Bst DNA polymerase mutant and use thereof

Abstract

Amino acid mutations at one or more different sites are carried out on the basis of a wild-type Bst-LF, and an obtained Bst DNA polymerase mutant has higher DNA polymerase activity, strand displacement activity, and / or thermal stability.

Description

Bst DNA polymerase mutant and its application

[0001] Related applications

[0002] This application claims priority to the Chinese patent application filed on December 19, 2023, with application number CN202311743966.X and titled “High-temperature-resistant Bst DNA polymerase mutants and their applications”, and the Chinese patent application filed on December 19, 2023, with application number CN202311743948.1 and titled “Bst DNA polymerase mutants with improved enzyme activity and their applications”, the entire text of which is hereby incorporated by reference. Technical Field

[0003] The present application belongs to the field of biotechnology and relates to a Bst DNA polymerase mutant and its application, and further relates to a Bst DNA polymerase mutant with high temperature resistance and / or improved enzyme activity and its application. Background Art

[0004] Since Kornberg discovered and detailed the DNA polymerase I from Escherichia coli in 1958, various DNA polymerases have been isolated and characterized from both prokaryotes and eukaryotes, garnering widespread attention and application in the biomedical community. A large fragment of DNA polymerase from Geobacillus stearothermophilus (Bst) possesses strong strand-displacement activity and thermostability, possessing both 5'-3' DNA polymerase activity and lacking 5'-3' exonuclease activity. It has been widely used in isothermal amplification experiments such as multiple strand displacement amplification (MDA), rolling circle amplification (RCA), and loop-mediated isothermal amplification (LAMP). LAMP is an extremely powerful nucleic acid amplification method that allows the continuous amplification of target nucleic acids at a single temperature using four to six primers and a DNA polymerase with strand-displacement activity. Similar to PCR, LAMP exponentially amplifies the target nucleic acid. However, unlike PCR, LAMP does not require heating for melting and cooling for annealing. Primer binding and amplification occur at a single temperature, utilizing the DNA polymerase's inherent strand displacement activity to achieve template melting. Therefore, LAMP does not require complex, specialized laboratory equipment and is much faster, making it extremely widely used in medical testing and the food industry. RT-LAMP, an extension of LAMP technology, directly uses mRNA as a template for the LAMP reaction. DNA polymerases with reverse transcription activity or multiple enzymes can be used in the reaction. Bst DNA polymerase Large Fragment has become the most popular enzyme in both LAMP and RT-LAMP techniques due to its superior DNA polymerase activity, strand displacement activity, and thermal stability. In recent years, the trend toward faster and more sensitive assays has posed new challenges to the performance of Bst DNA polymerase.

[0005] Although LAMP and RT-LAMP technologies are widely used in many fields, they still face some technical difficulties. For example, the current LAMP and RT-LAMP reaction temperatures are generally 60-65°C, which is less effective for amplifying target nucleic acids with high GC content. A common strategy to address the difficulty of amplifying high GC-rich templates is to increase the reaction temperature, but the large fragment of wild-type Bst DNA polymerase is not capable of higher reaction temperatures. In addition, nonspecific amplification of LAMP and RT-LAMP is also a technical difficulty. Increasing the reaction temperature can also reduce some nonspecific amplification. Therefore, a heat-resistant Bst DNA polymerase is urgently needed to adapt to higher reaction temperatures to achieve better detection results.

[0006] In view of this, the present application proposes a mutant Bst DNA polymerase with better thermal stability and / or higher enzymatic activity. Summary of the Invention

[0007] The purpose of the present application is to provide a Bst DNA polymerase mutant with high thermal stability; and also to provide a Bst DNA polymerase mutant with higher enzyme activity than the truncated wild-type Bst DNA polymerase.

[0008] The technical solutions adopted in this application are:

[0009] A thermostable Bst DNA polymerase mutant, which is a protein described in any one of a1-a4 below:

[0010] a1: A protein obtained by mutating the wild-type Bst DNA polymerase large fragment as shown in SEQ ID No. 1, wherein the mutation sites include any one or a combination of at least two of F67, F81, A114, D119, Q128, R133, I194, V281, N283, L320, T321, Q334, R339, L340, I359, S365, Y429, G430, R439, F445, E461, T487, T500, M502, N503, R553, and A565;

[0011] a2: a protein having substantially the same enzymatic activity and thermal stability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutation in the amino acid sequence shown in a1;

[0012] a3: A protein that has more than 90% sequence homology with the protein in a1 and has enzyme activity and thermal stability that are basically equivalent to those of the protein in a1;

[0013] a4: A fusion protein obtained by connecting the N-terminus and / or C-terminus of any amino acid sequence described in a1-a3 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

[0014] In some embodiments, the mutation sites include any one of F67, A114, Q128, R133, I194, V281, N283, L320, T321, Q334, R339, L340, I359, Y429, G430, R439, F445, E461, T487, M502, N503, R553, and A565, or a combination of at least two thereof.

[0015] In some embodiments, the mutation is any one of F67S, F81L, A114L, D119N, Q128G, R133Y, I194V, V281I or L, N283L, L320P, T321A, Q334C or H, R339C or H, L340R, I359L, S365D or I, Y429W, G430E, R439C, F445L or I, E461K, T487N, T500A, M502V, N503S, R553H, A565V, or a combination of at least two thereof. In some embodiments, the mutation is any one or a combination of at least two of F67S, A114L, Q128G, R133Y, I194V, V281I or L, N283L, L320P, T321A, Q334C or H, R339C or H, L340R, I359L, Y429W, G430E, R439C, F445L or I, E461K, T487N, M502V, N503S, R553H, A565V. In some embodiments, the mutation may be further selected from any one or a combination of at least two of F81L, D119N, S365D or I, and T500A.

[0016] In some embodiments, the protein is any one of the following b1-b3:

[0017] b1: A protein obtained by subjecting the wild-type Bst DNA polymerase large fragment with the amino acid sequence shown in SEQ ID No. 1 to any combination of the following mutations:

[0018] (1)V16R / I359L / E461K; (2)S65P / D428K; (3)R133Y / L320P; (4)A193M / N283L; (5)N204R / Q128G; (6)N283L; (7)A409P / N462K; (8)S65P / I359L; (9)L68K; (10)L68V; (11)A114L; (12)Q128G; (13)Q182E / L340R; (14)V2 81I; (15)V281L; (16)T321A / N503S; (17)S365D; (18)T487N; (19)F445L ;(20)F67S / F445L;(21)N204S / S365I;(22)I194V;(23)M33V / F81L / M50 2V; (24)M502V / R553H; (25)R339C; (26)D377G / R439C; (27)A565V; (28) N204K / T487N; (29) N204R / L320P / F445L; (30) Q128G / N204R / F445I; (31 )Q334C; (32) Q334H; (33) G430E; (34) D119N / R339H / I422T; (35) T500A;

[0019] b2: a protein having substantially the same enzyme activity and thermal stability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutation in the amino acid sequence shown in b1;

[0020] b3: A fusion protein obtained by connecting the N-terminus and / or C-terminus of any of the amino acid sequences described in b1-b2 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

[0021] In some embodiments, the protein is any one of the following c1-c3:

[0022] c1: A protein obtained by subjecting the wild-type Bst DNA polymerase large fragment with the amino acid sequence shown in SEQ ID No. 1 to any combination of the following mutations:

[0023] (1)N204R / Q128G; (2)S65P / I359L; (3)F67S / F445L; (4)N204S / S365I; (5)R339C; (6 )D377G / R439C; (7)N204K / T487N; (8)N204R / L320P / F445L; (9)Q128G / N204R / F445I;

[0024] c2: A protein having substantially the same enzyme activity and thermal stability obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in c1 except for the aforementioned mutations

[0025] c3: A fusion protein obtained by connecting the N-terminus and / or C-terminus of any amino acid sequence described in c1-c2 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

[0026] In some embodiments, the tag is a His tag, and the enzyme cleavage site is a thrombin cleavage site.

