T7 rna polymerase mutants and uses thereof

Abstract

The present application relates to the technical field of biology, and in particular to a T7 RNA polymerase mutant and application thereof. The present application provides a T7 RNA polymerase mutant and application thereof in IVT reaction. The mutant can significantly reduce the content of dsRNA by-products in in vitro transcription of T7 RNA polymerase, has higher transcription activity, can improve the integrity of products, and has good thermal stability.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to T7 RNA polymerase mutants and their applications. Background Technology

[0002] Ribonucleic acid (RNA) is a crucial biomolecule for transmitting genetic information in life processes. With the deepening research into RNA, RNA therapies for disease prevention based on RNA molecules are gradually emerging. Especially with the successful development of COVID-19 mRNA vaccines, this emerging vaccine, with its high target specificity and low genotoxicity, has rapidly become a research hotspot. In vitro RNA synthesis mainly involves two methods: chemical synthesis and enzymatic synthesis. Chemical synthesis is primarily suitable for short-chain RNA; for long-chain RNA, the high synthesis cost limits its practicality. Since protein-encoding mRNA typically contains thousands of nucleotides, enzymatic synthesis is currently the most common and optimal method for long-chain mRNA. In vitro synthesis of long-chain mRNA generally uses a double-stranded DNA template, and mRNA is synthesized through transcription using RNA polymerase.

[0003] RNA polymerases are a class of enzymes that transcribe DNA templates into RNA. Phage-derived RNA polymerases, including T7, T3, and SP6 polymerases, specifically recognize their respective promoters. For example, T7 phage-derived RNA polymerase can only transcribe DNA sequences located downstream of the T7 promoter sequence. In a typical in vitro transcription (IVT) reaction, the designed DNA template contains a phage promoter sequence located upstream of the target gene sequence, which is transcribed by the corresponding RNA polymerase. Besides forming complete single-stranded RNA, IVT reactions also generate some contaminant RNAs, such as truncated transcripts, linked transcripts, and double-stranded RNA (dsRNA). These contaminants severely affect the use of downstream RNA, requiring additional purification methods to remove them.

[0004] Among all RNA polymerases, T7 RNA polymerase, derived from *E. coli* T7 phage, is the most commonly used. Identified in the 1970s, it has since been widely applied for in vivo protein expression and in vitro RNA synthesis. T7 RNA polymerase consists of 883 amino acids, approximately 99 kDa, and uses double-stranded DNA as a template to transcribe and synthesize mRNA by recognizing specific promoter sequences. T7 RNA polymerase has a mature and stable heterologous expression and purification process, and it can perform transcriptional activity normally without any other cofactors, producing high-fidelity full-length RNA transcripts, thus becoming the mainstream tool enzyme for in vitro transcription. Although T7 RNA polymerase has significant advantages as an in vitro RNA synthase, it also suffers from the drawback of forming contaminating RNA. While some purification methods, such as high-performance liquid chromatography (HPLC), can remove this immune activation caused by the heterogeneity of RNA products, these methods significantly increase production costs in large-scale production, and the increased purification steps are detrimental to the stability of RNA drugs. Therefore, an improved T7 RNA polymerase mutant is needed to reduce the amount of contaminating RNA during IVT, improve transcription efficiency, and reduce purification steps.

[0005] There are various mutants of T7 RNA polymerase in the existing technology, but further improvements are still needed in many aspects such as transcriptase activity, byproduct content, transcription integrity and thermostability. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a T7 RNA polymerase mutant and its application.

[0007] The present invention provides a mutant of T7 RNA polymerase, and the amino acid sequence of the wild-type T7 RNA polymerase is shown in SEQ ID NO:1.

[0008] The mutant is mutated at at least one of the following sites: position 64, position 65, position 69, position 73, position 74, position 123, position 125, position 128, position 133, position 134, position 136, position 140, position 143, position 154, position 159, position 166, position 170, position 176, position 179, position 183, position 202, position 206, position 231, position 232, position 241, position 242, position 254, position 299, ... 300th, 379th, 382nd, 383rd, 386th, 387th, 389th, 390th, 391st, 397th, 437th, 439th, 467th, 473rd, 568th, 623rd, 653rd, 654th, 656th, 657th, 660th, 742nd, 750th, 756th, 757th, 758th, 759th, 760th, 761st, 772nd, or 775th.

[0009] In some embodiments, the mutant of the T7 RNA polymerase has at least one of the following mutations: V64Q, A65N, A69G, L73A, I74G, L123T, C125S, S128T, T133S, V134A, A136G, A140G, R143K, I154T, A159G, V166S, L170S, H176E, K179D, M183T, S202N, K206Q, R231Q, Q232N, S241T, E242D, I254S, T299S, H300F, R379S, A382G, A383G, R386T, K387Q, K389N, A390L, R391S, S397T, N437C, M439V, C467T, V473I, A568G, Y623F, D653N, T654V, Q656K, L657I, D660E, P742A, M750S, R756I, L757V, Q758N, P759V, T760S, I761V, H772Y or E775N.

[0010] For example, the mutants of the T7 RNA polymerase described in this invention have 1 to 5 of the mutation sites mentioned above.

