T7 RNA polymerase mutant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of protein engineering, and discloses a T7 RNA polymerase mutant as well as a preparation method and application thereof. The T7 RNA polymerase mutant provided by the invention is a protein as shown in A1) or A2): A1) relative to a wild type T7 RNA polymerase, the T7 RNA polymerase mutant comprises an amino acid substitution site A124E / G47S; the amino acid sequence of the wild type T7 RNA polymerase is as shown in SEQ ID NO: 1; a2) is a fusion protein obtained by connecting a label to the N end or / and the C end of the amino acid sequence in A1). Compared with wild type T7 RNA polymerase, the T7 RNA polymerase mutant has the advantage of low yield of by-products.

Description

Technical Field

[0001] This invention relates to the field of protein engineering technology, and in particular to a T7 RNA polymerase mutant, its preparation method, and its application. Background Technology

[0002] T7 RNA polymerase (T7 RNAP) is a single-subunit RNA polymerase with a molecular weight of approximately 99 kDa, composed of 883 amino acids. It includes an N-terminal domain (1-300 aa, involved in promoter recognition) and a C-terminal domain (301-883 aa, containing the catalytic core), catalyzing RNA synthesis in the 5'→3' direction. This enzyme exhibits high specificity for the T7 promoter, completing the transcription cycle without the need for additional transcription factors. Its elongation rate (approximately 300 nt / s) is more than 5 times faster than that of E. coli RNA polymerase, enabling efficient synthesis of long RNA transcripts exceeding 10 kb.

[0003] Although T7 RNAP is widely used in in vitro transcription (IVT), it suffers from a high rate of byproduct generation (such as double-stranded RNA (dsRNA)). This severely limits its application in high-temperature amplification techniques (such as nucleic acid sequence-dependent amplification (NASBA) and isothermal RNA amplification (TMA)).

[0004] Therefore, there is an urgent need to find a T7 RNA polymerase with low byproduct yield. Summary of the Invention

[0005] This invention aims to solve one of the technical problems existing in the prior art. To this end, this invention proposes a T7 RNA polymerase mutant.

[0006] This invention also proposes a biomaterial associated with a T7 RNA polymerase mutant.

[0007] The present invention also proposes an enzyme preparation.

[0008] This invention also proposes a method for preparing a T7 RNA polymerase mutant.

[0009] This invention also proposes a method for in vitro amplification of RNA molecules.

[0010] This invention also proposes the application of a T7 RNA polymerase mutant in the preparation of nucleic acid transcription products.

[0011] This invention also proposes the application of a T7 RNA polymerase mutant or the above-mentioned enzyme preparation in nucleic acid transcription.

[0012] The present invention also proposes a kit for nucleic acid transcription.

[0013] In a first aspect, the present invention provides a T7 RNA polymerase mutant, which is the protein described in A1) or A2): A1) Compared to wild-type T7 RNA polymerase, the T7 RNA polymerase mutant contains the amino acid substitution site A124E / G47S; the amino acid sequence of the wild-type T7 RNA polymerase is shown in SEQ ID NO: 1; A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence described in A1).

[0014] The T7 RNA polymerase mutant according to embodiments of the present invention has at least the following beneficial effects: The T7 RNA polymerase mutant of the present invention can reduce the generation of byproducts such as double-stranded RNA (dsRNA), and is suitable for the development of in vitro diagnostic reagents and synthetic biology applications, especially for applications that efficiently synthesize full-length RNA and reduce immunogenic byproducts.

[0015] In some embodiments of the present invention, the T7 RNA polymerase mutant further comprises at least one of the following amino acid substitution sites: S192P, V174K, H161E, K163W, M46L, N165P, Q169D, M219L, K363P, A258D, and M190D.