[0027] A Bst DNA polymerase mutant with improved enzymatic activity, which is a protein described in any one of the following d1-d4:

[0028] d1: A protein obtained by mutating the wild-type Bst DNA polymerase large fragment as shown in SEQ ID No. 1, wherein the mutation sites include any one or a combination of at least two of D119, Q128, R133, V281, L320, T321, T323, S328, R339, Q348, I359, Y429, R439, E461, K468, T487, N503, and E552;

[0029] d2: a protein having substantially the same enzymatic activity and thermal stability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutation in the amino acid sequence shown in d1;

[0030] D3: A protein that has more than 90% sequence homology with the protein of D1 and has enzyme activity and thermal stability that are basically equivalent to those of the protein of D1;

[0031] d4: A fusion protein obtained by connecting the N-terminus and / or C-terminus of any of the amino acid sequences described in d1-d3 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

[0032] In some embodiments, the mutation is any one of D119N, Q128G, R133Y, V281I, L320P, T321A, T323A, S328V, R339C, Q348L, I359L, Y429W, R439C, E461K, K468E or N, T487N, N503S, E552G, or a combination of at least two thereof. In some embodiments, the mutation site includes any one of Q128, R133, V281, L320, T321, T323, S328, R339, Q348, I359, Y429, R439, E461, K468, T487, N503, and E552, or a combination of at least two thereof. In some embodiments, the mutation is any one of Q128G, R133Y, V281I, L320P, T321A, T323A, S328V, R339C, Q348L, I359L, Y429W, R439C, E461K, K468E or N, T487N, N503S, E552G, or a combination of at least two thereof.

[0033] In some embodiments, the protein is any one of the following e1-e3:

[0034] e1: A protein obtained by subjecting the wild-type Bst DNA polymerase large fragment with the amino acid sequence shown in SEQ ID No. 1 to any combination of the following mutations:

[0035] (1)D119N / R339H / I422T; (2)S134P; (3)V16R / S328V; (4)A329D; (5)E96G / F171S / H278R / Q348L / E552G; (6 )L68K / K468E; (7) L68K / K468N; (8) L68K / I359L / D428K / Y429W; (9) I422T; (10) S134P / V143A; (11) T323A;

[0036] e2: a protein having substantially the same enzymatic activity and thermal stability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutations in the amino acid sequence of e1;

[0037] e3: A fusion protein obtained by connecting the N-terminus and / or C-terminus of any of the amino acid sequences described in e1-e2 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

[0038] In some embodiments, the protein is any one of the following f1-f3:

[0039] f1: A protein obtained by subjecting the wild-type Bst DNA polymerase large fragment with the amino acid sequence shown in SEQ ID No. 1 to any combination of the following mutations:

[0040] (1)L68K / I359L / D428K / Y429W; (2)S134P / V143A; (3)S65P / I359L;

[0041] (4)A329D;

[0042] f2: A protein having substantially the same enzyme activity and thermal stability obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in f1 except for the aforementioned mutations

[0043] f3: A fusion protein obtained by connecting the N-terminus and / or C-terminus of any amino acid sequence described in f1-f2 to a tag or enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

[0044] Also provided is a Bst DNA polymerase mutant, which is a protein described in any one of the following g1-g4:

[0045] g1: A protein obtained by mutating the wild-type Bst DNA polymerase large fragment as shown in SEQ ID No. 1, wherein the mutation sites include any one or a combination of at least two of F67, F81, A114, D119, Q128, R133, I194, V281, N283, L320, T321, Q334, R339, L340, I359, S365, Y429, G430, R439, F445, E461, T487, T500, M502, N503, R553, A565, T323, S328, Q348, K468, and E552;

[0046] g2: a protein having substantially the same enzyme activity and thermal stability obtained by subjecting the amino acid sequence shown in g1 to one or more of the following substitutions, deletions, and additions of one or more amino acid residues other than the aforementioned mutations;

[0047] g3: a protein that has more than 90% sequence homology with the protein of g1 and has enzyme activity and thermal stability substantially equivalent to that of the protein of g1; and,

[0048] g4: A fusion protein obtained by connecting one or more of the N-terminus and C-terminus of any of the amino acid sequences described in g1-g3 to a tag or enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

[0049] In some embodiments, the mutation sites include any one of F67, A114, Q128, R133, I194, V281, N283, L320, T321, Q334, R339, L340, I359, Y429, G430, R439, F445, E461, T487, M502, N503, R553, A565, T323, S328, Q348, K468, and E552, or a combination of at least two thereof. In some embodiments, the mutation sites include any one of F67, A114, Q128, R133, I194, V281, L320, T321, Q334, R339, I359, Y429, G430, R439, F445, T487, N503, R553, A565, T323, S328, Q348, and K468, or a combination of at least two thereof.

[0050] In some embodiments, the mutation includes any one of F67S, F81L, A114L, D119N, Q128G, R133Y, I194V, V281I or L, N283L, L320P, T321A, Q334C or H, R339C or H, L340R, I359L, S365D or I, Y429W, G430E, R439C, F445L or I, E461K, T487N, T500A, M502V, N503S, R553H, A565V, T323A, S328V, Q348L, K468E or N, and E552G, or a combination of at least two thereof. In some embodiments, the mutation includes any one of F67S, A114L, Q128G, R133Y, I194V, V281I or L, N283L, L320P, T321A, Q334C or H, R339C or H, L340R, I359L, Y429W, G430E, R439C, F445L or I, E461K, T487N, M502V, N503S, R553H, A565V, T323A, S328V, Q348L, K468E or N, E552G, or a combination of at least two thereof. In some embodiments, the mutation includes any one of F67S, A114L, Q128G, R133Y, I194V, V281I or L, L320P, T321A, Q334C or H, R339C or H, I359L, Y429W, G430E, R439C, F445L or I, T487N, N503S, R553H, A565V, T323A, S328V, Q348L, K468E or N, or a combination of at least two thereof.

[0051] In some embodiments, the Bst DNA polymerase mutant is a protein described in any one of the following h1-h3:

[0052] h1: A protein obtained by subjecting the wild-type Bst DNA polymerase large fragment with the amino acid sequence shown in SEQ ID No. 1 to any combination of the following mutations:

[0053] (1)V16R / I359L / E461K; (2)S65P / D428K; (3)R133Y / L320P; (4)A193M / N283L; (5)N204R / Q128G; (6)N283L; (7)A409P / N462K; (8)S65P / I359L; (9)L68K; (10)L68V; (11)A114L; (12)Q128G; (13) Q182E / L340R; (14)V281I; (15)V281L; (16)T321A / N503S; (17)S365D; (18)T487N; (19)F445L; ( 20)F67S / F445L; (21)N204S / S365I; (22)I194V; (23)M33V / F81L / M502V; (24)M502V / R553H; (25) R339C; (26)D377G / R439C; (27)A565V; (28)N204K / T487N; (29)N204R / L320P / F445L; (30)Q128G / N204R / F445I; (31)Q334C; (32)Q334H; (33)G430E; (34)D119N / R339H / I422T; (35)T500A; (36)S 134P; (37) V16R / S328V; (38) A329D; (39) E96G / F171S / H278R / Q348L / E552G; (40) L68K / K468E; (41) L68K / K468N; (42) L68K / I359L / D428K / Y429W; (43) I422T; (44) S134P / V143A; and, (45) T323A;