[0011] As a feasible example, mutants at the two mutation sites include, but are not limited to: those including V64Q and A65N, or those including V64Q and A69G, or those including V64Q and L73A, or those including V64Q and I74G, or those including V64Q and L123T, or those including A65N and A69G, or those including A65N and L73A, or those including A65N and I74G, or those including A65N and L123T, or those including A69G and L73A, or those including A69G and I74G, or those including A69G and L123T, or those including L73A and I74G, or those including L73A and L123T, or those including I74G and L123T, or those including L123T and C125S, or those including L123T. And S128T, or including C125S and S128T, or including C125S and T133S, or including S128T and T133S, or including T133S and V134A, or including V134A and A136G, or including A136G and A140G, or including A140G and R143K, or including R143K and I154T, or including I154T and A159G, or including A159G and V166S, or including V166S and L170S, or including L170S and H176E, or including H176E and K179D, or including K179D and M183T, or including M183T and S202N, or including S202N and K206Q, This may include K206Q and R231Q, or R231Q and Q232N, or Q232N and S241T, or S241T and E242D, or E242D and I254S, or I254S and T299S, or T299S and H300F, or H300F and R379S, or R379S and A382G, or A382G and A383G, or A383G and R386T, or R386T and K387Q, or K387Q and K389N, or K389N and A390L, or A390L and R391S, or R391S and S397T, or S397 T and N437C, or including N437C and M439V, or including M439V and C467T, or including C467T and V473I, or including V473I and A568G, or including A568G and Y623F, or including Y623F and D653N, or including D653N and T654V, or including T654V and Q656K, or including Q656K and L657I, or including L657I and D660E, or including D660E and P742A, or including P742A and M750S, or including M750S and R756I, or including R756I and L757V, or including L757V and Q758N, or including Q758N and P759V.This may include mutants such as P759V and T760S, T760S and I761V, I761V and H772Y, or H772Y and E775N.

[0012] As a feasible example, mutants at the three mutation sites include, but are not limited to: V64Q, A65N, A69G, or L73A, I74G, L123T, or C125S, S128T, T133S, or V134A, A136G, A140G, or R143K, I154T, A159G, or V166S, L170S, H176E, or K179D, M183T, S202N, or K206Q, R231Q, Q232N, or S241T, E242D, I254S, or T299S, H300F, R379S, or A382G, A383G, R386T. This may include K387Q, K389N, A390L, or R391S, S397T, N437C, or M439V, C467T, V473I, or A568G, Y623F, D653N, or T654V, Q656K, L657I, or D660E, P742A, M750S, or R756I, L757V, Q758N, or P759V, T760S, I761V, or H772Y, E775N, V64Q, or A65N, L73A, I74G, or A69G, C125S, S128T, or L123T, T133S, V134A, or other related models. S128T, A136G, A140G, or including T133S, R143K, I154T, or including V134A, V166S, L170S, or including A136G, H176E, K179D, or including A140G, M183T, S202N, or including R143K, K206Q, R231Q, or including I154T, Q232N, S241T, or including A159G, E242D, I254S, or including V166S, T299S, H300F, or including L170S, R379S, A382G, or including H176E, A383G, R386T, or including K179D, K387Q, K389N, or This includes M183T, A390L, R391S, or S202N, S397T, N437C, or K206Q, M439V, C467T, or R231Q, V473I, A568G, or Q232N, Y623F, D653N, or S241T, T654V, Q656K, or E242D, L657I, D660E, or I254S, P742A, M750S, or T299S, R756I, L757V, or H300F, Q758N, P759V, or R379S, T760S, I761V, or A382G, H772Y, E775N.This may include mutants of A383G, V64Q, A65N, R386T, A69G, L73A, K387Q, I74G, C125S, K389N, S128T, L123T, A390L, T133S, V134A, R391S, A136G, A140G, S397T, R143K, I154T, N437C, A159G, V166S, M439V, L170S, H176E, C467T, K179D, M183T, V473I, S202N, K206Q, or A568G, R231Q, Q232N.

[0013] As a feasible example, mutants at the four mutation sites include, but are not limited to: those including V64Q, A65N, A69G, L73A, or those including V64Q, A65N, I74G, L123T, or those including V64Q, A65N, C125S, S128T, or those including V64Q, A69G, I74G, C125S, or those including V64Q, A69G, L123T, S128T, or those including V64Q, I74G, L123T, T133S, or those including V64Q, L73A, L123T, T133S, or those including A65N, A69G, L73A, I74G, or those including A65N, A69G, I74G, L123T, or those including A65N. L73A, I74G, C125S, or including A65N, L73A, C125S, S128T, or including A65N, I74G, C125S, T133S, or including A69G, L73A, I74G, L123T, or including A69G, L73A, L123T, C125S, or including A69G, I74G, L123T, S128T, or including L73A, I74G, L123T, T133S, or including I74G, L123T, C125S, S128T, or including I74G, L123T, T133S, V134A, or including L123T, C125S, S128T, T133S, or including L12 3T, C125S, T133S, A136G, or including C125S, S128T, T133S, A136G, or including S128T, T133S, A136G, A140G, or including T133S, A136G, A140G, R143K, or including A136G, A140G, R143K, I154T, or including A140G, R143K, I154T, A159G, or including R143K, I154T, A159G, V166S, or including I154T, A159G, V166S, L170S, or including A159G, V166S, L170S, H176E, or including V166S, L170 S, H176E, K179D, or including L170S, H176E, K179D, M183T, or including H176E, K179D, M183T, S202N, or including K179D, M183T, S202N, K206Q, or including M183T, S202N, K206Q, R231Q, or including S202N, K206Q, R231Q, Q232N, or including K206Q, R231Q, Q232N, S241T, or including R231Q, Q232N, S241T, E242D, or including Q232N, S241T, E242D, I254S, or including S241T, E242D, I254S.T299S, or including E242D, I254S, T299S, H300F, or including I254S, T299S, H300F, R379S, or including T299S, H300F, R379S, A382G, or including H300F, R379S, A382G, A383G, or including R379S, A382G, A383G, R386T, or including A382G, A383G, R386T, K387Q, or including A383G, R386T, K387Q, K389N, or including R386T, K387Q, K389N, A390L, or including K387Q, K389N, A390L, R391S, or including K389N, A390L, R391S, S397T, or including A390L, R39 1S, S397T, N437C, or including R391S, S397T, N437C, M439V, or including S397T, N437C, M439V, C467T, or including N437C, M439V, C467T, V473I, or including M439V, C467T, V473I, A568G, or including C467T, V473I, A568G, Y623F, or mutants including V473I, A568G, Y623F, D653N, or mutants including A568G, Y623F, D653N, T654V, or mutants including Y623F, D653N, T654V, Q656K, or mutants including D653N, T654V, Q656K, L657I, or mutants including T654V, Q656K, L657I, and D660E.

