Thermostable mutant of t7 RNA polymerase and use of thermostable mutant

Abstract

The present invention relates to a thermostable mutant of a T7 RNA polymerase and a use of the thermostable mutant. Specifically, the present invention uses deep learning to predict mutation sites of amino acids, quickly screens for mutants having high-temperature activity and stability, and measures the melting temperature and activity of the mutants to obtain a mutant having increased thermal stability and high-temperature activity relative to a wild-type T7 RNA polymerase. The mutant of the present invention can be subjected to a transcription reaction at high temperature to reduce the production of by-products during the preparation of mRNA vaccines and drugs, can be applied to nucleic acid isothermal amplification technology, and the like, and has application prospects.

Description

Thermostable mutants of T7 RNA polymerase and uses thereof TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to thermostable mutants of T7 RNA polymerase and uses thereof. BACKGROUND

[0002] T7 RNA polymerase is a monomeric enzyme from the double-stranded DNA virus T7 bacteriophage, which has a total of 883 amino acids. Its main function is to polymerize RNA by catalyzing the formation of phosphodiester bonds between nucleoside triphosphate substrates using DNA as a template, and it plays an important role in the process of genetic information transmission from DNA to RNA. Since it was first used for RNA synthesis in the early 1980s, T7 RNA polymerase has become an important tool in the fields of molecular biology and genetic engineering.

[0003] RNA polymerases exist in almost all organisms and can be broadly classified into bacterial, archaeal, eukaryotic, and viral RNA polymerases. The RNA polymerases of bacteria, archaea, and eukaryotes usually have complex subunit structures and require the participation of multiple auxiliary factors in the transcription process, which poses a great challenge to the study of the transcription mechanism of the enzyme itself. In contrast, viral RNA polymerases usually have only a single subunit and a relatively simple structure. Common examples include T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase. Compared with non-viral polymerases, T7 RNA polymerase has a simple structure, high promoter specificity, and a very low error rate. It only transcribes DNA downstream of the T7 promoter and does not require auxiliary factors. Its transcription rate is 5 times that of E. coli RNAP. Compared with other viral polymerases, T7 RNA polymerase has a higher polymerization rate and transcribable length than SP6 RNA polymerase and T3 RNA polymerase. Due to its promoter specificity and high RNA polymerase activity, T7 RNA polymerase has a wide range of applications in many fields of molecular biology. In the field of recombinant protein expression, T7 RNA polymerase can be used to express recombinant genes at high levels in E. coli and for protein production in cell-free expression systems.

[0004] In the field of mRNA vaccines and drugs, T7 RNA polymerase is often used to produce mRNA. However, some unnecessary by-products, such as double-stranded RNA, are produced during transcription. If mRNA vaccines containing double-stranded RNA are injected into the human body, unnecessary immunogenic reactions will be triggered, and it is necessary to remove double-stranded RNA during the production of mRNA vaccines. The New England Biolabs team found that by increasing the temperature of the reaction, the by-products produced in in vitro RNA synthesis can be reduced, which puts higher requirements on the thermal stability and activity of T7 RNA polymerase at high temperatures.

[0005] Therefore, there is an urgent need in the art to develop T7 RNA polymerase mutants with thermal stability.

[0006] SUMMARY

[0007] The purpose of the present application is to provide T7 RNA polymerase mutants with thermal stability.

[0008] In a first aspect of the present application, a T7 RNA polymerase mutant is provided, the polymerase mutant comprising Q786L and L446F mutations; the numbering of the mutation sites is based on the sequence shown in SEQ ID NO: 1; and the melting temperature of the polymerase mutant is increased by ≥ 4°C compared with the wild type.

[0009] In another preferred embodiment, the melting temperature of the polymerase mutant is increased by ≥ 5°C, preferably by ≥ 8°C, more preferably by ≥ 10°C compared with the wild type.

[0010] In another preferred embodiment, the melting temperature of the polymerase mutant is increased by 4-13°C, preferably by 6-13°C, more preferably by 8-13°C compared with the wild type.

[0011] In another preferred embodiment, the melting temperature of the polymerase mutant is 48-59°C, preferably 49-59°C, more preferably 50-59°C.

[0012] In another preferred embodiment, the catalytic activity of the polymerase mutant at a higher temperature is Q1, and the catalytic activity of the wild type at the same temperature is Q0, Q1 / Q0≥ 5, preferably Q1 / Q0≥ 10, more preferably Q1 / Q0≥ 20; the higher temperature refers to 37-58°C, preferably 40-55°C, more preferably 42-53°C.

[0013] In another preferred embodiment, the polymerase mutant further comprises one or more amino acid mutations selected from the group consisting of S633P, L665D, G618E, S430P, A124N, M369T, N529V.

[0014] In another preferred embodiment, the polymerase mutant comprises a combination of mutations selected from the group consisting of:

[0015] (a) Q786L, L446F, and S633P;

[0016] (b) Q786L, L446F, and L665D;

[0017] (c) Q786L, L446F, and G618E;

[0018] (d) Q786L, L446F, and S430P; or

[0019] (e) Q786L, L446F, G618E, and L665D.

[0020] In another preferred embodiment, the polymerase mutant further comprises one or more amino acid mutations selected from the group consisting of S397W, S606V, I217L, L534V, K642G, T375K, P476E, Y846R, R792M, W797L, N601E, Y312D, P20A, P72E, H523K, Q656L, C125A, H772K, A881F, P657K.

[0021] In another preferred embodiment, the polymerase mutant comprises a combination of mutations Q786L, L446F, S633P, L534V, and L665D.

[0022] In another preferred embodiment, the polymerase mutant further comprises n amino acid mutations selected from the group consisting of S430P, S606V, I217L, S397W, M369T, A124N, N529V, G618E; 4 < n < 8.

