored at -80°C.

[0268] 2.4 LC-MS analysis of mRNA capping products

[0269] LC–MS analysis was performed using an Agilent 1290 Infinity II UPLC system coupled to a TOF mass spectrometer (Agilent 6230) with an electrospray source operating in negative ionization mode. MS spectra were collected in the range of m / z 400 to 3000. All samples were separated on an AdvanceBio Oligonucleotides Column (2.1 mm × 150 mm; 2.7 μm particle size, Agilent) at a column temperature of 30 °C at a flow rate of 0.2 mL / min. Mobile phase A consisted of 89.3 mM hexafluoroisopropanol and 9.6 mM triethylamine, and mobile phase B was 100% MeOH. The gradient was maintained at 95% A for 2 min and then increased to 70% A in a linear curve over 18 min. The gradient was changed to 20% A at 20.4 min and then increased linearly to 95% A in 3.6 min. The mass spectrometry results were processed using the Bioconfirm program for deconvolution and quantification.

[0270] Example 3. Sequence activity analysis

[0271] As described above, the relative activities of the capping enzymes of the present invention were determined in CHO cells, HEK 293T cells and yeast cells (see Figure 1-3 ), and the activities of the capping enzymes of the present invention in different species were compared (see Figure 4A and 4B ,in Figure 4A The capping enzyme activities in yeast and HEK 293T cells were compared; Figure 4B Capping enzyme activities were compared in CHO cells and HEK 293T cells).

[0272] The following Table 1 ranks the activities of the capping enzymes of the present invention in different species in in vivo experiments:

[0273] Table 1

[0274]

[0275] The 1st and 2nd columns of the above table reflect the corresponding relationship between the capping enzyme abbreviations recorded in the accompanying drawings and the sequence numbers of the present invention. The activity rankings 1-29 of the capping enzyme SEQ ID NOs: 1-28 of the present invention and the control NP868R in HEK 293T cells recorded in the 3rd column of the above table are from Figure 1, assigned values ​​according to their activity ranking: 100 for the 1st to 5th, 80 for the 6th to 10th, 60 for the 11th to 15th, 40 for the 16th to 20th, 20 for the 21st to 25th, and 0 for the 26th to 29th; the activity rankings of the capping enzymes SEQ ID NOs: 1-28 of the present invention and the control NP868R in CHO cells recorded in the fourth column of the above table are 1-18 (those without data are ranked 18th) from Figure 2 , assigned values ​​according to their activity ranking: 100 for the 1st to 5th, 75 for the 6th to 10th, 50 for the 11th to 15th, and 25 for the 16th to 18th; the activities of the capping enzymes SEQ ID NOs: 1-28 of the present invention and the control NP868R recorded in the 5th column of the above table in yeast cells are ranked 1-9 (those without data are ranked 9th) from Figure 3 , assign values ​​to them according to their activity ranking: the first is 100, the second is 90, the third is 80, and so on, the ninth is 20; the universality index of the capping enzyme SEQ ID NO: 1-28 of the present invention and the control NP868R in different species recorded in the sixth column of the above table is the average value of the values ​​assigned in the third to fifth columns. It can be seen that the capping enzyme of the present invention shows particularly excellent activity in yeast. In addition, some capping enzymes of the present invention have significantly higher cross-species universality than the control.

[0276] In addition, if Figure 5-6 As shown, the capping enzyme of the present invention has a significantly higher relative abundance of Cap0 in an in vitro reaction than a commercially available capping enzyme (NEB, New England Biolabs). It can be seen that the capping enzyme of the present invention has a better capping effect in vitro.

[0277] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the specific implementation of the present invention is described above, it should be understood by those skilled in the art that this is only an example, and the protection scope of the present invention is defined by the attached claims. Those skilled in the art may make various changes or modifications to the technical contents disclosed above without departing from the scope of the technical solution of the present invention, and these changes and modifications all fall within the protection scope of the present invention.

Claims

1. A capping enzyme, comprising or consisting of: (i) an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in any one of SEQ ID NOs: 1-28; or (2) An amino acid sequence encoded by a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the nucleotide sequence shown in any one of SEQ ID NOs: 29-56.

2. The capping enzyme according to claim 1, which is expressed in (a) in vitro, (b) in human cells, (c) in mouse cells or (d) in yeast cells, and has the activities of RNA 5' triphosphatase (RT), guanylate transferase (GT) and RNA guanine-N7 methyltransferase (RNMT).

3. The capping enzyme according to claim 1 or 2, wherein the capping enzyme comprises amino acid substitution, addition and / or deletion of any one of SEQ ID NOs: 1-28 or the capping enzyme is a mutant obtained by amino acid substitution, addition and / or deletion of any one of SEQ ID NOs: 1-28.

4. The capping enzyme according to any one of claims 1 to 3, comprising a deletion of any one of SEQ ID NOs: 1 to 28 or being a mutant obtained by deleting any one of SEQ ID NOs: 1 to 28, wherein the deletion is an N-terminal truncation and / or a C-terminal truncation.

5. The capping enzyme according to claim 3 or 4, wherein the deletion is a deletion of residues 1-6 of SEQ ID NO: 4, a deletion of residues 1-24 of SEQ ID NO: 7, or a deletion of residues 813-1003 of SEQ ID NO:

5.