[0014] As a feasible example, mutants at the five mutation sites include, but are not limited to: those including V64Q, A65N, L73A, I74G, L123T, or those including A69G, S128T, T133S, V134A, A136G, or those including A140G, R143K, I154T, A159G, V166S, or those including L170S, H176E, K179D, M183T, S202N, or those including K206Q, R231Q, Q232N, S241T, E242D, or those including I254S, T299S, H300F, R379S, A382G, or those including A383G, R386T, K387Q, K389N, A390L. Or including R391S, S397T, N437C, M439V, C467T, or including V473I, A568G, Y623F, D653N, T654V, or including Q656K, L657I, D660E, P742A, M750S, or including R756I, L757V, Q758N, P759V, T760S, or including I761V, H772Y, E775N, V64Q, A65N, or including A69G, L73A, I74G, L123T, C125S, or including S128T, T133S, V134A, A136G, A140G, or including R143K, I154T, A159G, V1 66S, L170S, or including H176E, K179D, M183T, S202N, K206Q, or including R231Q, Q232N, S241T, E242D, I254S, or including T299S, H300F, R379S, A382G, A383G, or including R386T, K387Q, K389N, A390L, R391S, or including S397T, N437C, M439V, C467T, V473I, or including A568G, Y623F, D653N, T654V, Q656K, or including L657I, D660E, P742A, M750S, R756I, or including L757V. Q758N, P759V, T760S, I761V, or including H772Y, E775N, V64Q, A65N, A69G, or including L73A, I74G, L123T, C125S, S128T, or including T133S, V134A, A136G, A140G, R143K, or including I154T, A159G, V166S, L170S, H176E, or including K179D, M183T, S202N, K206Q, R231Q, or including Q232N, S241T, E242D, I254S, T299S, or including H300F, R379S, A382G, A383G, R386T.This may include K387Q, K389N, A390L, R391S, S397T, or N437C, M439V, C467T, V473I, A568G, or Y623F, D653N, T654V, Q656K, L657I, or D660E, P742A, M750S, R756I, L757V, or Q758N, P759V, T760S, I761V, H772Y, or E775N, V64Q, A65N, A69G, L73A, or I74G, L123T, C125S, S128T, T133S, or V134A, A1 36G, A140G, R143K, I154T, or including A159G, V166S, L170S, H176E, K179D, or including M183T, S202N, K206Q, R231Q, Q232N, or including S241T, E242D, I254S, T299S, H300F, or including R379S, A382G, A383G, R386T, K387Q, or including K389N, A390L, R391S, S397T, N437C, or including M439V, C467T, V473I, A568G, Y623F, or including D653N, T654V, Q65 6K, L657I, D660E, or including P742A, M750S, R756I, L757V, Q758N, or including P759V, T760S, I761V, H772Y, E775N, or including V64Q, A65N, A69G, L73A, I74G, or including L123T, C125S, S128T, T133S, V134A, or including A136G, A140G, R143K, I154T, A159G, or including V166S, L170S, H176E, K179D, M183T, or including S202N, K206Q, R231Q, Q232N, S2 41T, or mutants including E242D, I254S, T299S, H300F, R379S, or mutants including A382G, A383G, R386T, K387Q, K389N, or mutants including A390L, R391S, S397T, N437C, M439V, or mutants including C467T, V473I, A568G, Y623F, D653N, or mutants including T654V, Q656K, L657I, D660E, P742A, or mutants including M750S, R756I, L757V, Q758N, P759V, or mutants including T760S, I761V, H772Y, E775N, V64Q.

[0015] In some specific embodiments, the mutants described in this invention have S128T, C467T, and M750S mutations.

[0016] In other specific embodiments, the mutants described in this invention have T133S and N437C mutations;

[0017] In other specific embodiments, the mutant described in this invention has the I761V mutation;

[0018] In other specific embodiments, the mutant described in this invention has a T133S mutation;

[0019] In other specific embodiments, the mutants described in this invention have D660E and T760S mutations;

[0020] In other specific embodiments, the mutant described in this invention has the S241T mutation;

[0021] In other specific embodiments, the mutant described in this invention has the D660E mutation;

[0022] In other specific embodiments, the mutants described in this invention have H176E, R391S, and D660E mutations;

[0023] In other specific embodiments, the mutants described in this invention have I154T and S241T mutations;

[0024] In other specific embodiments, the mutants described in this invention have T299S, T760S, and Q758N mutations;

[0025] In other specific embodiments, the mutants described in this invention have K387Q and A390L mutations;

[0026] In other specific embodiments, the mutants described in this invention have A69G and P742A mutations;

[0027] In other specific embodiments, the mutants described in this invention have V473I, A568G, M750S, and Q758N mutations;

[0028] In other specific embodiments, the mutants described in this invention have S128T, K179D, and E775N mutations;

[0029] In other specific embodiments, the mutants described in this invention have C125S and H300F mutations;

[0030] In other specific embodiments, the mutants described in this invention have A136G, V166S, Q232N, and A383G mutations;

[0031] In other specific embodiments, the mutants described in this invention have K179D, A383G, and R756I mutations;

[0032] In other specific embodiments, the mutants described in this invention have R143K, T299S, R386T, A568G, and P742A mutations;

[0033] In other specific embodiments, the mutants described in this invention have I254S and V473I mutations;

[0034] In other specific embodiments, the mutants described in this invention have M183T, K387Q, and A568G mutations;

[0035] In other specific embodiments, the mutants described in this invention have C125S, R143K, V166S, Y623F, and H772Y mutations;

[0036] In other specific embodiments, the mutants described in this invention have R143K, Q232N, E242D, S397T, and P759V mutations;

[0037] In other specific embodiments, the mutants described in this invention have K206Q, A390L, and Y623F mutations;

[0038] In other specific embodiments, the mutants described in this invention have A159G and E242D mutations;

[0039] In other specific embodiments, the mutants described in this invention have V166S, T299S, and S397T mutations;

[0040] In other specific embodiments, the mutants described in this invention have T133S and K387Q mutations;

[0041] In other specific embodiments, the mutants described in this invention have A140G, V166S, L170S, and Y623F mutations;