[0054] h2: a protein having substantially the same enzyme activity and thermal stability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutations in the amino acid sequence shown in h1; and

[0055] h3: A fusion protein obtained by connecting one or more of the N-terminus and C-terminus of any of the amino acid sequences described in h1-h2 to a tag or enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

[0056] In some embodiments, the Bst DNA polymerase mutant is a protein described in any one of the following i1-i3:

[0057] i1: A protein obtained by subjecting the wild-type Bst DNA polymerase large fragment with the amino acid sequence shown in SEQ ID No. 1 to any combination of the following mutations:

[0058] (1) N204R / Q128G; (2) S65P / I359L; (3) F67S / F445L; (4) N204S / S365I; (5) R339C; (6) D377G / R439C; (7) N204K / T487N; (8) N204R / L320P / F445L; (9) Q128G / N204R / F445I; (10) L68K / I359L / D428K / Y429W; (11) S134P / V143A; and, (12) A329D;

[0059] i2: a protein having substantially the same enzymatic activity and thermal stability obtained by substituting and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutation in the amino acid sequence shown in i1; and

[0060] i3: A fusion protein obtained by connecting one or more of the N-terminus and C-terminus of any of the amino acid sequences described in i1-i2 to a tag or enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

[0061] In some embodiments, the tag is a His tag, and the enzyme cleavage site is a thrombin cleavage site. The present application also discloses the encoding gene of the above-mentioned Bst DNA polymerase mutant.

[0062] The present application also discloses an expression vector of the above-mentioned Bst DNA polymerase mutant.

[0063] The present application also discloses an expression host bacterium for the above-mentioned Bst DNA polymerase mutant.

[0064] The present application also discloses the use of the above-mentioned Bst DNA polymerase mutant in MDA, RCA, LAMP or RT-LAMP.

[0065] The present application also discloses a kit containing the above-mentioned Bst DNA polymerase mutant, which can be used for MDA, RCA, LAMP or RT-LAMP technology.

[0066] The present application performs amino acid mutations at one or more different sites on the basis of the wild-type Bst-LF, and the resulting Bst DNA polymerase mutant has higher DNA polymerase activity and / or strand displacement activity. The DNA polymerase activity of the Bst DNA polymerase mutant can be increased to up to 216%, and the strand displacement activity can be increased to up to 255%. In the embodiments of the present application, 65°C RT-LAMP can be advanced by up to about 15 Ct values. The Bst DNA polymerase mutant with improved DNA polymerase activity and / or strand displacement activity of the present application has better application performance and can meet the needs of faster and more sensitive detection.

[0067] The present application provides a Bst DNA polymerase mutant, which is formed by amino acid mutations at one or more different sites on the basis of a truncated wild-type Bst DNA polymerase, has higher thermal stability, and its Tm value is significantly higher than that of the wild-type Bst DNA polymerase. Compared with the wild-type Bst DNA polymerase, the Bst DNA polymerase mutant of the present application has significantly improved residual activity after incubation at 65°C for 30 minutes, and can perform RT-LAMP reactions at a maximum temperature of 70°C. The Bst DNA polymerase mutant with improved thermal stability of the present application has a wider range of application temperatures and can adapt to more complex application scenarios. The mutant of the present application also includes improvements in one or at least two of the following properties: optimal reaction temperature, storage stability, transportation stability, continuous synthesis ability, amplification ability of high GC templates, product specificity, reaction sensitivity, etc.

[0068] Compared with the existing technology, this application has the following beneficial effects:

[0069] (1) The present application can significantly improve the thermal stability of BstDNA polymerase by designing mutations at one or more different amino acid sites, and the protein melting temperature (Tm) is increased by more than 0.1°C, more than 0.2°C, more than 0.5°C, or more than 1°C compared with the wild type; the increase can be more than 2°C, 3°C, or more than 4°C; the maximum increase is more than 5°C.

[0070] (2) The Bst DNA polymerase mutant of the present application has improved thermal stability, and the residual activity can still reach more than 80% after incubation at 65°C for 30 minutes.

[0071] (3) The Bst DNA polymerase mutant of the present application can work in high-temperature RT-LAMP and can complete the RT-LAMP reaction at 70°C.

[0072] (4) The present application obtains a Bst DNA polymerase mutant by designing mutations at one or more different amino acid sites, and its DNA polymerase activity and / or strand displacement activity are improved compared to the wild type. The DNA polymerase activity can be increased by up to 216%, and the strand displacement activity can be increased by up to 120% to 255%.

[0073] (5) The Bst DNA polymerase mutant of the present application has improved performance in LAMP and / or RT-LAMP, can significantly shorten the peak time in 65°C RT-LAMP, has a smaller Ct value, and can be advanced by up to 15 Ct values.

[0074] (6) The Bst DNA polymerase mutant of the present application has improved reaction sensitivity, thermal stability and other properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] FIG1 is a ΔTm comparison of Bst-LF and Bst DNA polymerase mutants in one embodiment of the present application.

[0076] FIG2 is a comparison of the residual activities of Bst-LF and Bst DNA polymerase mutants after incubation at 65° C. for 30 minutes in one embodiment of the present application.

[0077] FIG3 is an RT-LAMP curve of Bst-LF at 68° C. in one embodiment of the present application.

[0078] FIG4 is an RT-LAMP curve of ME5 at 68° C. in one embodiment of the present application.

[0079] FIG5 is an RT-LAMP curve of ME8 at 68° C. in one embodiment of the present application.

[0080] FIG6 is an RT-LAMP curve of ME20 at 68° C. in one embodiment of the present application.

[0081] FIG7 is an RT-LAMP curve of ME21 at 68° C. in one embodiment of the present application.

[0082] FIG8 is an RT-LAMP curve of ME25 at 68° C. in one embodiment of the present application.

[0083] FIG9 is an RT-LAMP curve of ME26 at 68° C. in one embodiment of the present application.

[0084] FIG10 is an RT-LAMP curve of ME28 at 68° C. in one embodiment of the present application.

[0085] FIG11 is an RT-LAMP curve of ME29 at 68° C. in one embodiment of the present application.

[0086] FIG12 is an RT-LAMP curve of ME30 at 68° C. in one embodiment of the present application.

[0087] FIG13 is an RT-LAMP curve of ME28 at 70° C. in one embodiment of the present application.

[0088] FIG14 is an RT-LAMP curve of ME29 at 70° C. in one embodiment of the present application.

[0089] FIG15 is an RT-LAMP curve of ME30 at 70° C. in one embodiment of the present application.

[0090] FIG16 is a comparison of the DNA polymerase activities of Bst-LF and Bst DNA polymerase mutants in one embodiment of the present application.

[0091] FIG17 is a comparison of the strand displacement activities of Bst-LF and Bst DNA polymerase mutants in one embodiment of the present application.

[0092] FIG18 is a comparison of Bst-LF, ME9 and ME51 at 65° C. RT-LAMP in one embodiment of the present application.

[0093] FIG19 is a comparison of Bst-LF and ME12, ME16, and ME54 at 65° C. RT-LAMP in one embodiment of the present application.

[0094] FIG20 is a comparison of Bst-LF and ME26 at 65° C. RT-LAMP in one embodiment of the present application. DETAILED DESCRIPTION

[0095] The specific implementation methods of the present application will be further described below in conjunction with the accompanying drawings, but the description of the embodiments does not limit the scope of protection of the present application.