[0023] In another preferred embodiment, the polymerase mutant comprises a combination of mutations selected from the group consisting of:

[0024] (1) Q786L, L446F, S430P, S606V, T375K, S633P, I217L, S397W, L534V, M369T, N529V, A124N, G618E, L665D;

[0025] (2) Q786L, L446F, S606V, T375K, S633P, I217L, S397W, L534V, M369T, N529V, A124N, G618E, L665D;

[0026] (3) Q786L, L446F, S606V, T375K, S633P, I217L, S397W, L534V, M369T, A124N, G618E, L665D;

[0027] (4) Q786L, L446F, S430P, S606V, S633P, I217L, S397W, L534V, M369T, A124N, L665D;

[0028] (5) Q786L, L446F, S430P, S606V, S633P, I217L, L534V, A124N, G618E, L665D;

[0029] (6) Q786L, L446F, S430P, S606V, S633P, S397W, L534V, M369T, G618E, L665D;

[0030] (7) Q786L, L446F, S430P, S606V, S633P, S397W, L534V, G618E, L665D;

[0031] (8) Q786L, L446F, S430P, S633P, S397W, L534V, N529V, G618E, L665D;

[0032] (9) Q786L, L446F, S430P, S633P, S397W, L534V, M369T, G618E, L665D;

[0033] (10) Q786L, L446F, S430P, S606V, S633P, I217L, S397W, L534V, L665D;

[0034] (11) Q786L, L446F, S430P, S633P, I217L, S397W, L534V, M369T, L665D;

[0035] (12) Q786L, L446F, S430P, Y846R, S606V, I217L, S397W, S633P;

[0036] (13) Q786L, L446F, S430P, Y846R, I217L, S397W, S633P;

[0037] (14) Q786L, L446F, S430P, W797L, Y846R, S606V, S397W, S633P;

[0038] (15) Q786L, L446F, S430P, W797L, S606V, I217L, S397W, S633P;

[0039] (16) Q786L, L446F, S430P, Y846R, S606V, S397W, S633P;

[0040] (17) Q786L, L446F, S430P, W797L, S606V, S397W, S633P;

[0041] (18) Q786L, L446F, S430P, Y846R, N601E, S606V, S397W, S633P;

[0042] (19) Q786L, L446F, S430P, N601E, S606V, I217L, S397W, S633P;

[0043] (20) Q786L, L446F, S430P, W797L, Y846R, I217L, S397W, S633P;

[0044] (21) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, P20A;

[0045] (22) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, A124N;

[0046] (23) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, R792M;

[0047] (24) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, G618E;

[0048] (25) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, L655D;

[0049] (26) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, P657K;

[0050] (27) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, N529V;

[0051] (28) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, M369T;

[0052] (29) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, H523K;

[0053] (30) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, Q656L;

[0054] (31) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, P72E;

[0055] (32) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, Y312D;

[0056] (33) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, R792M, L665D;

[0057] (34) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, A124N, L665D;

[0058] (35) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, Y312D, L665D;

[0059] (36) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, R792M, A881F;

[0060] (37) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, A124N, G618E;

[0061] (38) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, N529V, L665D;

[0062] (39) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, M369T, L665D;

[0063] (40) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, R792M, M369T;

[0064] (41) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, R792M, A124N;

[0065] (42) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, G618E, L665D;

[0066] (43) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, R792M, H772K;

[0067] (44) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, R792M, C125A;

[0068] (45) Q786L, L446F, S430P, S606V, K642G, S633P, I217L, S397W, L534V, A124N, G618E, L665D;

[0069] (46) Q786L, L446F, S430P, S606V, K642G, S633P, I217L, S397W, L534V, G618E, L665D;

[0070] (47) Q786L, L446F, S430P, S606V, K642G, S633P, S397W, L534V, G618E, L665D;

[0071] (48) Q786L, L446F, S430P, S606V, K642G, S633P, I217L, S397W, L534V, M369T, A124N, G618E, L665D;

[0072] (49) Q786L, L446F, S606V, K642G, S633P, I217L, S397W, L534V, M369T, A124N, G618E, L665D;

[0073] (50) Q786L, L446F, S430P, N601E, S606V, S633P, I217L, S397W, L534V, M369T, N529V, A124N, G618E, L665D;

[0074] (51) Q786L, L446F, S430P, W797L, Y846R, S606V, S633P, I217L, S397W, L534V, N529V, A124N, G618E, L665D;

[0075] (52) Q786L, L446F, S430P, W797L, Y846R, S606V, S633P, I217L, S397W, L534V, M369T, N529V, G618E, L665D;

[0076] (53) Q786L, L446F, S430P, S606V, S633P, I217L, S397W, L534V, A124N, G618E, L665D;

[0077] (54) Q786L, L446F, S430P, S633P, S397W, L534V, A124N, G618E, L665D;

[0078] (55) Q786L, L446F, S430P, S606V, S633P, I217L, S397W, A124N, G618E, L665D;

[0079] (56) Q786L, L446F, S430P, S606V, S633P, S397W, A124N, G618E, L665D;

[0080] (57) Q786L, L446F, S430P, S606V, S633P, I217L, S397W, G618E, L665D;

[0081] (58) Q786L, L446F, S430P, S606V, S633P, S397W, M369T, G618E, L665D; or

[0082] (59) Q786L, L446F, S430P, S606V, S633P, I217L, S397W, N529V, G618E, L665D.