[0042] In other specific embodiments, the mutants described in this invention have S128T, V134A, and E775N mutations;

[0043] In other specific embodiments, the mutants described in this invention have K179D, K206Q, E242D, and I761V mutations;

[0044] In other specific embodiments, the mutants described in this invention have T133S, T760S, and I761V mutations;

[0045] In other specific embodiments, the mutants described in this invention have A69G, A159G, R231Q, Q656K, and P742A mutations;

[0046] In other specific embodiments, the mutants described in this invention have A140G, Y623F, A65N, and V166S mutations;

[0047] In other specific embodiments, the mutants described in this invention have R231Q, Q232N, K387Q, M439V and R756I mutations;

[0048] In other specific embodiments, the mutants described in this invention have R143K, A159G, and Q232N mutations;

[0049] In other specific embodiments, the mutants described in this invention have A159G, L170S, H176E and V473I mutations;

[0050] In other specific embodiments, the mutants described in this invention have Q232N, K389N, N437C, and R756I mutations;

[0051] In other specific embodiments, the mutants described in this invention have V166S, M183T, R231Q, K389N, and H772Y mutations;

[0052] In other specific embodiments, the mutants described in this invention have L73A, I254S, and A383G mutations;

[0053] In other specific embodiments, the mutant described in this invention has the P742A mutation;

[0054] In other specific embodiments, the mutants described in this invention have A69G and Q758N mutations;

[0055] In other specific embodiments, the mutants described in this invention have T299S, R379S, and D660E mutations;

[0056] In other specific embodiments, the mutants described in this invention have R143K, V64Q, L657I, and R756I mutations;

[0057] In other specific embodiments, the mutants described in this invention have L73A and P742A mutations;

[0058] In other specific embodiments, the mutants described in this invention have V166S, I74G, C467T, and T654V mutations;

[0059] In other specific embodiments, the mutants described in this invention have H176E and H300F mutations;

[0060] In other specific embodiments, the mutants described in this invention have V64Q and S397T mutations;

[0061] In other specific embodiments, the mutants described in this invention have I154T, S202N, H300F, and D660E mutations;

[0062] In other specific embodiments, the mutants described in this invention have I154T, M183T, A383G, and Q758N mutations;

[0063] In other specific embodiments, the mutants described in this invention have I761V, I254S, and H300F mutations;

[0064] In other specific embodiments, the mutants described in this invention have A69G, I254S, and D653N mutations;

[0065] In other specific embodiments, the mutants described in this invention have R756I and T760S mutations;

[0066] In other specific embodiments, the mutant described in this invention has the A140G mutation;

[0067] In other specific embodiments, the mutants described in this invention have V166S, S202N, and T299S mutations;

[0068] In other specific embodiments, the mutants described in this invention have K206Q, R379S, and Q758N mutations;

[0069] In other specific embodiments, the mutants described in this invention have R391S and A382G mutations;

[0070] In other specific embodiments, the mutants described in this invention have M183T and H772Y mutations;

[0071] In other specific embodiments, the mutants described in this invention have R379S and K387Q mutations;

[0072] In other specific embodiments, the mutants described in this invention have A140G and L170S mutations;

[0073] In other specific embodiments, the mutants described in this invention have K179D, T299S, A390L, and T654V mutations;

[0074] In other specific embodiments, the mutants described in this invention have A65N and A136G mutations;

[0075] In other specific embodiments, the mutants described in this invention have Q232N and L757V mutations;

[0076] In other specific embodiments, the mutants described in this invention have T133S and R391S mutations;

[0077] In other specific embodiments, the mutants described in this invention have A136G, E242D, M439V and L657I mutations;

[0078] In other specific embodiments, the mutants described in this invention have A140G and A568G mutations;

[0079] In other specific embodiments, the mutants described in this invention have C125S, R143K, R379S, and P742A mutations;

[0080] In other specific embodiments, the mutants described in this invention have L123T, S241T, E242D, T654V, and L757V mutations;

[0081] In other specific embodiments, the mutant described in this invention has the A159G mutation;

[0082] In other specific embodiments, the mutants described in this invention have R379S and P742A mutations;

[0083] In other specific embodiments, the mutants described in this invention have A69G, R391S, S397T and R756I mutations;

[0084] In other specific embodiments, the mutants described in this invention have A65N, A140G, R379S, and P759V mutations.

[0085] The present invention mutates T7 RNA polymerase, and the resulting mutant has at least one of the following advantages: (1) reduced dsRNA production, (2) increased transcriptase activity, (3) improved transcription integrity, and (4) improved thermal stability.

[0086] The present invention also provides nucleic acids encoding the mutants described above.

[0087] The nucleic acid described in this invention can be mutated at the corresponding site in the coding sequence of the wild type shown in SEQ ID NO:2, or it can be codon optimized for the entire host. This invention does not limit this. In this invention, the nucleic acid encoding T7 RNA polymerase can be either DNA or RNA. The nucleic acid encompasses nucleotide sequences with different functions, such as coding regions and non-coding regions (e.g., regulatory sequences, which include promoters or transcription terminators, etc.).

[0088] Specifically, when the nucleic acid is DNA, it can be cDNA, genomic DNA, or DNA obtained through artificial synthesis. The nucleic acid described in this invention can be single-stranded or double-stranded; from a topological perspective, the nucleic acid can be linear or circular. Nucleic acids can also exist in various forms, such as as part of a vector (like an expression vector or cloning vector), or simply as a nucleic acid fragment. The nucleic acid described in this invention can be obtained directly from natural sources or prepared with the assistance of recombinant methods, enzymatic methods, or chemical techniques.

[0089] Furthermore, the present invention also provides an expression unit comprising the nucleic acid and promoter as described above.

[0090] In this invention, the promoter can be a prokaryotic promoter or a eukaryotic promoter, and this invention does not limit the type. For example, the promoter can be the T7 promoter, SP6 promoter, CMV promoter, SV40 promoter, U6 promoter, H1 promoter, CaMV 35S promoter, lac promoter, trp promoter, or araBAD promoter.