[0016] In some embodiments of the present invention, the amino acid substitution sites of the T7 RNA polymerase mutant are selected from the following combinations of mutations: A124E / G47S, A124E / G47S / S192P, A124E / G47S / V174K, A124E / G47S / H161E, A124E / G47S / K163W, A124E / G47S / M46L, A12 4E / G47S / N165P, A124E / G47S / Q169D, A124E / G47S / M219L, A124E / G47S / K363P, A124E / G47S / A258D, A124E / G47S / M190D.

[0017] In some embodiments of the present invention, the tag includes at least one of the tags that facilitate the dissolution, purification and detection of T7 RNA polymerase mutants.

[0018] In some embodiments of the present invention, the tag is a histidine tag.

[0019] It is understood that the T7 RNA polymerase mutant of the present invention may contain one or more tags; multiple tags may include combinations of multiple identical tags, or combinations of multiple different tags. For example: tags that facilitate the dissolution of T7 RNA polymerase mutants include, but are not limited to, nus tags or maltose-binding protein tags; tags that facilitate the purification of T7 RNA polymerase mutants include, but are not limited to, strep tags, His tags, GST tags, pelB signal tags, or ompA signal tags; tags that facilitate the detection of T7 RNA polymerase mutants include, but are not limited to, horseradish peroxidase (HRP) tags, β-galactosidase tags, luciferase tags, green fluorescent protein (GFP) tags, HcRed tags, DsRed tags, or cyan fluorescent protein (CFP) tags. Specifically, the tag may be a His tag.

[0020] A second aspect of the invention provides biological material related to the T7 RNA polymerase mutant described in the first aspect, said biological material being any one of B1) to B4): B1) A nucleic acid molecule encoding the T7 RNA polymerase mutant described in the first aspect embodiment; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) Recombinant biological cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

[0021] In some embodiments of the present invention, the nucleic acid sequence of the nucleic acid molecule is shown as any one of B1-1) to B1-14): B1-1) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GGC” to “AGC” and nucleotides 397-399 from “GCG” to “GAA”, from positions 28-2676; B1-2) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GGC” to “AGC”, nucleotides 397-399 from “GCG” to “GAA”, and nucleotides 601-603 from “AGC” to “CCG”, is the nucleotides 28-2676 of the resulting nucleic acid molecule. B1-3) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GGC” to “AGC”, nucleotides 397-399 from “GCG” to “GAA”, and nucleotides 547-549 from “GTG” to “AAA”, is the nucleotides 28-2676 of the resulting nucleic acid molecule; B1-4) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GGC” to “AGC”, nucleotides 397-399 from “GCG” to “GAA”, and nucleotides 508-510 from “CAT” to “GAA”, consists of nucleotides 28-2676. B1-5) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GGC” to “AGC”, nucleotides 397-399 from “GCG” to “GAA”, and nucleotides 514-516 from “AAA” to “TGG”, is the nucleotides 28-2676 of the resulting nucleic acid molecule. B1-6) The nucleic acid molecule obtained by mutating nucleotides 163-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from “ATGGGC” to “CTGAGC” and nucleotides 397-399 from “GCG” to “GAA”, and nucleotides 28-2676 of the resulting nucleic acid molecule; B1-7) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GGC” to “AGC”, nucleotides 397-399 from “GCG” to “GAA”, and nucleotides 520-522 from “AAC” to “CCG”, is the nucleotides 28-2676 of the resulting nucleic acid molecule. B1-8) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GGC” to “AGC”, nucleotides 397-399 from “GCG” to “GAA”, and nucleotides 532-534 from “CAG” to “GAT”, consists of nucleotides 28-2676. B1-9) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GGC” to “AGC”, nucleotides 397-399 from “GCG” to “GAA”, and nucleotides 682-684 from “ATG” to “CTG”, is the nucleotides 28-2676 of the resulting nucleic acid molecule. B1-10) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GGC” to “AGC”, nucleotides 397-399 from “GCG” to “GAA”, and nucleotides 1114-1116 from “AAA” to “CCG”, is the 28th-2676th nucleotide; B1-11) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GGC” to “AGC”, nucleotides 397-399 from “GCG” to “GAA”, and nucleotides 799-801 from “GCG” to “GAT”, is the nucleotides 28-2676 of the resulting nucleic acid molecule. B1-12) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from “GGC” to “AGC”, nucleotides 397-399 from “GCG” to “GAA”, and nucleotides 555-597 from “ATG” to “GAT”, is the nucleotides 28-2676 of the resulting nucleic acid molecule. The nucleic acid sequence shown in B1-13) has more than 70% homology with any of B1-1) to B1-12) and encodes the T7 RNA polymerase mutant; B1-14) hybridizes under stringent conditions with any one of the specified nucleic acid sequences from B1-1) to B14) and encodes the T7 RNA polymerase mutant.