6. The capping enzyme according to any one of claims 1 to 5, comprising a substitution of any one of SEQ ID NOs: 1 to 28 or being a mutant obtained by substituting any one of SEQ ID NOs: 1 to 28, wherein the substitution is selected from the group consisting of motif shuffling and site-directed mutagenesis.

7. The capping enzyme according to claim 6, wherein the motif rearrangement is to rearrange the motifs in SEQ ID NOs: 1-3, 7-9, 12-13, 16 and 21.

8. The capping enzyme according to claim 6 or 7, wherein the motif rearrangement is to rearrange the following motifs: (a) a motif selected from the group consisting of amino acids 37-41, 108-110, 355-361, 443-445, 495-497, 532-541, 649-653, and 677-684 in SEQ ID NO: 2; (b) a motif in SEQ ID NO:8 selected from the group consisting of amino acids 37-41, 108-110, 355-361, 443-445, 495-497, 532-541, 650-654, and 678-685; (c) a motif selected from the group consisting of amino acids 37-41, 109-111, 356-362, 444-446, 496-498, 533-542, 651-655, and 679-686 in SEQ ID NO:9; (d) a motif selected from the group consisting of amino acids 62-66, 133-135, 380-386, 468-470, 520-522, 557-566, 675-679, and 703-710 in SEQ ID NO:7; (e) a motif selected from the group consisting of amino acids 38-42, 110-112, 357-363, 445-447, 497-499, 534-543, 651-655, and 679-686 in SEQ ID NO: 1; (f) a motif selected from the group consisting of amino acids 38-42, 109-111, 356-362, 444-446, 499-501, 536-545, 653-657, and 681-688 in SEQ ID NO:3; (g) a motif in SEQ ID NO:16 selected from the group consisting of amino acids 63-71, 263-269, 452-459, and 759-763; (h) a motif selected from the group consisting of amino acids 63-71, 263-269, 452-459, and 759-763 of SEQ ID NO: 12; (i) a motif selected from the group consisting of amino acids 63-71, 263-269, 452-459, and 759-763 in SEQ ID NO: 13; or (j) a motif in SEQ ID NO:21 selected from the group consisting of amino acids 63-71, 263-269, 452-459 and 759-763.