[0083] In another preferred embodiment, the mutation sites of the polymerase mutant are in a combination selected from the group consisting of:

[0084] (1) Q786L, L446F, S430P, S606V, T375K, S633P, I217L, S397W, L534V, M369T, N529V, A124N, G618E, L665D;

[0085] (2) Q786L, L446F, S606V, T375K, S633P, I217L, S397W, L534V, M369T, N529V, A124N, G618E, L665D;

[0086] (3) Q786L, L446F, S606V, T375K, S633P, I217L, S397W, L534V, M369T, A124N, G618E, L665D;

[0087] (4) Q786L, L446F, S430P, S606V, S633P, I217L, S397W, L534V, M369T, A124N, L665D;

[0088] (5) Q786L, L446F, S430P, S606V, S633P, I217L, L534V, A124N, G618E, L665D;

[0089] (6) Q786L, L446F, S430P, S606V, S633P, S397W, L534V, M369T, G618E, L665D;

[0090] (7) Q786L, L446F, S430P, S606V, S633P, S397W, L534V, G618E, L665D;

[0091] (8) Q786L, L446F, S430P, S633P, S397W, L534V, N529V, G618E, L665D;

[0092] (9) Q786L, L446F, S430P, S633P, S397W, L534V, M369T, G618E, L665D;

[0093] (10) Q786L, L446F, S430P, S606V, S633P, I217L, S397W, L534V, L665D;

[0094] (11) Q786L, L446F, S430P, S633P, I217L, S397W, L534V, M369T, L665D;

[0095] In another preferred embodiment, the mutation sites of the polymerase mutant are in a combination selected from the group consisting of:

[0096] (1) Q786L, L446F, S430P, S606V, T375K, S633P, I217L, S397W, L534V, M369T, N529V, A124N, G618E, L665D;

[0097] (5) Q786L, L446F, S430P, S606V, S633P, I217L, L534V, A124N, G618E, L665D;

[0098] (6) Q786L, L446F, S430P, S606V, S633P, S397W, L534V, M369T, G618E, L665D;

[0099] (7) Q786L, L446F, S430P, S606V, S633P, S397W, L534V, G618E, L665D;

[0100] (9) Q786L, L446F, S430P, S633P, S397W, L534V, M369T, G618E, L665D.

[0101] In another preferred embodiment, the polymerase mutant is capable of reducing the formation of by-products in an in vitro RNA synthesis reaction, the by-products including double-stranded RNA (dsRNA).

[0102] In a second aspect of the present application, there is provided an isolated polynucleotide encoding the T7 RNA polymerase mutant as described in the first aspect of the present application.

[0103] In another preferred embodiment, the polynucleotide is DNA, RNA, cDNA, or a combination thereof.

[0104] In a third aspect of the present application, there is provided a vector comprising the polynucleotide as described in the second aspect of the present application.

[0105] In another preferred embodiment, the vector is an expression vector.

[0106] In a fourth aspect of the present application, there is provided a genetically engineered host cell comprising the vector as described in the third aspect of the present application, or the genome of which is integrated with the polynucleotide as described in the second aspect of the present application.

[0107] In another preferred embodiment, the host cell includes prokaryotic cells and eukaryotic cells.

[0108] In another preferred embodiment, the host cell is a prokaryotic cell.

[0109] In another preferred embodiment, the host cell comprises a cell derived from a microorganism selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipites, Candida shehatae, Candida tropicalis, Escherichia coli.

[0110] In another preferred embodiment, the host cell is Escherichia coli.

[0111] In a fifth aspect of the present application, a method for preparing the T7 RNA polymerase mutant as described in the first aspect of the present application is provided, comprising the steps of:

[0112] (1) introducing the polynucleotide as described in the second aspect of the present application into a host cell to obtain a genetically engineered host cell;

[0113] (2) culturing the genetically engineered host cell under conditions suitable for protein expression, thereby obtaining a culture comprising the T7 RNA polymerase mutant;

[0114] (3) isolating and / or purifying the T7 RNA polymerase mutant from the culture.

[0115] In a sixth aspect of the present application, a use of the T7 RNA polymerase mutant as described in the first aspect of the present application for the preparation of a reagent or a kit for the selection of a use selected from the group consisting of in vitro RNA synthesis, cell-free protein expression, nucleic acid detection, or a combination thereof is provided.

[0116] In another preferred embodiment, the RNA comprises an RNA drug.

[0117] In another preferred embodiment, the RNA drug comprises an RNA vaccine, a gene therapy drug, or a combination thereof.

[0118] In another preferred embodiment, the RNA is mRNA.

[0119] In another preferred embodiment, the RNA is non-coding RNA.

[0120] In another preferred embodiment, the non-coding RNA comprises sgRNA, tRNA, siRNA, snoRNA, or a combination thereof.

[0121] In a seventh aspect of the present application, there is provided a reagent for in vitro RNA synthesis reaction, the reagent comprising the T7 RNA polymerase mutant as described in the first aspect of the present application.

[0122] In an eighth aspect of the present application, there is provided a kit comprising the reagent as described in the seventh aspect of the present application.

[0123] In another preferred embodiment, the kit is for in vitro RNA synthesis reaction, which further comprises other reagents required for in vitro RNA synthesis reaction, including: DNA template, NTPs, in vitro RNA synthesis reaction buffer, and optionally including RNase inhibitor.

[0124] In a ninth aspect of the present application, there is provided a method for in vitro synthesis of RNA, comprising the step of: using the T7 RNA polymerase mutant as described in the first aspect of the present application to perform a catalytic reaction.

[0125] In another preferred embodiment, the catalytic reaction is performed at 37-58°C, preferably 40-55°C, more preferably 42-53°C.

[0126] In another preferred embodiment, the method has one or more advantages selected from the group consisting of:

[0127] (a) increasing the temperature of in vitro RNA synthesis reaction;

[0128] (b) reducing the formation of by-products in in vitro RNA synthesis reaction, the by-products including double-stranded RNA (dsRNA).

[0129] It should be understood that, within the scope of the present application, each of the technical features described above and in the following (such as the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0130] FIG. 1 shows a melting temperature column chart of T7 RNA polymerase single-site amino acid mutants.

[0131] Figure 2 shows the melting temperature column chart of T7 RNA polymerase wild type and mutants of the application.

[0132] Figure 3 shows the relative activity results of T7 RNA polymerase mutants transcribed at 52℃ for 1 hour, the relative activity refers to the ratio of mutant fluorescent signal to wild type fluorescent signal.

