A T7 RNA polymerase mutant and its preparation and application

Abstract

The present invention relates to the technical field of nucleic acid tool enzymes, and particularly to a T7 RNA polymerase mutant and its preparation and application. The T7 RNA polymerase mutant of the present invention has RNA polymerase activity based on the T7 promoter. Compared with the wild-type T7 RNA polymerase, it has the advantages of strong thermal stability, high transcriptional enzyme activity, and high transcriptional product expression rate. It can also use modified nucleotides such as (m<supgt;1< / supgt;φTP) instead of uridine triphosphate (UTP) as a substrate through in vitro co-transcription to efficiently synthesize mRNA with high expression efficiency. Compared with the existing T7 RNA polymerase, its performance is significantly improved, better meeting the requirements of current large-scale mRNA production, and providing an effective candidate enzyme tool for the research and application of RNA. Moreover, the method for preserving the T7 RNA polymerase mutant provided by the present invention can enable the lyophilized powder of the T7 RNA polymerase mutant to retain more than 90% of its enzyme activity after being stored at 4°C for 1 year.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid tool enzymes, and in particular to a T7 RNA polymerase mutant and the preparation and application thereof. Background Art

[0002] With the rapid development of biotechnology and theory, RNA, as a biological macromolecule, has become increasingly clear about its role in organisms. RNA-related research has gradually demonstrated its important application value in scientific research, drug development, and disease treatment. siRNA and mRNA can be used as key means of RNA targeted therapy to precisely regulate gene expression and bring hope of cure to congenital genetic diseases. In addition, mRNA is being promoted and applied as a new vaccine, with the advantages of high efficiency of transient expression of antigens in the body and safety of not being retained in the body for a long time. Finally, RNA can be used as a guide RNA and is widely used in gene knockout modification and editing.

[0003] At present, the in vitro synthesis of RNA is mainly divided into chemical synthesis and enzymatic synthesis. Chemical synthesis is only applicable to short RNA of dozens of nucleotides. For long RNA, the cost of chemical synthesis rises sharply and its application is very limited. Enzymatic synthesis is not limited by length and can transcribe RNA of thousands of nucleotides. It is widely used in the synthesis and production of mRNA, miRNA and siRNA, and is the current mainstream RNA synthesis method.

[0004] T7 RNA polymerase (T7 RNA Polymerase, T7RNAP) is a 99kda (883 amino acids) single subunit protein found in bacteriophage T7. It is one of the simplest enzymes that catalyze RNA synthesis. In addition, T7 RNA polymerase has a high promoter specificity and only transcribes the double-stranded DNA sequence located downstream of the T7 promoter, allowing it to accurately transcribe and synthesize the required RNA. Therefore, it plays an increasingly important role in synthetic biology and is widely used in in vitro RNA synthesis and in vivo protein expression (bacterial high expression system). The construction and expression research of T7 RNA polymerase with high stability, high enzyme activity and high expression rate of transcription products has become one of the research hotspots.

[0005] However, the wild-type T7 RNA polymerase has the disadvantages of low enzyme activity, poor thermal stability and many byproducts during in vitro transcription (IVT), which cannot meet the requirements of actual scientific research and production. At present, the use of enzyme engineering to mutate the amino acids of T7 RNA polymerase to improve the function of mutant strains is an important means of T7 RNA polymerase product development. Patent document CN 117070493 A discloses that by replacing the 468th alanine in the amino acid sequence of wild-type T7 RNA polymerase with other phenylpropanoid acids, its thermal stability and catalytic activity are improved, but it is limited to improving the tolerance of mutant strains to heat treatment, and its own optimal reaction temperature (37°C) is not improved; Patent CN 115960860 A discloses that by replacing the 240th aspartic acid and the 762nd asparagine of the wild-type T7 RNA polymerase, the mutated T7 RNA polymerase effectively reduces the interrupted RNA products and double-stranded RNA produced during in vitro transcription while maintaining transcription activity, but the thermal stability of the mutant and the ability to use modified nucleotides are not involved. In addition, at higher reaction temperatures, the stability of RNA molecules decreases during the in vitro transcription of T7 RNA polymerase, which reduces the content of non-specific dimers and by-products in the form of dsRNA, thereby reducing the subsequent purification cost and immunogenicity of its transcription products. However, in current related studies, while improving the reaction temperature and stability of T7 RNA polymerase, it is often accompanied by a decrease in related properties such as enzyme activity and transcription product expression rate.