[0022] In some embodiments of the present invention, the expression cassette refers to DNA capable of expressing the T7 RNA polymerase mutant in host cells. This DNA may include not only a promoter to initiate transcription of the T7 RNA polymerase mutant gene, but also a terminator to terminate transcription of the T7 RNA polymerase mutant gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0023] In some embodiments of the present invention, the vector may be a plasmid, a granule, a bacteriophage, or a viral vector. For example, it may be a PET-28a vector.

[0024] In some embodiments of the present invention, the recombinant vector may be a recombinant vector obtained by inserting a DNA molecule encoding the T7 RNA polymerase mutant into the multiple cloning site of the vector.

[0025] In some embodiments of the present invention, the biological cells include prokaryotic cells and eukaryotic cells. The prokaryotic cells include bacteria or algae. The eukaryotic cells include fungi, mammalian cells, or insect cells. The bacteria may be *Escherichia coli*, such as... E. coli DH5α or E. coli BL21. The recombinant organism does not contain reproductive material.

[0026] In some embodiments of the present invention, the recombinant biological cell is a recombinant biological cell obtained by introducing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3) into a biological cell. Specifically, it can be introduced into a biological cell... E. coli DH5α or E. coli Recombinant Escherichia coli obtained by introducing a recombinant vector into BL21.

[0027] A third aspect of the present invention provides an enzyme preparation comprising the T7 RNA polymerase mutant described in any one of the first aspects.

[0028] In some embodiments of the present invention, the enzyme preparation further includes a reaction premix or an enzyme preservation solution.

[0029] In some embodiments of the present invention, the reaction premix includes RNase inhibitors, spermidine, Tris-HCl, DTT, NTPs, and Mg. 2+ At least one of them.

[0030] In some embodiments of the present invention, the enzyme preservation solution contains at least one of KPB, NaCl, DTT, EDTA, and glycerol.

[0031] It is understood that the reaction premix and enzyme preservation solution should preferably not affect the activity of the T7 RNA polymerase mutant.

[0032] A fourth aspect of the present invention provides a method for preparing the T7 RNA polymerase mutant according to any one of the first aspects, comprising: The coding gene of the T7 RNA polymerase mutant described in the first aspect embodiment is introduced into a biological cell to express the coding gene, thereby obtaining the T7 RNA polymerase mutant.

[0033] In some embodiments of the present invention, the biological cells include prokaryotic cells and eukaryotic cells.

[0034] In some embodiments of the present invention, the prokaryotic cells include bacteria or algae. The bacteria may be *Escherichia coli* (e.g., *Escherichia coli*). E. coli BL21).

[0035] In some embodiments of the present invention, the eukaryotic cells include fungi (such as yeast), mammalian cells (such as HEK293 cells), or insect cells.

[0036] A fifth aspect of the present invention provides a method for in vitro amplification of RNA molecules, comprising the following steps: RNA molecules were amplified using the T7 RNA polymerase mutant described in the first aspect embodiment or the enzyme preparation described in the third aspect embodiment.