[0091] In addition to the promoter, the expression unit described in this invention may also include at least one of an enhancer, a transcription start site, a poly(A) signal, or a terminator. The terminator includes at least one of the following: T7 terminator, T4 terminator, SP6 terminator, SV40 terminator, CMV terminator, lacZ terminator, trpA terminator, rho-dependent terminator, rho-independent terminator, rpsL terminator, rplT terminator, rpoB terminator, rpsD terminator, rpsE terminator, and rpsF terminator.

[0092] The enhancer is at least one of the following: SV40 enhancer, CMV enhancer, SV-1 enhancer, ROSA26 enhancer, EF1α enhancer, HARE5 enhancer, UBC enhancer, EF1A enhancer, PGK enhancer, CAGG enhancer, COPIA enhancer, or ACT5C enhancer.

[0093] Furthermore, the present invention also provides a plasmid vector comprising the nucleic acid or expression unit as described above.

[0094] The plasmid vectors provided by this invention are used for the storage, amplification, or expression of the nucleic acid or expression unit, and this invention does not limit the specific uses. In a particular embodiment, the backbone vector of the plasmid vector is the pColdI vector. Alternatively, it can be the pColdII vector, pColdIII vector, pColdTF vector, pET series vectors, pGEX series vectors, pMAL series vectors, pBAD vector, pBADHis vector, pBADmycHis series vectors, pQE series vectors, pTrc99a vector, pTrcHis series vectors, pBV220 vector, pBV221 vector, pBV222 vector, pTXB series vectors, pLLP-ompA vector, pIN-III-ompA vector, pQBI63 vector, or pACYCduet-1 vector.

[0095] Furthermore, the present invention also provides a host,

[0096] Its transformation or transfection is performed using the plasmid vector described above;

[0097] Or its genome contains the aforementioned nucleic acids or expression units.

[0098] In this invention, the host is used for the storage, amplification, or expression of the mutant of the plasmid vector. In a specific embodiment, it is *Escherichia coli*. For example, *E. coli* DH5α is used for plasmid amplification or storage, and *E. coli* BL21 is used as the host for expressing the mutant. In addition, yeast, mammalian cells, insect cells, and *Bacillus subtilis* can also be used for the expression of the mutant described in this invention.

[0099] Furthermore, the present invention also provides a method for preparing the mutant as described above, which includes: culturing the host as described above, and inducing the expression of the mutant.

[0100] As a feasibility example, the host in the preparation method is *Escherichia coli*, and the culture medium is LB liquid medium. The bacterial strain is cultured until the OD600 value reaches 0.6–0.8, and then IPTG is used to induce the expression of the mutant. The concentration of IPTG used in the IPTG induction is 0.1–1.0 mM, preferably 0.5 mM. The induction conditions include incubation at 15°C and 250 rpm for approximately 20–24 hours.

[0101] After the induction of expression, the process also includes an extraction and purification step. Specifically, this involves taking wet bacterial bodies and resuspending them in 50mM Tris-HCl buffer at pH 8.0, adding PMSF to a final concentration of 1mM, sonicating the bacterial cells at 150W power with a 3-second on-time and 3-second off-time interval at 0-4℃, and then centrifuging at 15000rpm for 30 minutes at 4℃. The supernatant is then collected and loaded onto a nickel column for purification.

[0102] Furthermore, the present invention also provides the application of the mutants described above in the IVT response.

[0103] The mutant provided by this invention has good thermal stability and good activity in IVT reaction. It can not only maintain high transcriptional activity and preserve the integrity of the product, but also reduce the content of dsRNA.

[0104] Furthermore, the present invention provides an IVT reaction reagent comprising the mutant as described above or the mutant prepared by the method described above.

[0105] In some embodiments, the IVT reaction reagent further includes: Cap Analog, CTP / GTP / ATP / N1-Me-pUTP, inorganic pyrophosphatase, RNase inhibitor, transcription buffer, and water.

[0106] In the IVT reaction reagent of the present invention, the enzyme activity of the mutant is 200-300 U / μL, for example, 200 U / μL, 210 U / μL, 220 U / μL, 230 U / μL, 240 U / μL, 250 U / μL, 260 U / μL, 270 U / μL, 280 U / μL, 290 U / μL or 300 U / μL.

[0107] Furthermore, the present invention provides an in vitro transcription method, which uses the mutants described above or the IVT reaction reagents described above to convert double-stranded DNA into mRNA.

[0108] In this embodiment of the invention, the length of the double-stranded DNA is 1k to 8k. For example, the length of the double-stranded DNA is 1k, 2k, 3k, 4k, 5k, 6k, 7k, or 8k.

[0109] In some specific embodiments, each 20 μL of the conversion reaction system comprises:

[0110] 100mM Cap Analog 2μL; 10× transcription buffer 2μL; 100mM CTP / GTP / ATP / N1-Me-pUTP 2 μL each; Template DNA 1-2 μg; 250 U / μL mutant 1μL; 1 U / μL inorganic pyrophosphatase 0.04 μL; 40U / μL Nase inhibitor 1μL; <![CDATA[RNase free H2O]]> Make up to 20 μL.

[0111] In this embodiment of the invention, the reaction conditions for the transformation include a reaction at 35-40°C for 1-5 hours. For example, the reaction temperature for the transformation is 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C; and the reaction time for the transformation is 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or 5 hours.

[0112] This invention provides a T7 RNA polymerase mutant and its application in IVT reactions. The mutant can significantly reduce the content of dsRNA byproducts in in vitro transcription of T7 RNA polymerase, has high transcriptional activity, can improve the integrity of the product, and has good thermal stability. Attached Figure Description

[0113] Figure 1 The stability of different mutants after 1 day of storage was demonstrated.

[0114] Figure 2 The stability of different mutants after 3 days of storage was demonstrated.

[0115] Figure 3 The stability of different mutants after 7 days of storage was demonstrated. Detailed Implementation

[0116] This invention provides a T7 RNA polymerase mutant and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0117] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0118] In this invention, the terms "comprising," "including," and "having" are used interchangeably to indicate the inclusiveness of a solution, meaning that the solution may contain elements other than those listed. It should also be understood that the use of "comprising," "including," and "having" herein also provides for solutions "consisting of...".