9. The capping enzyme according to any one of claims 6 to 8, wherein the motif is rearranged as follows: (a) performing a motif rearrangement on SEQ ID NO:2 selected from the group consisting of: Replace amino acids 37-41 with DRHQR, ERHQN, GKHKS or ERQQV, Replace amino acids 108-110 with ERS, ERN or DRN, Replace amino acids 355-361 with QEAKVIA, QSAHIIS or EELGIVS, Replace amino acids 443-445 with AHQ, AEQ, ENL or AEL, Replace amino acids 495-497 with ENS or EHQ, Replace amino acids 532-541 with IKTVLSVWGD, VKTVLSVWAN, STTVVSVWND or MTTVLSVWGN, Substitution of amino acids 649-653 with VDMAS, VDFVS, MDAAS, VDAST, or VDLSS, and Amino acids 677-684 are replaced with SLSRVGAL, SLSKNGAM, ALLETGSM, LLLEKEAV or SLSKQGSL; (b) performing a motif rearrangement on SEQ ID NO:8 selected from the group consisting of: Replace amino acids 37-41 with DRHQR, DRHQK, ERHQN or ERQQV, Replace amino acids 108-110 with ERS, DRS or ERN, Replace amino acids 355-361 with QEAKVIA, QEARVIA or EELGIVS, Replace amino acids 443-445 with AHQ, ADQ, AEQ or AEL, Replace amino acids 495-497 with SHP or EHQ, Replace amino acids 532-541 with IKTVLSVWGD, VKTVLSVWGN, VKTVLSVWAN or MTTVLSVWGN, Replace amino acids 650-654 with VDMAS, VDFSS, VDFVS, MDAAS or VDLSS, Amino acids 678-685 are replaced with SLSRVGAL, SMSSDKAM, SLSKNGAM, ALLETGSM or SLSKQGSL; (c) rearranging SEQ ID NO:9 by a motif selected from the group consisting of: Replace amino acids 37-41 with DRHQR, DRHQK, ERHQN or GKHKS, Replace amino acids 109-111 with ERS, DRS or DRN, Replace amino acids 356-362 with QEAKVIA, QEARVIA or QSAHIIS, Replace amino acids 444-446 with AHQ, ADQ, AEQ or ENL, Replace amino acids 496-498 with SHP or ENS, Replace amino acids 533-542 with IKTVLSVWGD, VKTVLSVWGN, VKTVLSVWAN or STTVVSVWND, Replace amino acids 651-655 with VDMAS, VDFSS, VDFVS, MDAAS or VDAST, Amino acids 679-686 are replaced with SLSRVGAL, SMSSDKAM, SLSKNGAM, ALLETGSM or LLLEKEAV; (d) performing a motif rearrangement on SEQ ID NO:7 selected from the group consisting of: Replace amino acids 62-66 with DRHQR, DRHQK, ERHQN or ERQQV, Replace amino acids 133-135 with ERS, DRS or ERN, Replace amino acids 380-386 with QEAKVIA, QEARVIA or EELGIVS, Replace amino acids 468-470 with AHQ, ADQ, AEQ or AEL, Replace amino acids 520-522 with SHP or EHQ, Replace amino acids 557-566 with IKTVLSVWGD, VKTVLSVWGN, VKTVLSVWAN or MTTVLSVWGN, Replace amino acids 675-679 with VDMAS, VDFSS, VDFVS, VDAST or VDLSS, Amino acids 703-710 are replaced with SLSRVGAL, SMSSDKAM, SLSKNGAM, LLLEKEAV or SLSKQGSL; (e) rearranging SEQ ID NO:1 by a motif selected from the group consisting of: Replace amino acids 38-42 with DRHQK, ERHQN, GKHKS or ERQQV, Replace amino acids 110-112 with DRS, ERN or DRN, Replace amino acids 357-363 with QEARVIA, QSAHIIS or EELGIVS, Replace amino acids 445-447 with ADQ, AEQ, ENL or AEL, Replace amino acids 497-499 with ENS or EHQ, Replace amino acids 534-543 with VKTVLSVWGN, VKTVLSVWAN, STTVVSVWND or MTTVLSVWGN, Replace amino acids 651-655 with VDFSS, VDFVS, MDAAS, VDAST or VDLSS, Amino acids 679-686 were replaced with SMSSDKAM, SLSKNGAM, ALLETGSM, LLLEKEAV or SLSKQGSL; (f) rearranging SEQ ID NO:3 by a motif selected from the group consisting of: Replace amino acids 38-42 with DRHQR, DRHQK, GKHKS or ERQQV, Replace amino acids 109-111 with ERS, DRS or DRN, Replace amino acids 356-362 with QEAKVIA, QSAHIIS or EELGIVS, Replace amino acids 444-446 with AHQ, ADQ, ENL or AEL, Replace amino acids 499-501 with ENS or EHQ, Amino acids 536-545 are replaced with IKTVLSVWGD, VKTVLSVWGN, STTVVSVWND or MTTVLSVWGN, Replace amino acids 653-657 with VDMAS, VDFSS, MDAAS, VDAST or VDLSS, Amino acids 681-688 are replaced with SLSRVGAL, SMSSDKAM, ALLETGSM, LLLEKEAV or SLSKQGSL; (g) performing a motif rearrangement on SEQ ID NO: 16 selected from the group consisting of: Replace amino acids 63-71 with AIEIQSLEE, AIALQSLEE, AIEIQKIDE, AVEIQSPDE, AVEVQGIEE, AVQVDNDAE or SLIAINKDK, Amino acids 263-269 are replaced with MNVITTI, LNVLTAI, LNVVTTI, LNVITTI, LNVITTV or CNVLSTI, Amino acids 452-459 are replaced with TSMSDYIV, VLNDSYIV, TLKNEYVI, TLPNDYIV, KMKNEYIV, PVNNSYIV or PTN-KFVV, Amino acids 759-763 are replaced with SDGIY, SDEVY, SDGIH, TDDVY or TDSIY; (h) rearranging SEQ ID NO:12 by a motif selected from the group consisting of: Replace amino acids 63-71 with AIALQSLEE, AVEIQEMDE, AIEIQKIDE, AVEIQSPDE, AVEVQGIEE, AVQVDNDAE or SLIAINKDK, Amino acids 263-269 are replaced with LNVLTAI, LNVLTTL, LNVVTTI, LNVITTI, LNVITTV or CNVLSTI, Amino acids 452-459 are replaced with VLNDSYIV, TLRNEYVV, TLKNEYVI, TLPNDYIV, KMKNEYIV, PVNNSYIV or PTN-KFVV, Amino acids 759-763 are replaced with SDEIY, SDEVY, SDGIH, TDDVY or TDSIY; (i) rearranging SEQ ID NO: 13 by a motif selected from the group consisting of: Replace amino acids 63-71 with AIEIQSLEE, AIALQSLEE, AVEIQEMDE, AIEIQKIDE, AVEVQGIEE, AVQVDNDAE or SLIAINKDK, Amino acids 263-269 are replaced with MNVITTI, LNVLTAI, LNVLTTL, LNVITTI, LNVITTV or CNVLSTI, Amino acids 452-459 are replaced with TSMSDYIV, VLNDSYIV, TLRNEYVV, TLKNEYVI, KMKNEYIV, PVNNSYIV or PTN-KFVV, Amino acids 759-763 are replaced with SDGIY, SDEIY, SDEVY, TDDVY or TDSIY; or (j) rearranging SEQ ID NO:21 by a motif selected from the group consisting of: Replace amino acids 63-71 with AIEIQSLEE, AIALQSLEE, AVEIQEMDE, AVEIQSPDE, AVEVQGIEE, AVQVDNDAE or SLIAINKDK, Amino acids 263-269 are replaced with MNVITTI, LNVLTAI, LNVVTTI, LNVITTI, LNVITTV or CNVLSTI, Amino acids 452-459 are replaced with TSMSDYIV, VLNDSYIV, TLRNEYVV, TLPNDYIV, KMKNEYIV, PVNNSYIV or PTN-KFVV, Amino acids 759-763 are replaced with SDGIY, SDEIY, SDGIH, TDDVY or TDSIY. 10 . The capping enzyme according to claim 6 , wherein the site-directed mutagenesis is performed on the sequences of SEQ ID NOs: 1-4, 6-9 and 12.