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application belongs. The terms used herein and in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0097] The description of the mutation in this application is a description of the mutation recognized by those skilled in the art. Taking the mutation of one site as an example, F67S, refers to the mutation of phenylalanine (F) at position 67 of the amino acid sequence shown in SEQ ID NO: 1 to serine (S), that is, phenylalanine (F) at position 67 is replaced by serine (S).

[0098] "Homology" (percentage (%) of sequence identity) of an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical with the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the percentage (%) of sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) relative to the reference sequence to which it is compared by the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence, whichever is shorter. Conservative substitutions of amino acid residues may or may not be considered identical residues. For example, publicly available tools such as BLASTN, BLASTp (available on the website of the US National Center for Biotechnology Information (NCBI), see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute) can be used. Those skilled in the art can use the default parameters provided by the tools or can appropriately customize the parameters according to the needs of the comparison, for example by selecting a suitable algorithm.

[0099] As used herein, the term "amino acid" refers to an organic compound that includes amino (-NH2) and carboxyl (-COOH) functional groups and side chains unique to each amino acid. Amino acid names are also represented in this disclosure as standard single-letter or three-letter codes, which are summarized below.

[0100] In this application, unless otherwise specified, "nucleic acid" has the commonly known meaning in the technical field, also known as "polynucleotide", which is a molecule formed by multiple nucleotide monomers.

[0101] Unless otherwise specified, the term "vector" in this application refers to a vehicle into which a genetic element (e.g., the aforementioned nucleic acid molecule) can be operatively inserted and into which the genetic element is expressed. A vector can be, for example, a plasmid, a cosmid, a virus (e.g., a slow virus, a retrovirus, adenovirus, and an adeno-associated virus), an RNA vector, or a linear or circular DNA or RNA molecule, which can include a chromosome, a non-chromosomal, semisynthetic, or synthetic nucleic acid molecule. The term includes vectors as self-replicating nucleic acid structures and vectors incorporated into the host cell genome into which they are introduced. Certain vectors can direct the expression of nucleic acids to which they are operably connected.

[0102] Unless otherwise specified, the term "expression host bacteria" in this application can be used interchangeably with "host cell".

[0103] Host cells can include bacterial, fungal, plant, or animal cells. Easily transformed bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable fungi include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0104] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

[0105] Example 1

[0106] This example provides a Bst DNA polymerase mutant with improved thermal stability, and a vector construction and expression purification method thereof.

[0107] The wild-type Bst DNA polymerase is derived from a truncated version of Geobacillus stearothermophilus DNA polymerase I and is designated Bst-LF in this application. The amino acid sequence of Bst-LF is shown in SEQ ID NO. 1, and the nucleic acid sequence encoding Bst-LF is shown in SEQ ID NO. 2.

[0108] The Bst DNA polymerase mutants with improved thermostability provided in this embodiment include substitutions, deletions, or additions of one or more amino acids in the amino acid sequence of SEQ ID NO. 1 to form mutant proteins with similar functions to SEQ ID NO. 1. Specifically, the mutation sites of the Bst DNA polymerase mutants are shown in Table 1.

[0109] Table 1 Bst DNA polymerase mutants

[0110] In the present embodiment, the nucleotide sequence encoding Bst-LF and Table 1 Bst DNA polymerase mutants can be obtained by point mutation PCR or gene synthesis, and the nucleotide sequence can be optimized for E. coli codons if necessary. The nucleotide sequence of the correct coding Bst DNA polymerase obtained is connected to the vector pET28a by means of Nde I and Xho I double digestion or homologous recombination, and the N-terminus of Bst-LF and its mutant is connected to the His tag by the thrombin cleavage site. The vector carrying the Bst DNA polymerase coding sequence is then transformed into the expression host E. coli Rosetta.

[0111] The engineered bacteria containing the nucleic acid sequence encoding the Bst DNA polymerase mutant were cultured at 37°C until the OD 600 When the pH value is 0.7-0.8, 0.5 mM IPTG is added for induction, and then culture is continued at 16°C for 16-20 hours. The fermented E. coli cells are collected by centrifugation, resuspended and ultrasonically disrupted, and purified using a nickel column. Finally, the cells are concentrated and the stock solution is replaced to obtain purified Bst-LF and Bst DNA polymerase mutant proteins.

[0112] The Bst DNA polymerase mutant with improved thermal stability described in this application is not limited to the above-mentioned vectors, hosts, culture and purification conditions. Any method that can obtain a Bst DNA polymerase mutant protein with normal structure and function is also applicable to this application.

[0113] Example 2

[0114] The DNA polymerase activities of Bst-LF and the Bst DNA polymerase mutants obtained in Example 1 were detected.

[0115] Experimental Principle: The Bst-LF and Bst DNA polymerase mutant enzyme solutions obtained in Example 1 were diluted to a certain concentration using a stock solution and then tested for DNA polymerase activity. In this example, the template and primer used to evaluate the DNA polymerase activity of the Bst DNA polymerase mutant were derived from the same single-stranded deoxynucleotide sequence, designated Pol-PT in this application. The sequence of this single strand is: 5'-ACAACCATTTATGTAGCATTTATGAAATTTTTAAATCAATTTACTATTGGCTACTGCATACGCGAAAGCGTATGCAGTAGCC-3' (Seq ID No. 3). After annealing, the Pol-PT single strand forms a monocyclic substrate, which serves as both a template and primer for the DNA polymerization reaction. In the reaction system, Bst DNA polymerase extends the monocyclic nucleic acid substrate to form a double-stranded structure through DNA polymerase activity at 37°C for 5 minutes. The double-stranded yield is then measured using a dsDNA nucleic acid dye to assess Bst DNA polymerase activity. The DNA polymerase activity of Bst DNA polymerase was compared with wild-type Bst-LF.

[0116] The detailed reaction process and system are as follows: First, prepare the primer annealing system according to Table 2, incubate the prepared primer annealing system in a 95°C metal bath for 2 minutes, and then let it stand on ice for 5 minutes. Then, prepare the polymerization reaction system according to Table 3, and incubate the prepared polymerization reaction system in a 37°C metal bath for 5 minutes. After the reaction is completed, dsDNA nucleic acid dye (Yishen product Cat#12641) and the reaction solution are prepared according to the instructions using a full black 96-well ELISA plate. After incubation at room temperature for 5 minutes, the fluorescence value is measured under the conditions of Ex / Em=480nm / 520nm.

[0117] Table 2 Primer annealing system

[0118] Table 3 Polymerization reaction system

[0119] The DNA polymerase activity of the Bst DNA polymerase mutant was compared with the wild-type Bst-LF. The results are shown in Table 4, indicating that the obtained Bst DNA polymerase has basic polymerization activity.

[0120] Table 4 DNA polymerase activities of Bst-LF and Bst DNA polymerase mutants

[0121] Example 3

[0122] The strand displacement activities of Bst-LF and the Bst DNA polymerase mutants obtained in Example 1 were detected.