[0133] Figure 4 shows the relative activity results of T7 RNA polymerase mutants transcribed at 55℃ for 1 hour, the relative activity refers to the ratio of mutant fluorescent signal to wild type fluorescent signal.

[0134] Figure 5 shows the relative activity results of wild type and mutant T7 RNA polymerase at different incubation temperatures.

[0135] Figure 6 shows the RNA yield of T7 RNA polymerase mutants transcribed 1K or 4K length template.

[0136] Figure 7 shows the content of byproduct dsRNA in the transcription product of wild type and mutant T7 RNA polymerase. DETAILED DESCRIPTION

[0137] After extensive and in-depth research, the inventors used deep learning to predict the mutation site of amino acid, and screened out T7 RNA polymerase mutants with high temperature activity and stability through extensive experiments, which comprise a mutation combination of Q786L and L446F, and optionally one or more mutations at other sites. The mutant has thermal stability, and the melting temperature is greater than 48℃, which is significantly improved compared with the melting temperature of 45.5℃ of wild type T7 RNA polymerase. In addition, the catalytic activity of the mutant of the application at 52℃ is at least about 20 times that of the wild type, and the catalytic performance is significantly improved. The mutant of the application can perform in vitro transcription reaction at a higher temperature to reduce the production of byproducts in the preparation of mRNA vaccine and drugs, and is suitable for nucleic acid isothermal amplification technology. On this basis, the present application is completed.

[0138] TERMS

[0139] For easier understanding of the present application, certain technical and scientific terms are defined in detail below. Unless otherwise clearly defined in this text, all other technical and scientific terms used in this text have the meanings generally understood by those of ordinary skill in the art to which the present application belongs. Before describing the present application, it should be understood that the present application is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, and the scope of the present application will only be limited by the appended claims.

[0140] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this application belongs.

[0141] As used herein, the term "wild-type T7 RNA polymerase" or "WT T7 RNA polymerase" each includes a protein having the amino acid sequence as set forth in SEQ ID NO: 1.

[0142] As used herein, the terms "T7 RNA polymerase mutant", "mutant T7 RNA polymerase", "T7 RNA polymerase mutant protein" are used interchangeably and each refers to a mutant protein having at least two or more amino acid substitutions, deletions or insertions compared to the amino acid sequence of wild-type T7 RNA polymerase.

[0143] As used herein, the terms "melting temperature", "Tm" are used interchangeably and refer to the temperature corresponding to 50% of the protein unfolded.

[0144] The term "corresponding to" as used herein has the meaning commonly understood by one of ordinary skill in the art. In particular, "corresponding to" means that after homology or sequence identity alignment, a position in one sequence corresponds to a specified position in another sequence. Thus, for example, "corresponding to wild-type T7 RNA polymerase" means that an amino acid sequence is aligned with the amino acid sequence of wild-type T7 RNA polymerase to find the position in the amino acid sequence that corresponds to wild-type T7 RNA polymerase.

[0145] Mutants of the present application

[0146] As used herein, the term "mutants of the present application" refers to bacteriophage T7 RNA polymerase mutants.

[0147] In the present application, a preferred class of mutants is T7 RNA polymerase mutants having Q786L and L446F mutations corresponding to wild-type T7 RNA polymerase (the amino acid sequence is set forth in SEQ ID NO: 1). Such mutants have significantly improved performance compared to wild-type T7 RNA polymerase and other single-site mutants, as evidenced by: a melting temperature higher than 48°C, significantly higher than the 45.5°C of wild-type, with excellent thermal stability; in the use of in vitro RNA synthesis reactions, the mutants can reduce the formation of by-products in the reaction, including double-stranded RNA (dsRNA); at a temperature of 52°C, the catalytic activity of the mutants is at least about 20 times that of wild-type, up to about 60 times.

[0148] The sequence of wild-type T7 RNA polymerase is as follows (SEQ ID NO: 1):

[0149] The sequence of the Q786L+L446F T7 RNA polymerase mutant is as follows (SEQ ID NO: 2):

[0150] In one embodiment, the mutant is a Q786L+L446F T7 RNA polymerase mutant, which comprises an amino acid sequence as set forth in SEQ ID NO: 2. The term also includes variants and derivatives of the sequence of SEQ ID NO: 2 having the same function as the polypeptide shown. These variants include, but are not limited to, deletion, insertion and / or substitution of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or more (typically within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus.

[0151] More preferably, the mutant of the present application is further comprises a mutation of an amino acid residue selected from the group consisting of S633P, L665D, G618E, S430P, A124N, M369T, N529V, based on the wild-type T7 RNA polymerase sequence set forth in SEQ ID NO: 1. These single-site mutants do not have significantly improved melting temperature, and even have a lower melting temperature than the wild-type, but when combined with the Q786L+L446F mutation, the melting temperature of the multi-site mutant is significantly improved. For example, the melting temperature of the mutant with only G618E single mutation is 44°C, which is lower than the wild-type 45.5°C, but the melting temperature of the Q786L+L446F+G618E triple-site mutant is 49.4°C. The melting temperature of the single-site or multi-site mutants of T7 RNA polymerase is shown in Table 1.

[0152] Table 1. Partial T7 RNA polymerase mutants and their melting temperatures

[0153] More preferably, the mutant of the present application is based on Q786L+L446F T7 RNA polymerase mutant, comprising one or more of S633P, L665D, G618E, S430P, A124N, M369T, N529V, and further comprising mutation of amino acid residue selected from the group consisting of S397W, S606V, I217L, L534V, K642G, T375K, P476E, Y846R, R792M, W797L, N601E, Y312D, P20A, P72E, H523K, Q656L, C125A, H772K, A881F, P657K; wherein the positions are based on the wild-type T7 RNA polymerase sequence set forth in SEQ ID NO: 1. Such mutants have further improved performance, with melting temperature all higher than 49℃, and the highest melting temperature can reach 58.3℃. The mutation sites and melting temperatures of the multi-site mutants of the present application are shown in Table 2 below.