11. The capping enzyme according to claim 6 or 10, wherein the site-directed mutagenesis is to mutate the following sites: (a) a position selected from the group consisting of amino acids 21, 23, 37, 45, 100, 137, 139, 164, 180, 192, 235, 242, 255, 264, 303, 322, 354, 372, 397, 413, 417, 457, 493, 514, 567, 570, 574, 600, 603, 610, 628, 643, 653, 654, 675, 684, 710, and 759 of SEQ ID NO: 1; (b) a position selected from the group consisting of amino acids 20, 22, 36, 44, 98, 135, 137, 162, 178, 190, 233, 240, 253, 262, 301, 320, 352, 370, 395, 411, 415, 455, 491, 512, 565, 568, 572, 598, 601, 608, 626, 641, 651, 652, 673, 682, 708, and 757 of SEQ ID NO:2; (c) a position selected from the group consisting of amino acids 21, 23, 37, 45, 99, 136, 138, 163, 179, 191, 234, 241, 254, 263, 302, 321, 353, 371, 396, 412, 416, 456, 495, 516, 569, 572, 576, 602, 605, 612, 630, 645, 655, 656, 677, 686, 712, and 761 of SEQ ID NO:3; (d) a position selected from the group consisting of amino acids 45, 47, 61, 69, 123, 160, 162, 187, 203, 215, 258, 265, 278, 287, 326, 345, 377, 395, 420, 436, 440, 480, 516, 537, 590, 593, 597, 624, 627, 634, 652, 667, 677, 678, 699, 708, 734, and 782 of SEQ ID NO:7; (e) a position selected from the group consisting of amino acids 20, 22, 36, 44, 98, 135, 137, 162, 178, 190, 233, 240, 253, 262, 301, 320, 352, 370, 395, 411, 415, 455, 491, 512, 565, 568, 572, 599, 602, 609, 627, 642, 652, 653, 674, 683, 709, and 757 of SEQ ID NO:8; (f) a position selected from the group consisting of amino acids 20, 22, 36, 44, 99, 136, 138, 163, 179, 191, 234, 241, 254, 263, 302, 321, 353, 371, 396, 412, 416, 456, 492, 513, 566, 569, 573, 599, 602, 609, 627, 642, 653, 654, 675, 684, 710, and 759 of SEQ ID NO:9; (g) a position selected from the group consisting of 9, 11, 13, 19, 23, 24, 40, 46, 47, 50, 51, 52, 63, 67, 70, 71, 77, 78, 83, 88, 98, 99, 111, 113, 115, 116, 119, 123, 129, 130, 147, 155, 161, 162, 166, 167, 171, 172, 174, 175, 182, 183, 184, 191, 203, 205, 206, 207, 219, 222, 224, 226, 229, 230, 231 1, 234, 236, 237, 238, 240, 242, 249, 250, 251, 263, 268, 275, 276, 279, 281, 294, 297, 298, 299, 300, 302, 304, 311, 316, 320, 321, 324, 328, 332, 333, 334, 346, 347, 348, 351, 355, 356, 358, 360, 362, 363, 369, 370, 373, 374, 382, ​​394, 395, 396, 400, 404, 406, 409, 413, 414, 417, 420, 421, 426, 430, 432, 437, 447, 453, 455, 463, 468, 477, 478, 482, 495, 496, 497, 498, 501, 504, 505, 509, 515, 520, 526, 528, 530, 532, 533, 536, 542, 550, 551, 552, 553, 554, 555, 556, 558, 559, 564, 567, 581, 585, 596, 597, 602, 608, 62 amino acids 5, 629, 633, 643, 650, 653, 660, 663, 664, 670, 674, 676, 680, 682, 686, 688, 690, 702, 707, 713, 715, 716, 721, 729, 730, 733, 734, 739, 744, 745, 746, 749, 751, 763, 768, 770, 773, 779, 780, 785, 787, 802, 804, 810, 811, 819, 821, 827, 829, 830, 834, and 835; (h) a position selected from the group consisting of amino acids 12, 17, 20, 35, 37, 77, 118, 132, 158, 159, 178, 179, 188, 194, 195, 200, 212, 223, 280, 309, 314, 324, 343, 441, 485, 534, 558, 594, 599, 624, 654, 656, 657, 658, 659, 682, 683, 697, 700, 733, 761, 769, 774, 777, 798, and 821 of SEQ ID NO:6; or (i) a position in SEQ ID NO:4 selected from the group consisting of 250, 259, 281, 285, 322, 337, 367, 368, 382, ​​383, 386, 393, 395, 409, 410, 415, 435, 444, 451, 455, 458, 472, 479, 481, 487, 491, 496, 513, 544, 545, 550, 569, 576, 583, 585, 597, 604, 609, 614, 627, 631, 632, 642, 659, 66 1, 673, 676, 683, 697, 727, 729, 746, 748, 749, 767, 777, 791, 794, 805, 822, 826, 829, 834, 850, 870, 904, 905, 906, 919, 920, 921, 923, 925, 927, 944, 948, 956, 960, 962, 970, 1002, 1006, 1007, 1008, 1011, 1020, 1027, 1040 and 1045 amino acids.