[0037] A sixth aspect of the invention provides the use of any one of C1) to C3) in the preparation of nucleic acid transcription products; C1) The T7 RNA polymerase mutant described in the first aspect embodiment; C2), the biomaterials described in the second aspect embodiment; C3), the enzyme preparation described in the third aspect embodiment.

[0038] A seventh aspect of the present invention provides the application of the T7 RNA polymerase mutant described in the first aspect embodiment or the enzyme preparation described in the third aspect embodiment in nucleic acid transcription.

[0039] An eighth aspect of the present invention provides a kit for nucleic acid transcription, comprising the T7 RNA polymerase mutant described in the first aspect embodiment or the enzyme preparation described in the third aspect embodiment.

[0040] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0041] Figure 1 To compare the transcriptional performance of wild-type T7 RNA polymerase and T7 RNA polymerase mutants M1-M12 in Example 1 at different temperatures; where 1: 37℃, 2: 42℃, 3: 45℃; Figure 2 To detect the standard curve constructed in Example 2; Figure 3 The results were analyzed to determine the percentage of dsRNA, a transcriptional byproduct of the T7 RNA polymerase mutant M1-M12 in Example 2. Detailed Implementation

[0042] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0043] In the description of this invention, the term "amino acid" refers to the basic unit that constitutes a protein, giving the protein a specific molecular structure and morphology, and endowing its molecules with biochemical activity. For example, the "amino acid" used in this invention includes the following 20 natural amino acids: alanine (Ala or A), glycine (Gly or G), isoleucine (Ile or I), asparagine (Asn or N), arginine (Arg or R), lysine (Lys or K), lysine (Lys or K), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), leucine (Leu or L), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), valine (Val or V), and tyrosine (Tyr or Y). The "*" in the amino acid sequence is a stop codon.

[0044] In the description of this invention, each amino acid substitution is represented by a triplet: letter-number-letter; wherein the number indicates the position of the mutated amino acid, the letter before the number corresponds to the amino acid involved in the mutation, and the letter after the number indicates the amino acid used to replace the amino acid before the number.

[0045] The following detailed description is provided with reference to specific embodiments. Unless otherwise specified, conditions in the following embodiments are performed under conventional conditions (e.g., conditions described in literature or books) or conditions recommended by the manufacturer. Reagents or instruments used in the embodiments, unless otherwise specified by the manufacturer, are all commercially available conventional products.

[0046] Example 1: Obtaining the T7 RNA polymerase mutant The amino acid sequence of wild-type T7 RNA polymerase is shown in SEQ ID NO: 1. Compared to wild-type T7 RNA polymerase, the mutation sites of T7 RNA polymerase mutant M1 are A124E and G47S; those of T7 RNA polymerase mutant M2 are A124E, G47S, and S192P; those of T7 RNA polymerase mutant M3 are A124E, G47S, and V174K; those of T7 RNA polymerase mutant M4 are A124E, G47S, and H161E; those of T7 RNA polymerase mutant M5 are A124E, G47S, and K163W; those of T7 RNA polymerase mutant M6 are A124E, G47S, and M46L; those of T7 RNA polymerase mutant M7 are A124E, G47S, and N165P; and those of T7 RNA polymerase mutant M8 are A124E, G47S, and Q169D. The mutation sites for RNA polymerase mutant M9 are A124E, G47S, and M219L; the mutation sites for T7 RNA polymerase mutant M10 are A124E, G47S, and K363P; the mutation sites for T7 RNA polymerase mutant M11 are A124E, G47S, and A258D; and the mutation sites for T7 RNA polymerase mutant M12 are A124E, G47S, and M190D.

[0047] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA (SEQ ID NO: 1).