[0154] Table 2 T7 RNA polymerase mutants of the present application and their melting temperatures

[0155] Among the mutants of the present application shown in Table 2, the melting temperature is all improved compared with the wild type, with the maximum improvement of 12.8℃.

[0156] The present application also includes fragments, derivatives and analogs of the protein (or polypeptide). As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially retains the same biological function or activity of the polypeptide.

[0157] The polypeptide fragments, derivatives or analogs of the present application can be (i) a polypeptide having a substitution group in one or more amino acid residues, or (ii) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a fusion protein / tag sequence used to purify the polypeptide). These fragments, derivatives and analogs are within the scope of those skilled in the art according to the teachings herein.

[0158] As used herein, "isolated protein (or polypeptide)" refers to the protein substantially free of other proteins, lipids, carbohydrates or other materials with which it can be associated in nature. Those skilled in the art can purify the protein using standard protein purification techniques. A substantially pure protein produces a single major band on a non-reducing polyacrylamide gel. The purity of the protein can also be further analyzed using techniques such as amino acid sequencing.

[0159] The amino- or carboxyl-terminus of the protein of the present application can further comprise one or more polypeptide fragments as a protein tag. Any suitable tag can be used in the present application. For example, the tag can be FLAG, HA, c-Myc, Poly-His, Poly-Arg, Strep II, etc. These tags can be used for purification of the protein.

[0160] Polynucleotides, vectors and host cells of the present application

[0161] The polynucleotides of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0162] The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0163] The present application also relates to variants of the above polynucleotides, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present application. The variants of the polynucleotides can be naturally occurring isovariants or non-naturally occurring variants, but do not substantially alter the function of the encoded polypeptides.

[0164] The polypeptides and polynucleotides of the present application are preferably provided in isolated form, and more preferably purified to homogeneity. The full-length nucleotide sequence of the T7 RNA polymerase mutant of the present application or a fragment thereof can generally be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequence disclosed herein, and a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art can be used as a template to amplify the relevant sequence.

[0165] Once the relevant sequence is obtained, a recombinant protein can be prepared using bioengineering methods. This is generally achieved by cloning the polynucleotide sequence encoding the recombinant protein into an expression vector, and then transforming the expression vector into cells for expression, harvesting and purifying the recombinant protein.

[0166] At present, the DNA sequence encoding the protein (or fragment thereof, or derivative thereof) of the present application can be obtained entirely by chemical synthesis. The DNA sequence can then be introduced into various existing plasmids (or vectors) known in the art, and transformed into cells for expression and purification to obtain the protein.

[0167] The present application also relates to vectors comprising the appropriate DNA sequence described above and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express the protein.

[0168] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0169] The transformation of host cells with recombinant DNA and the recombinant expression of proteins are well known to those skilled in the art. The recombinant expressed proteins can be isolated and purified by conventional techniques well known to those skilled in the art, which will not be described here.

[0170] Applications

[0171] The present application also provides the use of the T7 RNA polymerase mutants and derivatives thereof of the present application, for preparing reagents or kits for in vitro RNA synthesis, cell-free protein expression, nucleic acid detection, etc.

[0172] In an embodiment of the present application, the use of the mutants of the present application in in vitro RNA synthesis is provided. The mutants of the present application can be used for synthesizing RNA drugs, including RNA vaccines, RNA interference agents, RNA inhibitors, gene editing tools, etc. In an embodiment of the present application, the in vitro RNA synthesis reaction can be used for synthesizing mRNA, typically including mRNA vaccines. In an embodiment of the present application, the in vitro RNA synthesis reaction can be used for synthesizing non-coding RNAs, including sgRNA, tRNA, siRNA, snoRNA, etc. The in vitro RNA synthesis reaction has a wide temperature range, and can be particularly suitable for reactions at a relatively high temperature of about 48-58℃, thereby reducing the generation of byproducts during the reaction, including double-stranded RNA (dsRNA) and the like.

[0173] In an embodiment of the present application, the thermostability and high melting temperature of the mutants of the present application can be suitable for nucleic acid isothermal amplification techniques, thereby being widely used in the field of nucleic acid detection, etc.

[0174] The main advantages of the present application include:

[0175] (1) The T7 RNA polymerase mutants screened by the present application have excellent thermostability, with a melting temperature of more than 49℃, and the highest being up to 58.3℃, which is significantly improved compared with the wild type of 45.5℃.

[0176] (2) The T7 RNA polymerase mutant of the present application has significantly improved catalytic activity at 52°C, which is about 60 times higher than that of the wild type.

[0177] (3) The T7 RNA polymerase mutant of the present application can perform transcription reaction at high temperature, thereby reducing the by-products of the transcription reaction and improving the safety in the process of preparing RNA vaccine and drugs.

[0178] (4) The T7 RNA polymerase mutant of the present application is suitable for nucleic acid isothermal amplification technology and has broad application prospects.

[0179] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally performed according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and fractions are weight percentages and weight fractions.

[0180] The experimental methods in the following examples, if not specifically stated, are all conventional methods. The test materials used in the following examples, if not specifically stated, are all purchased from conventional biochemical reagent companies. The quantitative tests in the following examples are all set up with three repeated experiments, and the results are averaged.

[0181] Example 1: Construction and synthesis of wild-type T7 RNA polymerase plasmid vector and selection of mutation sites

[0182] The amino acid sequence of the wild-type T7 RNA polymerase was searched in the NCBI database, and the encoding gene thereof was synthesized by GenScript Biotech Corporation. After codon optimization, the pQE-80L vector was selected, the SacI and HindIII double enzyme digestion sites were selected, and the synthesized plasmid product was transformed into the E. coli Ecoli-BL21(DE3) competent cells. The wild-type (WT) amino acid sequence of the T7 RNA polymerase involved in the present application is shown in SEQ ID NO. 1.