12. The capping enzyme according to any one of claims 6, 10-11, wherein the site-directed mutation is: (a) performing a site-directed mutagenesis of SEQ ID NO: 1 selected from the group consisting of H21R / K, K23R / H, T37S / N / Q, P45C / G, A100V / I / L / M / F / Y / W, N137S / T / Q, K139R / H, T164S / N / Q, V180A / I / L / M / F / Y / W, P192C / G, R235H / K, Y242A / V / I / L / M / F / W, K255R / H, L264A / V / I / M / F / Y / W, Q303S / T / N, S322T / N / Q, N354S / T / Q, P372C / G, G3 97C / P, D413E, K417R / H, K457R / H, E493D, K514R / H, I567A / V / L / M / F / Y / W, Q570S / T / N, G574C / P, G600C / P, M603A / V / I / L / F / Y / W, N 610S / T / Q, S628T / N / Q, L643A / V / I / M / F / Y / W, M653A / V / I / L / F / Y / W, A654V / I / L / M / F / Y / W, E675D, G684C / P, S710T / N / Q and K759R / H; (b) performing a site-directed mutagenesis of SEQ ID NO: 2 selected from the group consisting of Q20S / T / N, A22V / I / L / M / F / Y / W, T36S / N / Q, P44C / G, S98T / N / Q, N135S / T / Q, K137R / H, T162S / N / Q, A178V / I / L / M / F / Y / W, P190C / G, R233H / K, Y240A / V / I / L / M / F / W, D253E, L262A / V / I / M / F / Y / W, Q301S / T / N, S320T / N / Q, N352S / T / Q, S3 70T / N / Q, G395C / P, E411D, K415R / H, K455R / H, E491D, L512A / V / I / M / F / Y / W, K565R / H, Q568S / T / N, G572C / P, G598C / P, S601T / N / Q, N608S / T / Q, G626C / P, V641A / I / L / M / F / Y / W, F651A / V / I / L / M / Y / W, S652T / N / Q, N673S / T / Q, K682R / H, P708C / G and G757C / P; (c) performing site-directed mutagenesis on SEQ ID NO: 3 selected from the group consisting of H21R / K, K23R / H, T37S / N / Q, S45T / N / Q, S99T / N / Q, N136S / T / Q, K138R / H, T163S / N / Q, T179S / N / Q, P191C / G, L234A / V / I / M / F / Y / W, Y241A / V / I / L / M / F / W, D254E, T263S / N / Q, A302V / I / L / M / F / Y / W, Q321S / T / N, H353R / K, P371C / G, G396C / P, K412R / H, E416D, K456R / H, E495D, L516A / V / I / M / F / Y / W, T569S / N / Q, R572H / K, G576C / P, K602R / H, M605A / V / I / L / F / Y / W, D 612E, N630S / T / Q, N645S / T / Q, F655A / V / I / L / M / Y / W, V656A / I / L / M / F / Y / W, E677D, G686C / P, A712V / I / L / M / F / Y / W and N761S / T / Q; (d) performing a site-directed mutagenesis of SEQ ID NO:7 selected from the group consisting of H45R / K, K47R / H, T61S / N / Q, P69C / G, Q123S / T / N, N160S / T / Q, H162R / K, T187S / N / Q, V203A / I / L / M / F / Y / W, P215C / G, K258R / H, Y265A / V / I / L / M / F / W, K278R / H, V287A / I / L / M / F / Y / W, Q326S / T / N, Q345S / T / N, N377S / T / Q, P395C / G, G420C / P, E436D, K440R / H, K480R / H, E516D, K537R / H, V590A / I / L / M / F / Y / W, Q593S / T / N, G597C / P, G624C / P, L627A / V / I / M / F / Y / W, N63 4S / T / Q, N652S / T / Q, I667A / V / L / M / F / Y / W, A677V / I / L / M / F / Y / W, A678V / I / L / M / F / Y / W, E699D, G708C / P, S734T / N / Q and K782R / H; (e) performing a site-directed mutagenesis of SEQ ID NO: 8 selected from the group consisting of H20R / K, K22R / H, A36V / I / L / M / F / Y / W, S44T / N / Q, G98C / P, G135C / P, Q137S / T / N, T162S / N / Q, V178A / I / L / M / F / Y / W, S190T / N / Q, Q233S / T / N, H240R / K, K253R / H, I262A / V / L / M / F / Y / W, Q301S / T / N, L320A / V / I / M / F / Y / W, N352S / T / Q, P370 C / G, E395D, E411D, K415R / H, K455R / H, S491T / N / Q, K512R / H, I565A / V / L / M / F / Y / W, Q568S / T / N, E572D, G599C / P, F602A / V / I / L / M / Y / W, N609S / T / Q, S627T / N / Q, I642A / V / L / M / F / Y / W, A652V / I / L / M / F / Y / W, S653T / N / Q, E674D, E683D, S709T / N / Q and K757R / H; (f) performing a site-directed mutagenesis of