[0048] 1. Construction of expression vector: The codons of the wild-type T7 RNA polymerase encoding gene were optimized, and a nucleotide sequence encoding a 6×histidine tag (HHHHHH) and a linker peptide (GS) was designed at the 5' end of the coding sequence. The optimized nucleotide sequence is shown in SEQ ID NO: 2. Using PCR point mutagenesis, primers were designed based on the mutation sites of the T7 RNA polymerase mutant to perform point mutations, obtaining the nucleic acid molecule encoding the T7 RNA polymerase mutant.

[0049] The specific mutation details are as follows: T7 RNA polymerase mutant M1 (A124E / G47S): nucleotides 166-168 are mutated from “GGC” to “AGC”, and nucleotides 397-399 are mutated from “GCG” to “GAA”. T7 RNA polymerase mutant M2 (A124E / G47S / S192P): nucleotides 166-168 are mutated from “GGC” to “AGC”, nucleotides 397-399 are mutated from “GCG” to “GAA”, and nucleotides 601-603 are mutated from “AGC” to “CCG”. T7 RNA polymerase mutant M3 (A124E / G47S / V174K): nucleotides 166-168 are mutated from “GGC” to “AGC”, nucleotides 397-399 are mutated from “GCG” to “GAA”, and nucleotides 547-549 are mutated from “GTG” to “AAA”. T7 RNA polymerase mutant M4 (A124E / G47S / H161E): nucleotides 166-168 are mutated from “GGC” to “AGC”, nucleotides 397-399 are mutated from “GCG” to “GAA”, and nucleotides 508-510 are mutated from “CAT” to “GAA”. T7 RNA polymerase mutant M5 (A124E / G47S / K163W): nucleotides 166-168 are mutated from “GGC” to “AGC”, nucleotides 397-399 are mutated from “GCG” to “GAA”, and nucleotides 514-516 are mutated from “AAA” to “TGG”. T7 RNA polymerase mutant M6 (A124E / G47S / M46L): nucleotides 163-168 are mutated from “ATGGGC” to “CTGAGC”, and nucleotides 397-399 are mutated from “GCG” to “GAA”. T7 RNA polymerase mutant M7 (A124E / G47S / N165P): nucleotides 166-168 are mutated from “GGC” to “AGC”, nucleotides 397-399 are mutated from “GCG” to “GAA”, and nucleotides 520-522 are mutated from “AAC” to “CCG”. T7 RNA polymerase mutant M8 (A124E / G47S / Q169D): nucleotides 166-168 are mutated from “GGC” to “AGC”, nucleotides 397-399 are mutated from “GCG” to “GAA”, and nucleotides 532-534 are mutated from “CAG” to “GAT”. T7 RNA polymerase mutant M9 (A124E / G47S / M219L): nucleotides 166-168 are mutated from “GGC” to “AGC”, nucleotides 397-399 are mutated from “GCG” to “GAA”, and nucleotides 682-684 are mutated from “ATG” to “CTG”. T7 RNA polymerase mutant M10 (A124E / G47S / K363P): nucleotides 166-168 are mutated from “GGC” to “AGC”, nucleotides 397-399 are mutated from “GCG” to “GAA”, and nucleotides 1114-1116 are mutated from “AAA” to “CCG”. T7 RNA polymerase mutant M11 (A124E / G47S / A258D): nucleotides 166-168 are mutated from “GGC” to “AGC”, nucleotides 397-399 are mutated from “GCG” to “GAA”, and nucleotides 799-801 are mutated from “GCG” to “GAT”. T7 RNA polymerase mutant M12 (A124E / G47S / M190D): nucleotides 166-168 are mutated from “GGC” to “AGC”, nucleotides 397-399 are mutated from “GCG” to “GAA”, and nucleotides 555-597 are mutated from “ATG” to “GAT”.

[0050]

[0051] The corresponding DNA molecules were ligated into the PET-28a vector to obtain recombinant expression vectors PET-28a / WT, PET-28a / M1, PET-28a / M2, PET-28a / M3, PET-28a / M4, PET-28a / M5, PET-28a / M6, PET-28a / M7, PET-28a / M8, PET-28a / M9, PET-28a / M10, PET-28a / M11, and PET-28a / M12. Each recombinant expression vector was transformed into E. coli BL21 DE3 Star competent cells, and positive single colonies were screened for sequencing verification, obtaining single colonies with valid verification results.