[0119] In this invention, "and / or" as used herein includes the meaning of "and", "or", and "all or any other combination of elements linked by the term".

[0120] In this invention, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of a single item or a plurality of items.

[0121] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0122] IPTG: Isopropylβ-D-Thiogalactoside

[0123] LB: Luria-Bertani medium

[0124] PMSF: Phenylmethanesulfonylfluoride

[0125] Tris: Tris(hydroxymethyl)aminomethane (Tris(hydroxymethyl)aminomethane)

[0126] In this invention, the abbreviations of amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. Specifically, they are: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0127] The amino acid sequence of the wild-type T7 RNA polymerase involved in this invention (SEQ ID NO.1):

[0128] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0129] The nucleotide sequence (SEQ ID NO.2) encoding wild-type T7 RNA polymerase involved in the present invention:

[0130]

[0131] This invention, based on wild-type T7 RNA polymerase, identifies hotspot amino acids through virtual screening, performs virtual saturation mutations at corresponding sites, and obtains a series of T7 RNA polymerase mutants by identifying IVT yield, integrity, and dsRNA content. Compared with the wild type, the mutants have one or more of the following characteristics: higher transcriptional activity, higher integrity, lower dsRNA byproducts, and higher thermal stability.

[0132] The main steps of this invention include: first, synthesizing a wild-type T7 RNA polymerase plasmid; then, obtaining recombinant colonies with corresponding mutations through site-directed mutagenesis and semi-rational modification. After inducing expression and purification, the colonies are purified, and their purity and concentration are measured. Finally, the mutation effect is determined by IVT yield, integrity, dsRNA content, and thermostability.

[0133] It should be understood that in the various embodiments of this application, the sequence numbers of the above processes do not imply the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The present invention is further illustrated below with reference to embodiments:

[0134] Example 1:

[0135] 1. The wild-type T7 RNA polymerase sequence was constructed into the pColdI vector between NdeI and XbaI via whole-genome synthesis and transformed into E. coli BL21. The wild-type T7 RNA polymerase sequence is shown in Seq ID NO.1, and its corresponding nucleotide sequence is shown in Seq ID NO.2.

[0136] 2. The following mutants were obtained through directed evolution and semi-rational design.

[0137]

[0138]

[0139]

[0140] 3. Inoculate the mutant with the correct sequence into 1000 mL of fresh LB liquid medium and incubate in a shaker at 37°C and 200 rpm until the OD600 value is about 0.6-0.8. Then add 0.5 mM IPTG to the final concentration and incubate at 15°C and 250 rpm for about 20-24 h.

[0141] 4. Collect the bacterial cells after centrifugation at 4℃ and 8000 rpm for 10 min;

[0142] 5. Take 4g of wet bacterial body and suspend it in 40mL of 50mM Tris-HCl buffer at pH 8.0, and add it to a final concentration of 1mM PMSF;

[0143] 6. Place in an ice bath and sonicate at 150W power with a 3-second on-time and 3-second off-time interval until the solution is basically clear.

[0144] 7. After centrifuging the ultrasonically disrupted fluid at 4℃ and 15000rpm for 30min, collect the supernatant.

[0145] 8. Purify the mutant using a 20 mL nickel column. The purification procedure and purification buffer are shown in the table below:

[0146]

[0147]

[0148] Buffer 1 formulation: 100mM Tris-HCl, 200mM NaCl, 10% glycerol (pH 7.9);

[0149] Buffer 2 formulation: 100mM Tris-HCl, 200mM NaCl, 10% glycerol, 500mM imidazole (pH 7.9);

[0150] 9. Collect the eluent and place it in a dialysis bag with a molecular weight cutoff of 14000. Use Buffer 1 as the dialysate. Change the buffer four times, then remove the liquid from the bag. Add 50% glycerol, 10 mM DTT, 1 mM EDTA, and 0.1% (w / v) EDTA to a final concentration. After thoroughly mixing with X-100, filter through a 0.22μm filter membrane and determine the protein concentration using the TaKaRa Bradford Protein Assay Kit.

[0151] Example 2:

[0152] 1. Dilute each mutant protein to approximately 0.4 mg / mL, then serially dilute to approximately 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL;

[0153] 2. The T7 RNA polymerase in the Shenji Biotechnology commercial kit T7 co-transcription capping kit 10111 was serially diluted to 200U / μL, 100U / μL, 50U / μL, 25U / μL, 10U / μL, 5U / μL, 2U / μL, and 1U / μL for later use;

[0154] 3. According to the kit components and reaction conditions, perform in vitro transcription reactions using the enzymes described above. The template used for transcription is the 4K template produced by Shenji.

[0155] 4. Prepare working solution according to the requirements of AAT Bioquest pyrosequencing kit AAT-21611. Mix each gradient mutant and the serially diluted sample of commercial T7 RNA polymerase with the quantitative working solution of pyrosequencing, incubate at room temperature for 20 min, and measure the fluorescence value in the microplate reader under the conditions of Ex / Em = 316nm / 456nm (Cut-off 420nm).

[0156] 5. Plot a standard curve comparing the diluted commercial T7 RNA polymerase activity with the fluorescence signal, and calculate the diluted mutant enzyme activity based on the mutant signal value. The volumetric activity and specific activity of each mutant are shown below:

[0157]

[0158]

[0159]

[0160] Twenty mutants had specific enzyme activities comparable to or higher than wild-type T7 RNA polymerase, with Mut-25 having the highest specific enzyme activity, which was 1.3 times that of the wild type.

[0161] Example 3:

[0162] 1. Prepare the in vitro transcription system of T7 RNA polymerase according to the table below. Dilute wild-type T7 RNA polymerase or mutant to 250 U / μL, or add the corresponding volume according to the enzyme activity. React at 37℃ for 3 hours.