SEQ ID NO:9 selected from the group consisting of Q20S / T / N, D22E, E36D, P44C / G, S99T / N / Q, D136E, V138A / I / L / M / F / Y / W, I163A / V / L / M / F / Y / W, V179A / I / L / M / F / Y / W, K191R / H, R234H / K, Q241S / T / N, K254R / H, L263A / V / I / M / F / Y / W, Q302S / T / N, K321R / H, H353R / K, S371T / N / Q, K396R / H, D412E, V416A / I / L / M / F / Y / W, K456R / H, K492R / H, K513R / H, I566A / V / L / M / F / Y / W, Q569S / T / N, T573S / N / Q, G599C / P, M602A / V / I / L / F / Y / W, N 609S / T / Q, K627R / H, I642A / V / L / M / F / Y / W, L653A / V / I / M / F / Y / W, S654T / N / Q, L675A / V / I / M / F / Y / W, G684C / P, S710T / N / Q and K759R / H; (g) Perform site-directed mutagenesis on SEQ ID NO:12 selected from the following group: S9T / N / Q, A11V / I / L / M / F / Y / W, V13A / I / L / M / F / Y / W, V19A / I / L / M / F / Y / W, D23E, D24E, T40S / N / Q, F46A / V / I / L / M / Y / W, N47S / T / Q, V50A / I / L / M / F / Y / W, K51R / H, T52S / N / Q, A63V / I / L / M / F / Y / W, Q67S / T / N, E70D, E71D, R77H / K, C78G / P, D83E, V88A / I / L / M / F / Y / W, T98S / N / Q, N99S / T / Q, K111R / H, N113S / T / Q, E115D, E116D, L119A / V / I / M / F / Y / W, K123R / H, Y129A / V / I / L / M / F / W, N130S / T / Q, A147V / I / L / M / F / Y / W, I155A / V / L / M / F / Y / W, E161D, A162V / I / L / M / F / Y / W, Q166S / T / N, N167S / T / Q, K171R / H, K172R / H, K174R / H, E175D, P182C / G, I183A / V / L / M / F / Y / W, A184V / I / L / M / F / Y / W, F191A / V / I / L / M / Y / W, K194R / H, H203R / K, R205H / K, V206A / I / L / M / F / Y / W, I207A / V / L / M / F / Y / W, F219A / V / I / L / M / Y / W, F222A / V / I / L / M / Y / W, S224T / N / Q, R226H / K, S229T / N / Q, N230S / T / Q, G231C / P, L234A / V / I / M / F / Y / W, K236R / H, N237S / T / Q, T238S / N / Q, K240R / H, K242R / H, K249R / H, R250H / K, V251A / I / L / M / F / Y / W, M263A / V / I / L / F / Y / W, T268S / N / Q, L275A / V / I / M / F / Y / W, Y276A / V / I / L / M / F / W, K279R / H, I281A / V / L / M / F / Y / W, Y294A / V / I / L / M / F / W, R297H / K, Q298S / T / N, Y299A / V / I / L / M / F / W, S300T / N / Q, F302A / V / I / L / M / Y / W, F304A / V / I / L / M / Y / W, D311E, S316T / N / Q, K320R / H, E321D, F324A / V / I / L / M / Y / W,I328A / V / L / M / F / Y / W、K332R / H、D333E、N334S / T / Q、G346C / P、N347S / T / Q、V348A / I / L / M / F / Y / W、K351R / H、K355R / H、I356A / V / L / M / F / Y / W、E358D、Q360S / T / N、I362A / V / L / M / F / Y / W、N363S / T / Q、Y369A / V / I / L / M / F / W、K370R / H、A373V / I / L / M / F / Y / W、T374S / N / Q、V382A / I / L / M / F / Y / W、S394T / N / Q、A395V / I / L / M / F / Y / W、K396R / H、H400R / K、E404D、R406H / K、F409A / V / I / L / M / Y / W、L413A / V / I / M / F / Y / W、D414E、D417E、N420S / T / Q、V421A / I / L / M / F / Y / W、P426C / G、L430A / V / I / M / F / Y / W、S432T / N / Q、V437A / I / L / M / F / Y / W、P447C / G、S453T / N / Q、S455T / N / Q、A463V / I / L / M / F / Y / W、F468A / V / I / L / M / Y / W、L477A / V / I / M / F / Y / W、I478A / V / L / M / F / Y / W、K482R / H、K495R / H、D496E、E497D、P498C / G、G501C / P、N504S / T / Q、I505A / V / L / M / F / Y / W、T509S / N / Q、F515A / V / I / L / M / Y / W、A520V / I / L / M / F / Y / W、D526E、C528G / P、G530C / P、F532A / V / I / L / M / Y / W、G533C / P、I536A / V / L / M / F / Y / W、P542C / G、I550A / V / L / M / F / Y / W、L551A / V / I / M / F / Y / W、N552S / T / Q、G553C / P、554S / N / Q、D555E、K556R / H、Y558A / V / I / L / M / F / W、K559R / H、A564V / I / L / M / F / Y / W、T567S / N / Q、S581T / N / Q、Y585A / V / I / L / M / F / W、N596S / T / Q、N597S / T / Q、G602C / P、S608T / N / Q、S625T / N / Q、K629R / H、Y633A / V / I / L / M / F / W、E643D、H650R / K、I653A / V / L / M / F / Y / W、R660H / K、K663R / H、E664D、D670E、V674A / I / L / M / F / Y / W, R676H / K, E680D, E682D, N686S / T / Q, V688A / I / L / M / F / Y / W, S690T / N / Q, G702C / P, N707S / T / Q, D713E, K715R / H, V716A / I / L / M / F / Y / W, F721A / V / I / L / M / Y / W, N729S / T / Q, M730A / V / I / L / F / Y / W, D733E, V734A / I / L / M / F / Y / W, K739R / H, E744D, L745A / V / I / M / F / Y / W, F746A / V / I / L / M / Y / W, D749E, T751S / N / Q, Y763A / V / I / L / M / F / W, S768T / N / Q, V770A / I / L / M / F / Y / W, T773S / N / Q, N779S / T / Q, L780A / V / I / M / F / Y / W, Q785S / T / N, T787S / N / Q, F802A / V / I / L / M / Y / W, N804S / T / Q, I810A / V / L / M / F / Y / W, E811D, S819T / N / Q, Y821A / V / I / L / M / F / W, G827C / P, T829S / N / Q, M830A / V / I / L / F / Y / W, S834T / N / Q, and E835D; (h) performing site-directed mutagenesis on SEQ ID NO: 6 selected from the group consisting of N12S / T / Q, I17A / V / L / M / F / Y / W, S20T / N / Q, P35C / G, N37S / T / Q, N77S / T / Q, G118C / P, K132R / H, Q158S / T / N, R159H / K, A178V / I / L / M / F / Y / W, D179E, T188S / N / Q, T194S / N / Q, N195S / T / Q, V200A / I / L / M / F / Y / W, V212A / I / L / M / F / Y / W, F 223A / V / I / L / M / Y / W, A280V / I / L / M / F / Y / W, H309R / K, A314V / I / L / M / F / Y / W, V324A / I / L / M / F / Y / W, Y343A / V / I / L / M / F / W, Y441A / V / I / L / M / F / W, Q485S / T / N, N534S / T / Q , H558R / K, G594C / P, D599E, H624R / K, G654C / P, R656H / K, N657S / T / Q, M658A / V / I / L / F / Y / W, H659R / K, E682D, N683S / T / Q, H697R / K, C700G / P, P733C / G, A761V / I / L / M / F / Y / W, V769A / I / L / M / F / Y / W, S774T / N / Q, P777C / G, E798D, M821A / V / I / L / F / Y / W; or (i) performing site-directed mutagenesis on SEQ ID NO: 4 selected from the group consisting of A250V / I / L / M / F / Y / W, M259A / V / I / L / F / Y / W, G281C / P, E285D, Y322A / V / I / L / M / F / W, N337S / T / Q, P367C / G, S368T / N / Q, K382R / H, G383C / P, D386E, F393A / V / I / L / M / Y / W, E395D, V409A / I / L / M / F / Y / W, M 410A / V / I / L / F / Y / W, G415C / P, N435S / T / Q, F444A / V / I / L / M / Y / W, D451E, E455D, Y458A / V / I / L / M / F / W, A472V / I / L / M / F / Y / W, K479R / H, S481T / N / Q, K487R / H, D491E, Y496A / V / I / L / M / F / W, V513A / I / L / M / F / Y / W, K544R / H,D545E, L550A / V / I / M / F / Y / W, C569G / P, K576R / H, Q583S / T / N, T585S / N / Q, N597S / T / Q, E604D, D609E, N614S / T / Q, P627C / G, E631D, R632H / K, Y642A / V / I / L / M / F / W, I659A / V / L / M / F / Y / W, V661A / I / L / M / F / Y / W, I673A / V / L / M / F / Y / W, S676T / N / Q, K683R / H, K697R / H, A727V / I / L / M / F / Y / W, S729T / N / Q, C746G / P, P748C / G, M749A / V / I / L / F / Y / W, Q767S / T / N, V777A / I / L / M / F / Y / W, T791S / N / Q, I794A / V / L / M / F / Y / W, Q805S / T / N, G822C / P, N826S / T / Q, D829E, L834A / V / I / M / F / Y / W, N850S / T / Q, L870A / V / I / M / F / Y / W, V904A / I / L / M / F / Y / W, E905D, L906A / V / I / M / F / Y / W, N919S / T / Q, D920E, K921R / H, D923E, K925R / H, L927A / V / I / M / F / Y / W, G944C / P, P948C / G, F956A / V / I / L / M / Y / W, G960C / P, Y962A / V / I / L / M / F / W, K970R / H, F1002A / V / I / L / M / Y / W, A1006V / I / L / M / F / Y / W, Q1007S / T / N, Y1008A / V / I / L / M / F / W, N1011S / T / Q, N1020S / T / Q, S1027T / N / Q, R1040H / K and Y1045A / V / I / L / M / F / W., 13. A combination of capping enzymes, comprising at least two capping enzymes of different sequences according to any one of claims 1 to 12.