[0123] Experimental Principle: The Bst-LF and Bst DNA polymerase mutant enzyme solutions obtained in Example 1 were diluted to a certain concentration using a stock solution and then tested for strand displacement activity. In this example, the template and primers used to evaluate the strand displacement activity of the Bst DNA polymerase mutant were derived from the same single-stranded deoxynucleotide sequence. The 5' end of this single strand was modified with the fluorescent group FAM. This single strand was designated Strand F and had the following nucleic acid sequence: 5'-ACAACCATTTATGTAGCATTTATGAAATTTTTAAATCAATTTACTATTGGCTACTGCATACGCGAAAGCGTATGCAGTAGCC-3' (SEQ ID NO. 3). Separately, a primer complementary to the 5' end of Strand F was designed. This complementary primer was designated Strand Q and had the following sequence: 5'-TAAAAATTTCATAAATGCTACATAAATGGTTGT-3' (SEQ ID NO. 4). After annealing, Strand F forms a single-stranded loop substrate. Simultaneously, Strand Q binds complementary to the 5' end of Strand F, quenching the fluorescent group carried by Strand F. In the reaction system, Bst DNA polymerase briefly extends the single-loop nucleic acid substrate, Strand F, through its DNA polymerase activity at 37°C for 5 minutes. The strand displacement activity of Bst DNA polymerase then displaces Strand Q from Strand F, releasing it into the solution. This allows the fluorescent group carried by Strand F to emit light, which is then collected to characterize the strength of the strand displacement activity. Wild-type Bst-LF was used as a control for the strand displacement activity of Bst DNA polymerase.

[0124] The detailed reaction process and system are as follows: First, prepare the primer annealing system according to Table 5. Incubate the prepared primer annealing system in a 95°C metal bath for 2 minutes, then let it rest on ice for 5 minutes. Next, prepare the strand displacement reaction system according to Table 6. Incubate the prepared strand displacement reaction system in a 37°C metal bath for 5 minutes. After the reaction is completed, transfer 20 μL of the reaction solution to a black 96-well microtiter plate. Add 80 μL of DEPC HO, shake well, and measure fluorescence at Ex / Em = 485 nm / 535 nm.

[0125] Table 5 Primer annealing system

[0126] Table 6 Strand displacement reaction system

[0127] The strand displacement activity of the Bst DNA polymerase mutant was compared with the wild-type Bst-LF. The results are shown in Table 7, indicating that the obtained Bst DNA polymerase mutant has basic strand displacement activity.

[0128] Table 7 Strand displacement activity of Bst-LF and Bst DNA polymerase mutants

[0129] Example 4

[0130] The melting temperatures (Tm) of Bst-LF and the Bst DNA polymerase mutants obtained in Example 1 were detected.

[0131] In this example, the thermal stability of Bst DNA polymerase was reflected by measuring the melting temperature (Tm) of the Bst DNA polymerase protein.

[0132] The detailed principle is: the protein peptide chain folds in water, and its hydrophilic groups will wrap the hydrophobic groups inside. Under external pressure (high temperature), the protein will denature and unfold, gradually exposing the hydrophobic groups. At this time, the hydrophobic fluorescent dye in the system will combine with the protein hydrophobic groups to emit fluorescence. The Tm of different proteins can be calculated through the changes in the fluorescence curve, thereby realizing the comparison of the thermal stability of mutants.

[0133] The detailed method is as follows: the Bst DNA polymerase protein purified in Example 1 is diluted to an appropriate concentration, a hydrophobic fluorescent dye is mixed with the diluted protein solution, and the temperature is raised to 35°C at a constant rate of 1.6°C / s in a real-time fluorescence quantitative PCR instrument and held constant for 20 seconds, then gradually raised to 80°C and held constant for 1 second. During the heating process, a fluorescence signal is collected every 0.5 seconds, and Tm is calculated based on the collected signal analysis.

[0134] The Bst DNA polymerase Tm determination described in this application is not limited to the above method, and other methods suitable for protein Tm determination are applicable to this application.

[0135] The Bst DNA polymerase mutant with improved thermal stability obtained in this application has a significantly increased melting temperature (Tm) compared to the wild-type Bst-LF (Figure 1). In detail, it can be increased by more than 0.1°C, more than 0.2°C, more than 0.5°C, or more than 1.0°C compared to the wild type; the increase can be more than 2.0°C, 3.0°C, or 4.0°C; the maximum increase is more than 5.0°C. To a certain extent, the melting temperature (Tm) reflects the thermal stability of Bst DNA polymerase. The higher the melting temperature (Tm), the better the thermal stability. This shows that the Bst DNA polymerase mutant obtained in this application has better thermal stability than Bst-LF.

[0136] Example 5

[0137] The residual activities of Bst-LF and the Bst DNA polymerase mutants obtained in Example 1 after treatment at 65° C. for 30 minutes were detected.

[0138] Detailed method: The Bst-LF and Bst DNA polymerase mutant enzyme solutions obtained in Example 1 were diluted to a certain concentration with a stock solution, and the DNA polymerase activity was tested after incubation at 65°C for 30 minutes, wherein the same Bst DNA polymerase that was directly tested for DNA polymerase activity without being subjected to a 30-minute heat incubation treatment at 65°C was used as a control. The ratio of the DNA polymerization activity after heat incubation treatment to that before heat incubation treatment is the percentage of residual activity of the Bst DNA polymerase. In this embodiment, the method for detecting the DNA polymerase activity of the Bst DNA polymerase mutant before and after heat incubation treatment is the same as that in Example 2. The results show that the residual activity of the Bst DNA polymerase mutant obtained in this application after incubation at 65°C for 30 minutes is significantly improved compared with that of the wild type (Figure 2). In detail, the residual activities of mutation numbers ME1 to ME8, ME16, ME18 to ME31, and ME34 have been significantly improved, among which the residual activities of ME5, ME8, ME28, ME29, and ME30 are more than 50%, among which the highest residual activities of ME8 and ME29 can reach more than 80%.

[0139] Example 6: RT-LAMP reaction at 68°C using the mutant obtained in Example 1

[0140] The effectiveness of the RT-LAMP reaction of the Bst DNA polymerase mutant obtained in Example 1 at 68°C was tested.

[0141] The RT-LAMP test method in this example uses Yisheng's "RT-LAMP Dye Assay Kit (UDG plus)" (Cat#13762) and "RT-LAMP COVID-19 Primer Master Mix (N)" (Cat#13966). For detailed operating steps, please refer to the official website instructions.

[0142] Combining Examples 2 and 3, mutants with excellent thermal stability were selected for 68°C RT-LAMP reaction, with at least eight replicates performed for each mutant. The 68°C RT-LAMP test results are shown in Figures 3-12.

[0143] Results: As shown in Figures 3-12, Bst-LF is not competent for 68°C RT-LAMP reaction, while the Bst DNA polymerase mutants ME5, ME8, ME20, ME21, ME25, ME26, ME28, ME29 and ME30 with improved thermal stability described in this application have improved thermal stability compared to Bst-LF. Except for ME26 which is not very competent for 68°C RT-LAMP, the others can complete 68°C RT-LAMP well.

[0144] Example 7: RT-LAMP reaction at 70°C using the mutant obtained in Example 1

[0145] The effectiveness of the RT-LAMP reaction of the Bst DNA polymerase mutant obtained in Example 1 at 70°C was tested.

[0146] The RT-LAMP test method in this example uses Yisheng's "RT-LAMP Dye Assay Kit (UDG plus)" (Cat#13762) and "RT-LAMP COVID-19 Primer Master Mix (N)" (Cat#13966). For detailed operating steps, please refer to the official website instructions.

[0147] Based on Examples 2, 3, and 4, mutants with excellent thermal stability were selected for 70°C RT-LAMP reaction, with at least 8 replicates performed for each mutant. The test results of 70°C RT-LAMP are shown in Figures 13-15.

[0148] Results: In the 70°C RT-LAMP test, the thermostability-enhanced Bst DNA polymerase mutants ME5, ME8, ME20, ME21, ME25, ME26, ME28, ME29, and ME30 described herein exhibited distinct fluorescence peak increases. ME29 and ME30 performed best, demonstrating significantly improved thermostability and the ability to successfully complete the RT-LAMP reaction at 70°C.