[0006] When mRNA reaches the cell body through a delivery vector such as nanoliposomes (LNP), its expression efficiency in the cell is closely related to the capping rate, tailing rate of the mRNA itself, and whether it contains modified nucleotides. In the translation system of eukaryotic cells, elF4E (eukaryotic initiation factor 4E) is tightly bound to the mRNA 5' cap structure, and forms an eIF4A complex with eIF4G and eIF4A, participating in the initiation of protein translation. The poly (A) tail is composed of multiple adenylic acids, which can prevent exonucleases from degrading the 3' end and increase the stability of mRNA. It can also act as a translation enhancer and increase translation efficiency. Pseudouridine (φ) is the most abundant modified nucleoside on RNA and can pair with adenosine like uridine. Katalin Karikó et al. found that introducing pseudouridine into RNA can reduce its immunogenicity, improve the stability of mRNA and enhance its translation ability. Oliwia Andries et al. found that using m 1Complete replacement of uridine with φ (pseudouridine methylated at the N1 position) can reduce the immunogenicity of mRNA more than completely replacing uridine with φ, and can enhance the protein expression ability of mRNA. At present, in the process of in vitro transcription, the poly-A tail is mainly designed on the transcription template and formed along with in vitro transcription, while pseudouridine directly replaces uridine as a substrate for transcription. Capping is divided into co-transcriptional capping and enzymatic capping. The former uses cap analogs to be incorporated into the transcription system and forms a cap structure along with the transcription process. Due to its simple operation and simple process, in vitro co-transcriptional capping has gradually become the mainstream capping method. Therefore, the requirements of in vitro transcription on T7 RNA polymerase are becoming more and more stringent. The co-transcriptional capping effect, tailing rate, ability to utilize modified nucleotides, and expression effect of transcription products have become important indicators for evaluating the performance of T7 RNA polymerase. However, the existing T7 RNA polymerase still has deficiencies in cost, thermal stability, ability to utilize modified nucleotides, and expression effect of transcription products, and cannot fully meet the needs of production and research. Summary of the invention

[0007] In order to solve the above problems, the purpose of the present invention is to provide a T7 RNA polymerase mutant and its preparation and application. The T7 RNA polymerase mutant of the present invention has RNA polymerase activity based on the T7 promoter, and has the advantages of strong thermal stability, high transcription activity and high transcription product expression rate compared with the wild-type T7 RNA polymerase; it can also be modified by using modified nucleotides such as (m 1 The invention discloses a method for preserving a mutant of T7 RNA polymerase, which can efficiently synthesize mRNA with high expression efficiency by using uridine triphosphate (UTP) instead of uridine triphosphate (UTP) as a substrate through in vitro co-transcription. Compared with the existing T7 RNA polymerase, the performance is significantly improved, which better meets the requirements of large-scale production of mRNA and provides an effective candidate enzyme tool for RNA research and application. Moreover, the method for preserving a mutant of T7 RNA polymerase provided by the invention can make the enzyme activity of the mutant of T7 RNA polymerase remain higher than 90% after being stored at 4°C for one year.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] The first object of the present invention is to provide a T7 RNA polymerase mutant, wherein the T7 RNA polymerase mutant is obtained by causing a single point mutation or a combined mutation at the following sites of the amino acid sequence shown in SEQ ID NO.1:

[0010] S43Y, A49V, T118A, K206R, E252D, V297L, T299R, A304V, K333R, D388E, R394K, N406R, W415Y, N485 S, H486N, P508L, I543L, I573V, Q583H, T596A, N601D, V710I, Q744L, Q786L, Q788S, F807I, P818A.