14. A nucleic acid sequence encoding the capping enzyme according to any one of claims 1 to 12. A nucleic acid construct comprising the nucleic acid sequence of claim 14 .

16. The nucleic acid construct according to claim 15, further comprising a resistance gene selected from the group consisting of an ampicillin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, a spectinomycin resistance gene, a tetracycline resistance gene, a bleomycin resistance gene, a streptomycin resistance gene, a hygromycin resistance gene, a gentamicin resistance gene, an apramycin sulfate resistance gene, an erythromycin resistance gene, a nourseoinhibin resistance gene, a geneticin resistance gene, and a puromycin resistance gene.

17. The nucleic acid construct according to claim 15 or 16, which is obtained by introducing the nucleic acid sequence according to claim 14 into a plasmid vector, wherein the plasmid vector is selected from the group consisting of a prokaryotic expression vector, a yeast expression vector, a plant expression vector, an insect expression vector and a mammalian expression vector.

18. A recombinant host cell comprising the nucleic acid sequence of claim 14 or the expression construct of any one of claims 15-17.

19. The recombinant host cell according to claim 18, wherein the cell is prepared by introducing the nucleic acid sequence according to claim 14 or the nucleic acid construct according to any one of claims 14-16 into a host cell, preferably the host cell is Escherichia coli, a yeast cell or a mammalian cell, more preferably an Escherichia coli Top10 or DH5α cell, a Saccharomyces cerevisiae cell, a Chinese hamster ovary cell or a human embryonic kidney cell.

20. A kit for capping RNA, comprising: (i) the capping enzyme of any one of claims 1 to 12 or the combination of claim 13; (ii) a storage buffer in which the capping enzyme or combination is located; and (iii) a reaction buffer, The storage buffer is the same as or different from the reaction buffer.

21. The kit according to claim 20, further comprising: (iv) guanosine triphosphate (GTP) or modified GTP; (v) a methyl donor; and (vi) Optional target RNA to be capped.

22. The kit according to claim 20 or 21, wherein the storage buffer comprises or consists of: 20-100 mM Tris-HCl, 1-20 mM KCl, 0.5-5 mM MgCl2 and 0.5-10 mM dithiothreitol (DTT), preferably the storage buffer comprises or consists of: 50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2 and 1 mM DTT.

23. The kit according to any one of claims 20-22, wherein the reaction buffer comprises or consists of: 20-100 mM Tris-HCl, 1-20 mM KCl, 0.5-5 mM MgCl2 and 0.5-10 mM dithiothreitol (DTT), preferably the reaction buffer comprises or consists of: 50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2 and 1 mM DTT.

24. The kit according to any one of claims 21-23, wherein the modified GTP is selected from the group consisting of GTP modified on the base, GTP modified on the ribose, and GTP modified on the α-phosphate group; preferably, the modified GTP is selected from the group consisting of GTP modified on the 2'-OH of ribose and GTP modified on the 3'-OH of ribose; more preferably, the modified GTP is selected from the group consisting of GTP modified by O-methylation on the 3'-OH of ribose, GTP modified by NH2 on the 3'-OH of ribose, and dGTP modified by N3 on the 3'-OH of ribose.

25. The kit according to any one of claims 21-24, wherein the methyl donor is selected from the group consisting of choline, trimethylglycine, methionine, S-adenosylmethionine (SAM), methylfolate and methylcobalamin, preferably the methyl donor is SAM.

26. A method for capping RNA in vitro, comprising: (i) providing an uncapped RNA sample; (ii) adding the capping enzyme of any one of claims 1 to 12 or the combination of claim 13, guanosine triphosphate (GTP) or modified GTP, and a methyl donor to a reaction buffer to provide a capping reaction mixture; (iii) reacting the RNA sample with the mixture at a temperature of 30° C. to 60° C. for 10 to 200 minutes to form a reaction product, i.e., capped RNA.

27. The method according to claim 26, wherein in step (i), the uncapped RNA sample is incubated at 50-80°C for 3-15 minutes, preferably at 55-70°C for 5-10 minutes, and then ice-bathed, preferably the ice-bath is carried out for 5-10 minutes.

28. The method according to claim 26 or 27, wherein in step (ii) the capping enzyme or combination is added to the reaction buffer in an amount of 0.2-2.0, preferably 0.5-1.0 U / μL.

29. The method according to any one of claims 26 to 28, wherein in step (ii) the GTP or modified GTP is added to the reaction buffer in an amount of 0.2-5 mM, preferably 0.5-1 mM.

30. The method according to any one of claims 26-29, wherein the methyl donor is added to the reaction buffer in step (ii) in an amount of 0.1-0.8 mM.

31. The method according to any one of claims 26-30, wherein in step (ii) the buffer comprises or consists of 20-100 mM Tris-HCl, 1-20 mM KCl, 0.5-5 mM MgCl2 and 0.5-10 mM dithiothreitol (DTT), preferably the buffer comprises or consists of 50 mM Tris-HCl, 5 mM KCl, 1 mM MgCl2 and 1 mM DTT.

32. The method according to any one of claims 26 to 31, wherein step (iii) is reacted at 30-42°C for 45-90 minutes.

33. The method according to any one of claims 26 to 32, further comprising: (iv) purifying the reaction product and / or storing the reaction product or the purified reaction product at -80°C.

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