[0052] 2. Protein expression: The single colony verified in step 1 above was inoculated into 100 mL LB medium containing 50 μg / mL ampicillin and cultured overnight at 37°C with shaking. The seed culture from the overnight culture was then inoculated into 2 L LB medium containing 50 μg / mL ampicillin at a volume ratio of 1:100 and cultured at 37°C with shaking until 0D. 600 The concentration was 0.6~0.8; 10% IPTG was added to a final concentration of 0.4 mmol / L, and the cells were induced by shaking at 37℃ for 6~8 h; the cells were collected by centrifugation and weighed, and the wet weight of the cells was recorded and stored at -80℃.

[0053] 3. Protein purification: (1) High-pressure disruption of induced expression cells: After induction of expression, bacterial cells were frozen at -80℃. Based on the recorded wet weight of the bacterial cells, 5 mL of lysis buffer (50 mM Tris-HCl, pH 7.8 at 25℃, 300 mM NaCl, 50 mM Imidazole, 5% Glycerol) was added per gram of bacterial cells to resuspend the cells. The cells were then lysed using a high-pressure homogenizer at 650 bar for three cycles. The lysed cells were centrifuged at 12000 rpm for 30 min at 4℃, and the supernatant and precipitate were separated. The supernatant was transferred to a 200 mL sterile beaker. SDS-PAGE electrophoresis was performed on both the supernatant and the precipitate.

[0054] (2) Nickel ion affinity chromatography purification: The selected chromatography column was a Ni-NTA Purose 6 Fast Flow (purchased from Jiaxing Qianchun Biotechnology Co., Ltd.). The binding buffer was buffer A (50mM Tris-HCl, pH 7.8 at 25℃, 100mM NaCl, 80 mM Imidazole, 5% Glycerol); the elution buffer was buffer B (50mM Tris-HCl, pH 7.8 at 25℃, 100mM NaCl, 500 mM Imidazole, 5% Glycerol). The supernatant from step (1) was loaded into the chromatography column equilibrated with buffer A. After loading, the column was first rinsed with equilibration buffer A, and then eluted with buffer B at a gradient of 0%-100%. The eluted fraction was detected by SDS-PAGE protein electrophoresis. The eluent containing the target protease was collected according to the detection results, and the eluent was dialyzed into buffer A. The dialysate was collected for later use.

[0055] (3) Secondary nickel ion affinity chromatography purification: Repeat step (2) to perform SDS-PAGE protein electrophoresis on the eluted fraction. Collect the elution containing the target protease according to the detection results, and dialyze the elution into buffer C (50 mM Tris-HCl, pH 7.8 at 25℃, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA). Collect the dialysate for later use.

[0056] (4) Strong anion exchange chromatography purification: The selected chromatography column was a Q Purose 6 Fast Flow (purchased from Jiaxing Qianchun Biotechnology Co., Ltd.), with binding buffer C and elution buffer D (50 mM Tris-HCl, pH 7.8 at 25℃, 800 mM NaCl, 1 mM DTT, 0.1 mM EDTA). The dialysate obtained in step (3) was added to the chromatography column that had been equilibrated with buffer C. After the addition was complete, the column was first rinsed with buffer C, and then eluted with buffer D at a gradient of 0%-100%. The eluted fraction was detected by SDS-PAGE protein electrophoresis. The eluent was collected according to the detection results and dialyzed into a storage solution (20 mM KPB (pH 7.9), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% glycerol) for later use.