[0149] In summary, amino acid mutations at one or more different sites can significantly improve the thermal stability of Bst DNA polymerase and significantly increase the Tm of the Bst DNA polymerase mutant. Compared with wild-type Bst DNA polymerase, the residual activity after incubation at 65°C for 30 minutes is significantly increased, and RT-LAMP reactions can be performed at temperatures up to 70°C. The Bst DNA polymerase with improved thermal stability in this application has a wider range of application temperatures and can adapt to more complex application scenarios.

[0150] Example 8

[0151] This example provides a Bst DNA polymerase mutant with improved DNA polymerase activity, and a vector construction and expression purification method thereof.

[0152] The wild-type Bst DNA polymerase is obtained by truncating DNA polymerase I from Geobacillus stearothermophilus and is named Bst-LF in this application. The amino acid sequence of Bst-LF is SEQ ID NO. 1, and the nucleic acid sequence encoding Bst-LF is SEQ ID NO. 2.

[0153] The Bst DNA polymerase mutants with enhanced DNA polymerase activity provided in this example are obtained by substituting, deleting, or adding one or more amino acids in the amino acid sequence of SEQ ID NO. 1 to form mutant proteins with similar functions to SEQ ID NO. 1. Specifically, the mutation sites of the Bst DNA polymerase mutants are shown in Table 8.

[0154] Table 8 Bst DNA polymerase mutants

[0155] In this embodiment, the nucleic acid sequence encoding Bst-LF and the Bst DNA polymerase mutants in Table 8 can be obtained by point mutation PCR or gene synthesis, and the nucleic acid sequence can be optimized for Escherichia coli codons if necessary. The obtained nucleic acid sequence encoding the correct Bst DNA polymerase is connected to the vector pET28a by double digestion with Nde I and Xho I or homologous recombination, and the N-terminus of Bst-LF and its mutants is connected to the His tag through the thrombin cleavage site. The vector containing the correct Bst DNA polymerase coding sequence is then transferred to the expression host Escherichia coli Rosetta. The engineered bacteria containing the nucleic acid sequence encoding the Bst DNA polymerase mutant are cultured at 37°C to an OD of 0. 600 When the pH value is 0.7-0.8, 0.5 mM IPTG is added for induction, and then culture is continued at 16°C for 16-20 hours. The fermented E. coli cells are collected by centrifugation, resuspended and ultrasonically disrupted, and purified using a nickel column. Finally, the cells are concentrated and the stock solution is replaced to obtain purified Bst-LF and Bst DNA polymerase mutant proteins.

[0156] The Bst DNA polymerase mutant with improved DNA polymerase activity described in this application is not limited to the above-mentioned vectors, hosts, culture and purification conditions, and any method that can obtain a Bst DNA polymerase mutant protein with normal structure and function is also applicable to this application.

[0157] Example 9

[0158] The DNA polymerase activities of Bst-LF and the Bst DNA polymerase mutants obtained in Example 8 were detected.

[0159] Experimental Principle: The Bst-LF and Bst DNA polymerase mutant enzyme solutions obtained in Example 8 were diluted to a certain concentration using a stock solution and then tested for DNA polymerase activity. In this example, the template and primer used to evaluate the DNA polymerase activity of the Bst DNA polymerase mutant were derived from the same single-stranded deoxynucleotide sequence, designated Pol-PT in this application. The sequence of this single strand is: 5'-ACAACCATTTATGTAGCATTTATGAAATTTTTAAATCAATTTACTATTGGCTACTGCATACGCGAAAGCGTATGCAGTAGCC-3' (SEQ ID NO. 3). After annealing, the Pol-PT single strand forms a monocyclic substrate, which serves as both template and primer for the DNA polymerization reaction. In the reaction system, Bst DNA polymerase extends the monocyclic nucleic acid substrate to form a double-stranded structure through DNA polymerase activity at 37°C for 5 minutes. The double-stranded yield is then measured using a dsDNA nucleic acid dye to assess Bst DNA polymerase activity. The DNA polymerase activity of Bst DNA polymerase was compared with wild-type Bst-LF.

[0160] The detailed reaction process and system are as follows: First, prepare the primer annealing system according to Table 9, incubate the prepared primer annealing system in a 95°C metal bath for 2 minutes, and then let it stand on ice for 5 minutes. Then, prepare the polymerization reaction system according to Table 3, and incubate the prepared polymerization reaction system in a 37°C metal bath for 5 minutes. After the reaction is completed, dsDNA nucleic acid dye (Yishen product Cat#12641) and the reaction solution are prepared using a black 96-well ELISA plate according to the instructions. After incubation at room temperature for 5 minutes, the fluorescence value is measured under the conditions of Ex / Em=480nm / 520nm.

[0161] Table 9 Primer annealing system

[0162] Table 10 Polymerization reaction system

[0163] The DNA polymerase activity of the Bst DNA polymerase mutants was compared with the wild-type Bst-LF. The results are shown in Table 11 and Figure 16. The DNA polymerase activity of mutants ME3 and ME50 was increased by more than 20%, the DNA polymerase activity of mutants ME26 and ME44 was increased by more than 40%, the DNA polymerase activity of mutants ME8, ME9, ME12, and ME16 was increased by more than 60%, the DNA polymerase activity of mutant ME51 was increased by more than 80%, and the DNA polymerase activity of mutant ME54 was increased by 115%.

[0164] Table 11 DNA polymerase activities of Bst-LF and Bst DNA polymerase mutants

[0165] Example 10

[0166] The strand displacement activities of Bst-LF and the Bst DNA polymerase mutants obtained in Example 8 were detected.

[0167] Experimental Principle: The Bst-LF and Bst DNA polymerase mutant enzyme solutions obtained in Example 8 were diluted to a certain concentration using a stock solution and then tested for strand displacement activity. In this example, the template and primers used to evaluate the strand displacement activity of the Bst DNA polymerase mutant were derived from the same single-stranded deoxynucleotide sequence. The 5' end of this single strand was modified with the fluorescent group FAM. This single strand was designated Strand F and has the sequence: 5'-ACAACCATTTATGTAGCATTTATGAAATTTTTAAATCAATTTACTATTGGCTACTGCATACGCGAAAGCGTATGCAGTAGCC-3' (SEQ ID NO. 3). Separately, a primer complementary to the 5' end of Strand F was designed. This complementary primer, designated Strand Q, has a quencher group BHQ modified at its 3' end. The sequence of Strand Q is: 5'-TAAAAATTTCATAAATGCTACATAAATGGTTGT-3' (SEQ ID NO. 4). After annealing, Strand F forms a single-stranded loop substrate. Simultaneously, Strand Q binds complementary to the 5' end of Strand F, quenching the fluorescent group carried by Strand F. In the reaction system, Bst DNA polymerase briefly extends the single-loop nucleic acid substrate, Strand F, through its DNA polymerase activity at 37°C for 5 minutes. The strand displacement activity of Bst DNA polymerase then displaces Strand Q from Strand F, releasing it into the solution. This allows the fluorescent group carried by Strand F to emit light, which is then collected to characterize the strength of the strand displacement activity. Wild-type Bst-LF was used as a control for the strand displacement activity of Bst DNA polymerase.

[0168] The detailed reaction process and system are as follows: First, prepare the primer annealing system according to Table 12. Incubate the prepared primer annealing system in a 95°C metal bath for 2 minutes, then let it rest on ice for 5 minutes. Next, prepare the strand displacement reaction system according to Table 13. Incubate the prepared strand displacement reaction system in a 37°C metal bath for 5 minutes. After the reaction is completed, transfer 20 μL of the reaction solution to a black 96-well microtiter plate, add 80 μL of DEPC HO, shake to mix, and measure fluorescence at Ex / Em = 485 nm / 535 nm.