[0183] The target sites for the modification of the T7 RNA polymerase based on deep learning were scored, and the top 50 were selected as target sites each time to be synthesized by GenScript Biotech Corporation. The mutant T7 RNA polymerase of the present application was made and verified to be correct by introducing mutations into the wild-type T7 RNA polymerase gene.

[0184] The mutation sites of the obtained T7 RNA polymerase mutant relative to the wild type are shown in Table 2.

[0185] Example 2: Expression of single-site and multi-site combination mutant proteins and measurement of melting temperature

[0186] E. coli Ecoli-BL21(DE3) containing T7 RNA polymerase was inoculated into LB liquid medium containing 50 pg / mL ampicillin and cultured in a shaker at 37 °C, 220 rpm. When the OD600 of the bacteria reached 0.6-0.8, 1 mM IPTG was added to induce the expression of the target protein at 37 °C for 6 h. The bacteria were collected by centrifugation at 4000 rpm for 25 min, washed with Buffer A (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 3 mM imidazole, 0.1 mM EDTA), and resuspended in Buffer A. The bacteria were then disrupted by ultrasonic treatment. The supernatant was collected by centrifugation at 12,000 rpm for 60 min. The supernatant was filtered and subjected to Ni-NTA purification. The elution fractions containing the target protein were collected and ultrafiltrated into Buffer B (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1 mM EDTA) and stored at -20 °C with the addition of 75% glycerol.

[0187] To measure the melting temperature of the mutant, 0.2 mg / ml T7 RNA polymerase variant was prepared in buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1 mM EDTA) and 5x exogenous fluorescent SYPRO Orange was added. The melting temperature was measured using an Analytik Jena qTower3.

[0188] The results of the melting temperature measurement of the single-site and multi-site mutants are shown in Figures 1 and 2. The results of Figure 1 show that only some of the single-site mutants have a small increase in melting temperature, and some of the single-site mutants have a lower melting temperature than the wild type. The results of Figure 2 show that the multi-site mutants of the present application formed by the combination of single-site mutants have a significantly higher melting temperature than the wild type, with the lowest increase of about 4 °C and the highest increase of about 12.8 °C. The results show that the multi-site mutants of the present application have unexpected and significant thermal stability.

[0189] Example 3: Detection of transcriptional activity at 52 °C

[0190] A 99 bp template DNA sequence was designed and synthesized, and the sequence name was iSpinach-template, with the base sequence (5'-3') as shown in SEQ ID NO: 3:

[0191] The end-point reaction buffer (200 mM HEPES, pH 7.5, 30 mM MgCl2, 20 mM DTT, 0.4 U / μL RNase inhibitor, 5 mM NTPs mix, 0.04 mg / ml T7 RNA polymerase, 100 nM DNA template) was configured, and the reaction was carried out at 52°C for 1 hour. The reaction was terminated by adding 100 mM EDTA, and 100 μΜ DFHBI was added after 10 min of reaction. The fluorescence signal was detected at an excitation wavelength of 470 nm and an emission wavelength of 512 nm.

[0192] The results of the relative activity (relative to the activity of the wild type) of the wild type T7 RNA polymerase and the mutants at 52°C for 1 hour of transcription are shown in the graph of FIG. 3. The results show that the catalytic activity of the mutant T7 RNA polymerase of the present application at 52°C is significantly higher than that of the wild type, with an increase of at least about 20 times, and up to about 60 times.

[0193] Example 4: Detection of transcription activity at 55°C

[0194] A 99 bp template DNA sequence shown in SEQ ID NO: 3 (sequence name iSpinach-template) was designed and synthesized. The end-point reaction buffer (200 mM HEPES, pH 7.5, 30 mM MgCl2, 20 mM DTT, 0.4 U / μL RNase inhibitor, 5 mM NTPs mix, 0.04 mg / ml T7 RNA polymerase, 100 nM DNA template) was configured, and the reaction was carried out at 55°C for 1 hour. The reaction was terminated by adding 100 mM EDTA, and 100 μΜ DFHBI was added after 10 min of reaction. The fluorescence signal was detected at an excitation wavelength of 470 nm and an emission wavelength of 512 nm.

[0195] The results of the relative activity (relative to the activity of the wild type) of the wild type T7 RNA polymerase and the mutants at 55°C for 1 hour of transcription are shown in FIG. 4. The results show that the catalytic activity of the mutant T7 RNA polymerase of the present application at 55°C is significantly higher than that of the wild type, with an increase of at least 30 times. Mutants M40, M53, and M57 have an activity increase of more than 50 times.

[0196] Example 5: Change of transcription activity with incubation temperature

[0197] A 99 bp template DNA sequence (Sequence name iSpinach-template) shown in SEQ ID NO: 3 was designed and synthesized. T7 RNA polymerase mutants M40, M53 and M57 were incubated at 40°C, 42.5°C, 45°C, 47.5°C, 50°C, 52.5°C, 55°C, 57.5°C, 60°C, respectively, for 15 minutes. End-point reaction buffer (200 mM HEPES, pH 7.5, 30 mM MgCl2, 20 mM DTT, 0.4 U / μL RNase inhibitor, 5 mM NTPs mix, 0.04 mg / ml T7 RNA polymerase, 100 nM DNA template) was configured, and the reaction was carried out at 37°C for half an hour. The reaction was terminated by adding 100 mM EDTA, and 100 μM DFHBI was added after 10 minutes of reaction. The fluorescence signal was detected at an excitation wavelength of 470 nm and an emission wavelength of 512 nm.

[0198] The results are shown in Figure 5, and the results show that the mutants M40, M53 and M57 of the application still maintain high reaction activity after incubation at higher temperatures.