[0057] Detection Example 1 Two single-stranded DNA molecules (SEQ ID NO: 3 and SEQ ID NO: 4) were designed and annealed to create a 30 bp double-stranded DNA template containing the T7 promoter. T7 RNA polymerase in vitro transcription systems were prepared according to Table 1 using wild-type T7 RNA polymerase and T7 RNA polymerase mutants M1-M12, respectively, and transcribed at 37℃ / 42℃ / 45℃ for 3 h. The transcribed products were digested with DNase I, and the products were analyzed using 15% urea for Native Page assay.

[0058] DNA single strand 1: 5'-TTCTAATACGACTCACTATAGGGAAACATCTTGTTGAGAGCAGTATACAACTC-3' (SEQ ID NO: 3); DNA single strand 2: 5'-GAGTTGTATACTGCTCTCAACAAGATGTTTCCCTATAGTGAGTCGTATTAGAA-3' (SEQ ID NO: 4).

[0059] Table 1

[0060] The results are as follows Figure 1 As shown.

[0061] Wild-type T7 RNA polymerase and T7 RNA polymerase mutants M1-M12 all exhibit good transcription performance at 37℃, 42℃, and 45℃.

[0062] Detection Example 2 The nucleic acid sequence shown in SEQ ID NO: 5 was used as a template for detecting byproducts. T7 RNA polymerase in vitro transcription systems were prepared according to Table 1 using wild-type T7 RNA polymerase and T7 RNA polymerase mutants M1-M12. Transcription was performed at 37℃ for 2 h, followed by inactivation at 70℃ for 10 min. The transcribed product was digested with DNase I to remove the DNA template. The digested transcripts were recovered using an RNA purification kit (Aikerui Biotechnology, catalog number: AG21033), and the volume of the recovered product was brought to 100 μL with enzyme-free sterile water. The product concentration was recorded. The RNA product obtained from wild-type T7 RNA polymerase was diluted 5000× with enzyme-free sterile water, and the RNA product from the T7 RNA polymerase mutants was diluted 2000× for subsequent detection. The transcripts were quantified using a Qubit RNA Quantification Kit (Thermo Fisher Scientific). The diluted RNA products were detected using the NovoFast dsRNA ELISA Kit (nearshore protein, catalog number: RD017). A standard curve was first constructed using the standards in the kit. The diluted RNA products and standards were then processed in the same manner as described in the kit instructions. The absorbance values ​​at 450 nm were recorded, and the dsRNA content and percentage of the product were calculated (dsRNA content percentage = dsRNA yield / total RNA yield × 100%).

[0063] 5'--3' (SEQ ID NO: 5).

[0064] The constructed standard curve is as follows Figure 2 As shown. The results are as follows. Figure 3 (The ratio of dsRNA produced by transcription to total RNA) and Table 2 are shown.

[0065] Table 2

[0066] Compared to wild-type T7 RNA polymerase, the T7 RNA polymerase mutant M1-M12 has a lower dsRNA yield.

[0067] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A T7 RNA polymerase mutant, characterized in that, The T7 RNA polymerase mutant is the protein described in A1) or A2): A1) Compared to wild-type T7 RNA polymerase, the T7 RNA polymerase mutant contains the amino acid substitution site A124E / G47S; the amino acid sequence of the wild-type T7 RNA polymerase is shown in SEQ ID NO: 1; A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence described in A1).

2. The T7 RNA polymerase mutant according to claim 1, characterized in that, The T7 RNA polymerase mutant also contains at least one of the following amino acid substitution sites: S192P, V174K, H161E, K163W, M46L, N165P, Q169D, M219L, K363P, A258D, and M190D.