[0169] Table 12 Primer annealing system

[0170] Table 13 Strand displacement reaction system

[0171] The strand displacement activity of the Bst DNA polymerase mutants was compared with the wild-type Bst-LF. The results are shown in Table 14 and Figure 17. The strand displacement activity of mutants ME1, ME2, ME3, ME7, ME14, ME34, ME50, ME54, and ME55 can be increased by more than 20%, the strand displacement activity of mutants ME9, ME40, ME43, and ME46 can be increased by more than 40%, the strand displacement activity of mutant ME16 can be increased by more than 60%, the strand displacement activity of mutant ME26 can be increased by more than 80%, the strand displacement activity of mutant ME12 can be increased by 121%, and the strand displacement activity of mutant ME51 can be increased by 155%.

[0172] Table 14 Strand displacement activity of Bst-LF and Bst DNA polymerase mutants

[0173] Example 11

[0174] The mutant obtained in Example 8 was used to perform 65°C RT-LAMP reaction.

[0175] The effect of the Bst DNA polymerase mutant obtained in Example 8 on the RT-LAMP reaction at 65°C was tested.

[0176] The RT-LAMP assay in this example uses the Yisheng RT-LAMP Dye Assay Kit (UDG plus) (Cat#13762) and the RT-LAMP COVID-19 Primer Master Mix (N) (Cat#13966). For detailed steps, refer to the official instructions. To better compare the differences between Bst DNA polymerase mutants, the template input can be appropriately reduced.

[0177] In combination with Examples 9 and 10, mutants with superior DNA polymerase activity and / or strand displacement activity were selected for 65°C RT-LAMP reactions. Each mutant was subjected to at least four or eight replicate reactions. The results of the 65°C RT-LAMP assay are shown in Figures 18-20.

[0178] Results: As shown in Figures 18-20, ME9, ME12, ME16, ME26, ME51, and ME54 performed better than Bst-LF in RT-LAMP at 65°C, with earlier peak elution times and similar fluorescence peaks. ME12, ME51, and ME54 performed similarly, achieving the best performance among all mutants, with an advance of up to approximately 15 Ct values. ME9, ME16, and ME26 were second, but still performed better than Bst-LF, with an advance of approximately 8 Ct values. This suggests that the enhanced DNA polymerase activity and / or strand displacement activity of Bst DNA polymerase mutants can improve LAMP or RT-LAMP performance, resulting in earlier peak elution times. It is reasonable to speculate that these Bst DNA polymerase mutants with enhanced activity may possess higher sensitivity, enabling detection of even smaller amounts of template.

[0179] In summary, the present application performs amino acid mutations at one or more different sites on the basis of the wild-type Bst-LF, and the resulting Bst DNA polymerase mutant has higher DNA polymerase activity and / or chain displacement activity. The DNA polymerase activity of the Bst DNA polymerase mutant can be increased to up to 216%, and the chain displacement activity can be increased to up to 255%. In the embodiments of the present application, 65°C RT-LAMP can be advanced by up to about 15 Ct values. The Bst DNA polymerase mutant with improved DNA polymerase activity and / or chain displacement activity of the present application has better application performance and can meet the needs of faster and more sensitive detection.

[0180] The applicant declares that while the above-mentioned embodiments illustrate the detailed methods of the present application, the present application is not limited to the above-mentioned detailed methods, which does not mean that the present application must rely on the above-mentioned detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present application, equivalent replacements for the raw materials of the present application's products, addition of auxiliary ingredients, and selection of detailed methods, etc., fall within the scope of protection and disclosure of the present application.

Claims

1. A thermostable Bst DNA polymerase mutant, which is a protein described in any one of a1-a4 below: a1: a protein obtained by mutation based on the amino acid sequence of the wild-type Bst DNA polymerase large fragment as shown in SEQ ID No. 1, wherein the mutation sites include any one of F67, F81, A114, D119, Q128, R133, I194, V281, N283, L320, T321, Q334, R339, L340, I359, S365, Y429, G430, R439, F445, E461, T487, T500, M502, N503, R553, and A565, or a combination of at least two thereof; a2: a protein having substantially the same enzyme activity and thermal stability obtained by subjecting the amino acid sequence shown in a1 to one or more of substitution, deletion and addition of one or more amino acid residues other than the aforementioned mutation; a3: a protein that has a sequence homology of more than 90% with the protein of a1 and has an enzyme activity and thermal stability that are substantially equivalent to the protein of a1; and, a4: A fusion protein obtained by connecting one or more of the N-terminus and C-terminus of any of the amino acid sequences described in a1-a3 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

2. The Bst DNA polymerase mutant according to claim 1, wherein The mutation is any one of F67S, F81L, A114L, D119N, Q128G, R133Y, I194V, V281I or L, N283L, L320P, T321A, Q334C or H, R339C or H, L340R, I359L, S365D or I, Y429W, G430E, R439C, F445L or I, E461K, T487N, T500A, M502V, N503S, R553H, and A565V, or a combination of at least two thereof.

3. The Bst DNA polymerase mutant according to claim 1, which is a protein described in any one of the following b1-b3: b1: A protein obtained by causing any combination of the following mutations to occur on the basis of the amino acid sequence of the wild-type Bst DNA polymerase large fragment shown in SEQ ID No. 1: (1)V16R / I359L / E461K; (2) S65P / D428K; (3) R133Y / L320P; (4)A193M / N283L; (5)N204R / Q128G; (6)N283L; (7)A409P / N462K; (8) S65P / I359L; (9)L68K; (10) L68V; (11)A114L; (12)Q128G; (13)Q182E / L340R; (14) V281I; (15) V281L; (16)T321A / N503S; (17)S365D; (18)T487N; (19) F445L; (20) F67S / F445L; (21)N204S / S365I; (22)I194V; (23)M33V / F81L / M502V; (24)M502V / R553H; (25)R339C; (26)D377G / R439C; (27)A565V; (28)N204K / T487N; (29)N204R / L320P / F445L; (30)Q128G / N204R / F445I; (31)Q334C; (32)Q334H; (33) G430E; (34) D119N / R339H / I422T; and (35) T500A; b2: a protein having substantially the same enzyme activity and thermal stability obtained by replacing and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutation in the amino acid sequence shown in b1; and b3: A fusion protein obtained by connecting one or more of the N-terminus and C-terminus of any of the amino acid sequences described in b1-b2 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

4. The Bst DNA polymerase mutant according to claim 1, which is a protein described in any one of the following c1-c3: c1: A protein obtained by causing any combination of the following mutations based on the amino acid sequence of the wild-type Bst DNA polymerase large fragment shown in SEQ ID No. 1: (1)N204R / Q128G; (2)S65P / I359L; (3) F67S / F445L; (4)N204S / S365I; (5) R339C; (6)D377G / R439C; (7)N204K / T487N; (8) N204R / L320P / F445L; and (9)Q128G / N204R / F445I; c2: a protein having substantially the same enzyme activity and thermal stability obtained by replacing and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutation in the amino acid sequence shown in c1; and c3: A fusion protein obtained by connecting one or more of the N-terminus and C-terminus of any of the amino acid sequences described in c1-c2 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

5. A Bst DNA polymerase mutant with improved enzyme activity, which is a protein described in any one of the following d1-d4: d1: a protein obtained by mutation based on the amino acid sequence of the wild-type Bst DNA polymerase large fragment as shown in SEQ ID No. 1, wherein the mutation sites include any one of D119, Q128, R133, V281, L320, T321, T323, S328, R339, Q348, I359, Y429, R439, E461, K468, T487, N503, and E552, or a combination of at least two thereof; d2: a protein having substantially the same enzyme activity and thermal stability obtained by replacing and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutation in the amino acid sequence shown in d1; d3: a protein that has more than 90% sequence homology with the protein in d1 and has enzyme activity and thermal stability that are basically equivalent to those of the protein in d1; as well as, d4: A fusion protein obtained by connecting one or more of the N-terminus and the C-terminus of any of the amino acid sequences described in d1-d3 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

6. The Bst DNA polymerase mutant according to claim 5, wherein The mutation is any one of D119N, Q128G, R133Y, V281I, L320P, T321A, T323A, S328V, R339C, Q348L, I359L, Y429W, R439C, E461K, K468E or N, T487N, N503S, E552G, or a combination of at least two thereof.