[0199] Example 6: Yield change of different length templates

[0200] In order to study the yield change of the T7 RNA polymerase mutants of the application when catalyzing long templates, a 1K length EGFP template and a 4K length Cas9 template were selected for reaction, and the specific operation was as follows:

[0201] The total volume of the long template transcription system was 20 ul, Tris-HCl 40 mM ph 7.5, MgCl2 20 mM, NaCl 50 mM, spermidine 2 mM, DTT 1 mM, NTP mix 5 mM, RNase inhibitor 1 U / ul, Triton X-100 0.01%, T7 RNA polymerase 0.1 mg / ml, DNA template 15 ng / ul, and the reaction was carried out at 50°C for 30 minutes. After the reaction was completed, 2 U DNase I was added, and the reaction was incubated at 37°C for 30 minutes to digest the transcribed DNA template. The IVT solution after digestion was purified by using the RNA column purification kit of NEB.

[0202] The results are shown in Figure 6, and the T7 RNA polymerase mutants of the application have high levels of yield in the case of 1K or 4K long templates. It is shown that the T7 RNA polymerase mutants of the application have high levels of ability to catalyze long templates.

[0203] Example 7: Detection of by-product dsRNA

[0204] The by-product dsRNA is detected by sandwich ELISA, the dsRNA standard and the 1K-length sample obtained by transcription are added to the enzyme-labeled plate pre-coated with anti-dsRNA antibody, then biotin-labeled dsRNA detection antibody is added, and finally streptavidin-labeled horseradish peroxidase is added. After reaction, TMB color developing solution is added for color development. TMB is converted from colorless to blue under the catalysis of HRP enzyme and finally converted to yellow under the action of termination solution. The depth of yellow color is positively correlated with the amount of dsRNA detected in the sample.

[0205] As shown in Figure 7, the mutant M54, M55 and M57 of the present application have less by-product dsRNA in the transcription product, and the content of dsRNA is reduced by 50% or more compared with the wild-type T7 RNA polymerase, and the product has higher purity. The above results show that the transcription reaction catalyzed by the T7 RNA polymerase mutant of the present application produces less by-product, and is more suitable for use in the fields of vaccine and drug preparation.

[0206] All the documents mentioned in the present application are cited as references in the present application, as if each document is cited as a reference individually. In addition, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the above teaching of the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

Claims

1. A T7 RNA polymerase mutant, characterized in that, The polymerase mutant comprises Q786L and L446F mutations; the numbering of the mutation sites is based on the sequence set forth in SEQ ID NO: 1; and the melting temperature of the polymerase mutant is increased by > 4°C compared to wild type.

2. The T7 RNA polymerase mutant of claim 1, wherein, The catalytic activity of the polymerase mutant at a higher temperature is Q1, and the catalytic activity of wild type at the same temperature is Q0, Q1 / Q0 > 5, preferably Q1 / Q0 > 10, more preferably Q1 / Q0 > 20; the higher temperature refers to 37-58°C, preferably 40-55°C, more preferably 42-53°C.

3. The T7 RNA polymerase mutant of claim 1, wherein, The polymerase mutant further comprises one or more amino acid mutations selected from the group consisting of S633P, L665D, G618E, S430P, A124N, M369T, N529V.

4. The T7 RNA polymerase mutant of claim 1, wherein, The polymerase mutant comprises a combination of mutations selected from the group consisting of: (a) Q786L, L446F and S633P; (b) Q786L, L446F and L665D; (c) Q786L, L446F and G618E; (d) Q786L, L446F and S430P; or (e) Q786L, L446F, G618E and L665D.

5. The T7 RNA polymerase mutant of claim 3, wherein, The polymerase mutant further comprises one or more amino acid mutations selected from the group consisting of S397W, S606V, I217L, L534V, K642G, T375K, P476E, Y846R, R792M, W797L, N601E, Y312D, P20A, P72E, H523K, Q656L, C125A, H772K, A881F, P657K.

6. The T7 RNA polymerase mutant of claim 1, wherein, The polymerase mutant comprises a combination of mutations: Q786L, L446F, S633P, L534V and L665D.

7. The T7 RNA polymerase mutant of claim 6, wherein, The polymerase mutant further comprises n amino acid mutations selected from the group consisting of S430P, S606V, I217L, S397W, M369T, A124N, N529V, G618E; 4 < n < 8.