[0163] Components Volume (μL) <![CDATA[RNase free H2O]]> Up to 20 Cap Analog (100mM) 2 10×TranscriptionBuffer 2 CTP / GTP / ATP / N1-Me-pUTP(100mM each) 2each Template DNA 1-2μg T7 RNA Polymerase (250 U / μL) 1 Inorganic Pyrophosphatase(1U / μL) 0.04 Murine RNase inhibitor(40U / μL) 1

[0164] 2. After the reaction is complete, add 2 μL LDNase I (Shenji Bio 10201) to each tube and incubate at 37°C for 15 min to remove template DNA.

[0165] 3. The transcribed RNA was purified using RNACleaner magnetic beads (Shenji Bio 10511) to remove proteins and free nucleotides.

[0166] 4. The concentrations of transcripts in the wild type and each mutant were determined using Nanodrop. The RNA yields of the wild type and each mutant are shown below:

[0167]

[0168]

[0169]

[0170] Example 4:

[0171] Referring to Example 3, in vitro transcription and purification of RNA products were performed using Shenji's self-produced 1k, 2k, and 8k self-replicating templates. At the same time, the transcribed product of the 4k template in Example 3 was taken and quantitatively analyzed using Shenji Bio's dsRNA detection kit (DS0001).

[0172] Set up standard wells and sample wells. Add 100 μL of standard at different concentrations to the standard wells and add 100 μL of diluted test sample to the sample wells.

[0173] Seal the reaction wells with a sealing film, and react at room temperature for 60 minutes at 500 rpm.

[0174] Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (250uL), let stand for 30 seconds, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 4 times.

[0175] Add 100 μL of the working concentration of biotinylated detection antibody to each of the standard and sample wells, seal the reaction wells with sealing film, and react at room temperature (500 rpm) for 60 min.

[0176] Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (250uL), let stand for 30 seconds, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 4 times.

[0177] Add 100 μL of HRP-streptavidin to each well of the standard and sample, seal the reaction wells with a sealing film, and react at room temperature (500 rpm) for 30 min.

[0178] Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (250μL), let stand for 30s, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 4 times.

[0179] Add 100 μL of single-component substrate chromogenic solution to each well, seal the reaction wells with sealing film, and incubate at room temperature in the dark for 30 min. Add 50 μL of stop solution to each well and perform detection immediately. Set the wavelength of the microplate reader to 450 nm (it is recommended to use dual wavelengths 450 nm / 650 nm).

[0180] Calculate the dsRNA content in mutant and wild-type transcripts.

[0181] Example 5:

[0182] Sample pretreatment: The RNA from Example 4 was serially diluted to 10 ng / uL using dilution buffer. The diluted sample was then placed at 65–70 °C for 5–10 min for heat denaturation and immediately placed on ice.

[0183] Clip connection and calibration: Open the clip door on the top of the instrument, insert the clip, close the clip door, click "Clip Connection" to connect the clip, click "Clip Calibration", then click "Yes" to calibrate the clip. After successful calibration, click "OK".

[0184] Creation method: Click the blank cell below Method to enter the analysis method editing interface, select the appropriate Alignment Marker and Method, and then click "OK";

[0185] Sample Information Editing: Click the blank cell below “Sample Position” to bring up the sample information editing interface. You can select the position of the sample in the 96-well tray on the left. Double-click the corresponding position on the right to enter the Sample ID. Click OK when finished.

[0186] Sample testing: After setting the program number, click "Run" to start the sample separation and testing;

[0187] Data Analysis: Click "Fragment Distribution Analysis" in the upper right corner, drag the two vertical lines that define the main peak to both sides of the main peak; input the size of the fragments displayed on the right into the corresponding position on the left, check "Apply to All Selected File(S)inList" and click "Execute" to obtain the integrity value of each sample.

[0188]

[0189]

[0190] From the data above, we can see that:

[0191] In the 1K template system, the dsRNA content of mut-38, mut-153, mut-227, mut-306, mut-344, mut-528, mut-594, mut-835 and mut-948 was less than 1 / 10 of that of the wild type, with mut-594 having the lowest content.

[0192] In the 2K template system, the dsRNA content of mut-38, mut-153, mut-227, mut-306, mut-344, mut-594 and mut-835 was less than 1 / 10 of that of the wild type, with mut-594 having the lowest content.

[0193] In the 4K template system, the dsRNA content of mut-38, mut-153, mut-227, mut-306, mut-344, mut-528, mut-594 and mut-835 was less than 1 / 10 of that of the wild type, with mut-594 having the lowest content.

[0194] The dsRNA content of the 8K template systems mut-38, mut-153, mut-227, mut-306, mut-344, mut-594, and mut-835 was less than 1 / 10 of that of the wild type, with mut-594 having the lowest content. The integrity of the transcripts of all mutants was the same as that of the wild type.

[0195] Example 6:

[0196] The heat resistance of mut-38, mut-153, mut-227, mut-306, mut-344, mut-528, mut-594, mut-835 and mut-948 selected in Example 5 was tested.

[0197] Three portions of each of the wild type and the nine selected mutants were dispensed and placed at three different temperatures of 4°C, 37°C and 45°C. Samples were taken on the first, third and seventh days and then frozen at -20°C.

[0198] In vitro transcription was performed according to Example 3. After purification, the concentration of the product was determined using Nanodrop to confirm the transcription yield. The results are as follows:

[0199]

[0200] The results above show that mut-153, mut-344, mut-528, mut-594, mut-835, and mut-948 have better thermal stability than the wild type, with mut-594 having the best thermal stability and mut-528 being the second best.

[0201] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mutant of T7 RNA polymerase, characterized in that, The amino acid sequence of wild-type T7 RNA polymerase is shown in SEQ ID NO:

1. The mutant is mutated at at least one of the following sites: position 64, position 65, position 69, position 73, position 74, position 123, position 125, position 128, position 133, position 134, position 136, position 140, position 143, position 154, position 159, position 166, position 170, position 176, position 179, position 183, position 202, position 206, position 231, position 232, position 241, position 242, position 254, position 299, ... 300th, 379th, 382nd, 383rd, 386th, 387th, 389th, 390th, 391st, 397th, 437th, 439th, 467th, 473rd, 568th, 623rd, 653rd, 654th, 656th, 657th, 660th, 742nd, 750th, 756th, 757th, 758th, 759th, 760th, 761st, 772nd, or 775th.