7. The Bst DNA polymerase mutant according to claim 5, which is a protein described in any one of the following e1-e3: e1: A protein obtained by causing any combination of the following mutations based on the amino acid sequence of the wild-type Bst DNA polymerase large fragment shown in SEQ ID No. 1: (1)D119N / R339H / I422T; (2)S134P; (3) V16R / S328V; (4) A329D; (5)E96G / F171S / H278R / Q348L / E552G; (6)L68K / K468E; (7)L68K / K468N; (8)L68K / I359L / D428K / Y429W; (9)I422T; (10) S134P / V143A; and (11) T323A; e2: a protein having substantially the same enzyme activity and thermal stability obtained by replacing and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutation in the amino acid sequence shown in e1; and e3: A fusion protein obtained by connecting one or more of the N-terminus and the C-terminus of any of the amino acid sequences described in e1-e2 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

8. The Bst DNA polymerase mutant according to claim 5, which is a protein described in any one of the following f1-f3: f1: A protein obtained by causing any combination of the following mutations based on the amino acid sequence of the wild-type Bst DNA polymerase large fragment shown in SEQ ID No. 1: (1)L68K / I359L / D428K / Y429W; (2)S134P / V143A; (3) S65P / I359L; and (4) A329D; f2: a protein having substantially the same enzyme activity and thermal stability obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in f1 except for the aforementioned mutation; and f3: A fusion protein obtained by connecting one or more of the N-terminus and C-terminus of any of the amino acid sequences in f1-f2 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

9. A Bst DNA polymerase mutant, which is a protein described in any one of g1-g4 below: g1: a protein obtained by mutation based on the amino acid sequence of the wild-type Bst DNA polymerase large fragment as shown in SEQ ID No. 1, wherein the mutation sites include any one of F67, F81, A114, D119, Q128, R133, I194, V281, N283, L320, T321, Q334, R339, L340, I359, S365, Y429, G430, R439, F445, E461, T487, T500, M502, N503, R553, A565, T323, S328, Q348, K468, and E552, or a combination of at least two thereof; g2: a protein having substantially the same enzyme activity and thermal stability obtained by subjecting the amino acid sequence shown in g1 to one or more of the substitution, deletion and addition of one or more amino acid residues other than the aforementioned mutation; g3: a protein having a sequence homology of more than 90% with the protein of g1, and having an enzyme activity and a thermal stability substantially equivalent to that of the protein of g1; and, g4: A fusion protein obtained by connecting one or more of the N-terminus and the C-terminus of any of the amino acid sequences in g1-g3 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

10. The Bst DNA polymerase mutant according to claim 9, wherein the protein described in g1 is: The mutation sites include any one of F67, A114, Q128, R133, I194, V281, N283, L320, T321, Q334, R339, L340, I359, Y429, G430, R439, F445, E461, T487, M502, N503, R553, A565, T323, S328, Q348, K468, and E552, or a combination of at least two thereof.

11. BstDNA polymerase mutant according to claim 9, wherein the mutation comprises any one or a combination of at least two of F67S, F81L, A114L, D119N, Q128G, R133Y, I194V, V281I or L, N283L, L320P, T321A, Q334C or H, R339C or H, L340R, I359L, S365D or I, Y429W, G430E, R439C, F445L or I, E461K, T487N, T500A, M502V, N503S, R553H, A565V, T323A, S328V, Q348L, K468E or N, E552G.

12. The Bst DNA polymerase mutant according to claim 9, which is a protein described in any one of the following h1-h3: h1: A protein obtained by subjecting the wild-type Bst DNA polymerase large fragment shown in SEQ ID No. 1 to any of the following mutations: (1)V16R / I359L / E461K; (2) S65P / D428K; (3) R133Y / L320P; (4)A193M / N283L; (5)N204R / Q128G; (6)N283L; (7)A409P / N462K; (8)S65P / I359L; (9)L68K; (10) L68V; (11)A114L; (12)Q128G; (13)Q182E / L340R; (14) V281I; (15) V281L; (16)T321A / N503S; (17)S365D; (18)T487N; (19) F445L; (20) F67S / F445L; (21)N204S / S365I; (22)I194V; (23)M33V / F81L / M502V; (24)M502V / R553H; (25)R339C; (26)D377G / R439C; (27)A565V; (28)N204K / T487N; (29)N204R / L320P / F445L; (30)Q128G / N204R / F445I; (31)Q334C; (32)Q334H; (33) G430E; (34)D119N / R339H / I422T; (35) T500A; (36)S134P; (37) V16R / S328V; (38)A329D; (39)E96G / F171S / H278R / Q348L / E552G; (40)L68K / K468E; (41)L68K / K468N; (42) L68K / I359L / D428K / Y429W; (43)I422T; (44) S134P / V143A; and (45)T323A; h2: a protein having substantially the same enzyme activity and thermal stability obtained by replacing and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutation in the amino acid sequence shown in h1; and h3: A fusion protein obtained by connecting one or more of the N-terminus and C-terminus of any of the amino acid sequences described in h1-h2 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

13. The Bst DNA polymerase mutant according to claim 9, which is a protein described in any one of the following i1-i3: i1: A protein obtained by causing any combination of the following mutations based on the amino acid sequence of the wild-type Bst DNA polymerase large fragment shown in SEQ ID No. 1: (1)N204R / Q128G; (2)S65P / I359L; (3) F67S / F445L; (4)N204S / S365I; (5) R339C; (6)D377G / R439C; (7)N204K / T487N; (8)N204R / L320P / F445L; (9)Q128G / N204R / F445I; (10)L68K / I359L / D428K / Y429W; (11) S134P / V143A; and (12) A329D; i2: a protein having substantially the same enzyme activity and thermal stability obtained by replacing and / or deleting and / or adding one or more amino acid residues other than the aforementioned mutation in the amino acid sequence shown in i1; and i3: A fusion protein obtained by connecting one or more of the N-terminus and C-terminus of any of the amino acid sequences described in i1-i2 to a tag or an enzyme cleavage site, wherein the tag or enzyme cleavage site does not affect the function of the Bst DNA polymerase mutant.

14. The Bst DNA polymerase mutant according to any one of claims 1 to 13, wherein The tag is a His tag, and the enzyme cleavage site is a thrombin cleavage site.

15. A gene encoding the Bst DNA polymerase mutant according to any one of claims 1 to 13.

16. An expression vector for the Bst DNA polymerase mutant according to any one of claims 1 to 13.

17. An expression host bacterium for the Bst DNA polymerase mutant according to any one of claims 1 to 13.

18. Use of the Bst DNA polymerase mutant according to any one of claims 1 to 13 in MDA, RCA, LAMP or RT-LAMP.

19. A kit comprising the Bst DNA polymerase mutant according to any one of claims 1 to 13.

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