8. The T7 RNA polymerase mutant of claim 1, wherein, The mutation sites of the polymerase mutant are a combination selected from the group consisting of: (1) Q786L, L446F, S430P, S606V, T375K, S633P, I217L, S397W, L534V, M369T, N529V, A124N, G618E, L665D; (2) Q786L, L446F, S606V, T375K, S633P, I217L, S397W, L534V, M369T, N529V, A124N, G618E, L665D; (3) Q786L, L446F, S606V, T375K, S633P, I217L, S397W, L534V, M369T, A124N, G618E, L665D; (4) Q786L, L446F, S430P, S606V, S633P, I217L, S397W, L534V, M369T, A124N, L665D; (5) Q786L, L446F, S430P, S606V, S633P, I217L, L534V, A124N, G618E, L665D; (6) Q786L, L446F, S430P, S606V, S633P, S397W, L534V, M369T, G618E, L665D; (7) Q786L, L446F, S430P, S606V, S633P, S397W, L534V, G618E, L665D; (8) Q786L, L446F, S430P, S633P, S397W, L534V, N529V, G618E, L665D; (9) Q786L, L446F, S430P, S633P, S397W, L534V, M369T, G618E, L665D; (10) Q786L, L446F, S430P, S606V, S633P, I217L, S397W, L534V, L665D; (11) Q786L, L446F, S430P, S633P, I217L, S397W, L534V, M369T, L665D; (12) Q786L, L446F, S430P, Y846R, S606V, I217L, S397W, S633P; (13) Q786L, L446F, S430P, Y846R, I217L, S397W, S633P; (14) Q786L, L446F, S430P, W797L, Y846R, S606V, S397W, S633P; (15) Q786L, L446F, S430P, W797L, S606V, I217L, S397W, S633P; (16) Q786L, L446F, S430P, Y846R, S606V, S397W, S633P; (17) Q786L, L446F, S430P, W797L, S606V, S397W, S633P; (18) Q786L, L446F, S430P, Y846R, N601E, S606V, S397W, S633P; (19) Q786L, L446F, S430P, N601E, S606V, I217L, S397W, S633P; (20) Q786L, L446F, S430P, W797L, Y846R, I217L, S397W, S633P; (21) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, P20A; (22) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, A124N; (23) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, R792M; (24) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, G618E; (25) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, L655D; (26) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, P657K; (27) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, N529V; (28) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, M369T; (29) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, H523K; (30) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, Q656L; (31) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, P72E; (32) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, Y312D; (33) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, R792M, L665D; (34) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, A124N, L665D; (35) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, Y312D, L665D; (36) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, R792M, A881F; (37) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, A124N, G618E; (38) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, N529V, L665D; (39) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, M369T, L665D; (40) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, R792M, M369T; (41) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, R792M, A124N; (42) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, G618E, L665D; (43) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, R792M, H772K; (44) Q786L, L446F, S430P, P476E, S606V, T375K, K642G, S633P, I217L, S397W, L534V, R792M, C125A; (45) Q786L, L446F, S430P, S606V, K642G, S633P, I217L, S397W, L534V, A124N, G618E, L665D; (46) Q786L, L446F, S430P, S606V, K642G, S633P, I217L, S397W, L534V, G618E, L665D; (47) Q786L, L446F, S430P, S606V, K642G, S633P, S397W, L534V, G618E, L665D; (48) Q786L, L446F, S430P, S606V, K642G, S633P, I217L, S397W, L534V, M369T, A124N, G618E, L665D; (49) Q786L, L446F, S606V, K642G, S633P, I217L, S397W, L534V, M369T, A124N, G618E, L665D; (50) Q786L, L446F, S430P, N601E, S606V, S633P, I217L, S397W, L534V, M369T, N529V, A124N, G618E, L665D; (51) Q786L, L446F, S430P, W797L, Y846R, S606V, S633P, I217L, S397W, L534V, N529V, A124N, G618E, L665D; (52) Q786L, L446F, S430P, W797L, Y846R, S606V, S633P, I217L, S397W, L534V, M369T, N529V, G618E, L665D; (53) Q786L, L446F, S430P, S606V, S633P, I217L, S397W, L534V, A124N, G618E, L665D; (54) Q786L, L446F, S430P, S633P, S397W, L534V, A124N, G618E, L665D; (55) Q786L, L446F, S430P, S606V, S633P, I217L, S397W, A124N, G618E, L665D; (56) Q786L, L446F, S430P, S606V, S633P, S397W, A124N, G618E, L665D; (57) Q786L, L446F, S430P, S606V, S633P, I217L, S397W, G618E, L665D; (58) Q786L, L446F, S430P, S606V, S633P, S397W, M369T, G618E, L665D; or (59) Q786L, L446F, S430P, S606V, S633P, I217L, S397W, N529V, G618E, L665D.

9. The T7 RNA polymerase mutant of claim 1, wherein, The mutation site of the polymerase mutant is a combination selected from the group consisting of: (1) Q786L, L446F, S430P, S606V, T375K, S633P, I217L, S397W, L534V, M369T, N529V, A124N, G618E, L665D; (2) Q786L, L446F, S606V, T375K, S633P, I217L, S397W, L534V, M369T, N529V, A124N, G618E, L665D; (3) Q786L, L446F, S606V, T375K, S633P, I217L, S397W, L534V, M369T, A124N, G618E, L665D; (4) Q786L, L446F, S430P, S606V, S633P, I217L, S397W, L534V, M369T, A124N, L665D; (5) Q786L, L446F, S430P, S606V, S633P, I217L, L534V, A124N, G618E, L665D; (6) Q786L, L446F, S430P, S606V, S633P, S397W, L534V, M369T, G618E, L665D; (7) Q786L, L446F, S430P, S606V, S633P, S397W, L534V, G618E, L665D; (8) Q786L, L446F, S430P, S633P, S397W, L534V, N529V, G618E, L665D; (9) Q786L, L446F, S430P, S633P, S397W, L534V, M369T, G618E, L665D; (10) Q786L, L446F, S430P, S606V, S633P, I217L, S397W, L534V, L665D; (11) Q786L, L446F, S430P, S633P, I217L, S397W, L534V, M369T, L665D.

10. An isolated polynucleotide, comprising: The polynucleotide encodes the T7 RNA polymerase mutant of claim 1.

11. A vector, characterized in that, The vector contains the polynucleotide of claim 10.

12. A genetically engineered host cell, characterized in that, The host cell contains the vector of claim 11, or the genome of the host cell is integrated with the polynucleotide of claim 10.

13. A method of making the T7 RNA polymerase mutant of claim 1, wherein, Comprising the following steps: (1) introducing the polynucleotide of claim 10 into a host cell to obtain a genetically engineered host cell; (2) culturing the genetically engineered host cell under conditions suitable for protein expression, thereby obtaining a culture comprising the T7 RNA polymerase mutant; (3) isolating and / or purifying the T7 RNA polymerase mutant from the culture.

14. Use of a T7 RNA polymerase mutant according to claim 1, characterized in that, Use of the reagent or kit for in vitro RNA synthesis, cell-free protein expression, nucleic acid detection, or a combination thereof.

15. A reagent for an in vitro RNA synthesis reaction, characterized by, The reagent comprises the T7 RNA polymerase mutant of claim 1.

16. A kit comprising, The kit comprises the reagent of claim 15, and further comprises other reagents required for in vitro RNA synthesis reaction.

17. A method of performing in vitro synthesis of RNA, characterized by, The method comprises the step of performing a catalytic reaction using the T7 RNA polymerase mutant of the first aspect of the invention.

18. The method of claim 17, wherein, The catalytic reaction is performed at 37-58 °C.

Images