2. The mutant according to claim 1, characterized in that, Having at least one of the following mutations: V64Q, A65N, A69G, L73A, I74G, L123T, C125S, S128T, T133S, V134A, A136G, A140G, R143K, I154T, A159G, V166S, L170S, H176E, K179D, M183T, S202N, K206Q, R231Q, Q232N, S241T, E242D, I254S, T299S, H300F R379S, A382G, A383G, R386T, K387Q, K389N, A390L, R391S, S397T, N437C, M439V, C467T, V473I, A568G, Y623F, D653N, T654V, Q656K, L657I, D660E, P742A, M750S, R756I, L757V, Q758N, P759V, T760S, I761V, H772Y or E775N.

3. The mutant according to claim 1, characterized in that, It has S128T, C467T and M750S mutations; Or it may have T133S and N437C mutations; Or it may have the I761V mutation; Or it may have a T133S mutation; Or it may have D660E and T760S mutations; Or it may have the S241T mutation; Or it may have the D660E mutation; Or may have H176E, R391S and D660E mutations; Or it may have I154T and S241T mutations; Or it may have T299S, T760S and Q758N mutations; Or it may have K387Q and A390L mutations; Or it may have A69G and P742A mutations; Or it may have V473I, A568G, M750S and Q758N mutations; Or it may have S128T, K179D, and E775N mutations; Or it may have C125S and H300F mutations; Or it may have mutations in A136G, V166S, Q232N, and A383G; Or it may have K179D, A383G and R756I mutations; Or it may have the R143K, T299S, R386T, A568G and P742A mutations; Or it may have I254S and V473I mutations; Or it may have M183T, K387Q and A568G mutations; Or it may have C125S, R143K, V166S, Y623F, and H772Y mutations; Or it may have the R143K, Q232N, E242D, S397T and P759V mutations; Or it may have K206Q, A390L and Y623F mutations; Or it may have A159G and E242D mutations; Or may have V166S, T299S, and S397T mutations; Or it may have T133S and K387Q mutations; Or it may have A140G, V166S, L170S and Y623F mutations; Or may have S128T, V134A and E775N mutations; Or it may have K179D, K206Q, E242D and I761V mutations; Or may have T133S, T760S, and I761V mutations; Or it may have A69G, A159G, R231Q, Q656K and P742A mutations; Or it may have A140G, Y623F, A65N, and V166S mutations; Or may have R231Q, Q232N, K387Q, M439V and R756I mutations; Or it may have R143K, A159G and Q232N mutations; Or it may have mutations in A159G, L170S, H176E, and V473I; Or it may have Q232N, K389N, N437C and R756I mutations; Or may have V166S, M183T, R231Q, K389N and H772Y mutations; Or it may have L73A, I254S and A383G mutations; Or it may have the P742A mutation; Or it may have A69G and Q758N mutations; Or it may have T299S, R379S and D660E mutations; Or it may have R143K, V64Q, L657I, and R756I mutations; Or it may have L73A and P742A mutations; Or may have V166S, I74G, C467T, and T654V mutations; Or it may have H176E and H300F mutations; Or it may have V64Q and S397T mutations; Or may have I154T, S202N, H300F, and D660E mutations; Or may have I154T, M183T, A383G, and Q758N mutations; Or may have I761V, I254S and H300F mutations; Or it may have A69G, I254S and D653N mutations; Or it may have R756I and T760S mutations; Or it may have the A140G mutation; Or may have V166S, S202N, and T299S mutations; Or it may have K206Q, R379S, and Q758N mutations; Or it may have R391S and A382G mutations; Or it may have M183T and H772Y mutations; Or it may have R379S and K387Q mutations; Or it may have A140G and L170S mutations; Or may have K179D, T299S, A390L, and T654V mutations; Or it may have A65N and A136G mutations; Or it may have Q232N and L757V mutations; Or it may have T133S and R391S mutations; Or may have A136G, E242D, M439V and L657I mutations; Or it may have A140G and A568G mutations; Or it may have C125S, R143K, R379S and P742A mutations; Or it may have L123T, S241T, E242D, T654V and L757V mutations; Or it may have the A159G mutation; Or it may have R379S and P742A mutations; Or it may have A69G, R391S, S397T and R756I mutations; Or it may have A65N, A140G, R379S and P759V mutations.

4. Biomaterials, including at least one of the following: I) The nucleic acid encoding the mutant according to any one of claims 1 to 3; II) An expression unit, which includes the nucleic acid and promoter described in I); III) Plasmid vector, comprising the nucleic acid described in I) or the expression unit described in II); IV) The host, which is transformed or transfected with the plasmid vector described in III); or whose genome is integrated with the nucleic acid described in I) or the expression unit described in II).

5. The biomaterial according to claim 4, characterized in that, The backbone vector of the plasmid vector is: pColdI vector; The host is Escherichia coli.

6. The method for preparing the mutant according to any one of claims 1 to 3, characterized in that, include: The host described in claim 4 is cultured to induce the expression of the mutant.

7. The use of the mutant according to any one of claims 1 to 3 in the IVT response.

8. IVT reaction reagent, comprising the mutant according to any one of claims 1 to 3 or the mutant prepared by the method of claim 7.

9. The IVT reaction reagent according to claim 8, characterized in that, Also includes: Cap Analog, CTP / GTP / ATP / N1-Me-pUTP, inorganic pyrophosphatase, RNase inhibitor, transcription buffer, and water; The enzyme activity of the mutant is 250 U / μL.

10. A method for in vitro transcription, characterized in that, Double-stranded DNA is converted into mRNA using the mutants described in any one of claims 1 to 3 or the IVT reaction reagents described in claim 8 or 9.

11. The method according to claim 10, characterized in that, The length of the double-stranded DNA is 1k to 8k; The reaction conditions for the transformation include a reaction at 35–40°C for 1–5 hours; Each 20 μL of the conversion reaction system comprises:

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