3. Biomaterials associated with the T7 RNA polymerase mutant according to claim 1 or 2, characterized in that, The biomaterial is any one of B1) to B4): B1) A nucleic acid molecule encoding the T7 RNA polymerase mutant as described in claim 1 or 2; B2), an expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) Recombinant biological cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

4. The biomaterial of claim 3, wherein, The nucleic acid sequence of the nucleic acid molecule is shown in any one of B1-1) to B1-14): B1-1) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GGC" to "AGC" and nucleotides 397-399 from "GCG" to "GAA" is nucleotide 28-2676. B1-2) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GGC" to "AGC", nucleotides 397-399 from "GCG" to "GAA", and nucleotides 601-603 from "AGC" to "CCG" is nucleotide 28-2676. B1-3) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GGC" to "AGC", nucleotides 397-399 from "GCG" to "GAA", and nucleotides 547-549 from "GTG" to "AAA" is nucleotide 28-2676. B1-4) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GGC" to "AGC", nucleotides 397-399 from "GCG" to "GAA", and nucleotides 508-510 from "CAT" to "GAA" is nucleotide 28-2676. B1-5) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GGC" to "AGC", nucleotides 397-399 from "GCG" to "GAA", and nucleotides 514-516 from "AAA" to "TGG" is nucleotide 28-2676. B1-6) The nucleic acid molecule obtained by mutating nucleotides 163-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from "ATGGGC" to "CTGAGC" and nucleotides 397-399 from "GCG" to "GAA" is nucleotide 28-2676. B1-7) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GGC" to "AGC", nucleotides 397-399 from "GCG" to "GAA", and nucleotides 520-522 from "AAC" to "CCG" is nucleotide 28-2676. B1-8) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GGC" to "AGC", nucleotides 397-399 from "GCG" to "GAA", and nucleotides 532-534 from "CAG" to "GAT" is nucleotide 28-2676. B1-9) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GGC" to "AGC", nucleotides 397-399 from "GCG" to "GAA", and nucleotides 682-684 from "ATG" to "CTG" is nucleotide 28-2676. B1-10) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GGC" to "AGC", nucleotides 397-399 from "GCG" to "GAA", and nucleotides 1114-1116 from "AAA" to "CCG" is the nucleotides 28-2676. B1-11) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GGC" to "AGC", nucleotides 397-399 from "GCG" to "GAA", and nucleotides 799-801 from "GCG" to "GAT" is nucleotide 28-2676. B1-12) The nucleic acid molecule obtained by mutating nucleotides 166-168 of the nucleic acid sequence shown in SEQ ID NO: 2 from "GGC" to "AGC", nucleotides 397-399 from "GCG" to "GAA", and nucleotides 555-597 from "ATG" to "GAT" is nucleotide 28-2676. The nucleic acid sequence shown in B1-13) has more than 70% homology with any of B1-1) to B1-12) and encodes the T7 RNA polymerase mutant; B1-14) hybridizes under stringent conditions with any of the specified nucleic acid sequences from B1-1) to B14) and encodes the T7 RNA polymerase mutant.

5. An enzyme preparation, characterized in that, Includes the T7 RNA polymerase mutant as described in claim 1 or 2.

6. A method of producing the T7 RNA polymerase mutant of claim 1 or 2, characterized by, include: The coding gene of the T7 RNA polymerase mutant according to claim 1 or 2 is introduced into a biological cell to express the coding gene, thereby obtaining the T7 RNA polymerase mutant.

7. A method of amplifying an RNA molecule in vitro, characterized in that, Includes the following steps: RNA molecules are amplified using the T7 RNA polymerase mutant as described in claim 1 or 2 or the enzyme preparation as described in claim 5.

8. The application of any one of C1) to C3) in the preparation of nucleic acid transcription products; C1), the T7 RNA polymerase mutant according to claim 1 or 2; C2), the biomaterial as described in claim 3 or 4; C3), the enzyme preparation according to claim 5.

9. The use of the T7 RNA polymerase mutant of claim 1 or 2 or the enzyme preparation of claim 5 in nucleic acid transcription.

10. A kit for nucleic acid transcription, characterized in that, Includes the T7 RNA polymerase mutant of claim 1 or 2 or the enzyme preparation of claim 5.

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