[0011] In one embodiment of the present invention, the T7 RNA polymerase mutant is selected from one of St1, St3, St5, St7, St8, St9, St13, St14, St15 or St16;

[0012] The amino acid sequence of the T7 RNA polymerase mutant is compared with the amino acid sequence shown in SEQ ID NO.1, and the mutations are as follows:

[0013] St1: K206R, E252D, A304V;

[0014] St3: R395S, G645K;

[0015] St5: V710I;

[0016] St7: T118A, N485S;

[0017] St8: K333R, H486N;

[0018] St9: S43Y, Q744L, K333R, H486N, P508L;

[0019] St13: S43Y, Q744L, Q786L;

[0020] St14:G788S;

[0021] St15:D388E;

[0022] St16:A638P.

[0023] In one embodiment of the present invention, the T7 RNA polymerase mutant is St9.

[0024] The second object of the present invention is to provide a gene encoding the above-mentioned T7 RNA polymerase mutant.

[0025] The third object of the present invention is to provide a plasmid containing the above gene.

[0026] The plasmid of the invention can satisfy the subsequent expression and purification of the T7 RNA polymerase mutant without affecting the performance of the T7 RNA polymerase mutant itself.

[0027] The fourth object of the present invention is to provide a transformant containing the above plasmid.

[0028] A fifth object of the present invention is to provide a method for preparing a T7 RNA polymerase mutant, comprising the following steps:

[0029] (A1) homologously recombining the nucleotide sequence encoding the T7 RNA polymerase mutant with a vector to obtain a recombinant expression vector;

[0030] (A2) transforming the recombinant expression vector prepared in step (A1) into competent Escherichia coli cells, and screening positive transformants;

[0031] (A3) Cultivation step (A2) The positive transformants obtained are screened and induced to express the T7 RNA polymerase mutant. The bacterial cells are collected and then subjected to disruption treatment and protein purification treatment in sequence to obtain the T7 RNA polymerase mutant.

[0032] In one embodiment of the present invention, in step (A1), the vector is a Petlinker plasmid;

[0033] In step (A2), the E. coli competent cells are E. coli BL21 (DE3) pLysS competent cells;

[0034] In step (A3), 1 mM IPTG is used as an inducing agent, and during the induction process, the temperature is 18 to 37° C. and the time is 6 to 18 hours.

[0035] Chelliserrykattil et al. can achieve functional screening of T7 RNA polymerase mutants through a dual plasmid screening system based on the transcriptional activity of T7 RNA polymerase for T7 promoter (Chelliserrykattil, J., Ellington, A. Evolution of a T7 RNA polymerase variant that transcribes 2′-O-methyl RNA. Nat Biotechnol 22, 1155-1160 (2004).), but this screening system has defects such as the influence of plasmid copy number differences, the instability of chloramphenicol itself, and the interference of the host's own endogenous T7 RNA polymerase. Based on the defects of this screening system, the present invention has modified the system to a certain extent, specifically:

[0036] (1) The dual-plasmid screening system was changed to a single-plasmid dual-expression frame screening system to reduce the impact of the copy number differences between the dual-plasmids between different individuals on the screening results.

[0037] (2) At the same time, the reporter gene was changed from the chloramphenicol resistance gene (CAT) to the GFP expression gene to reduce false positive interference caused by the instability of chloramphenicol and other reasons;

[0038] (3) The screening cells were changed from BL21(DE3) to DH5α, and the promoter for expressing the T7 RNA polymerase library was changed to the constitutive promoter J23100 to eliminate the interference of endogenous T7 RNA polymerase in BL21(DE3) on the background expression of the reporter gene.

[0039] Through the above transformation, the single plasmid dual expression frame screening system can screen the T7 RNA polymerase mutant library with high efficiency, high throughput and high specificity without other obvious defects. Through this system, a T7 RNA polymerase mutant with high transcription activity, high thermal stability and high expression rate of transcription products was screened. The thermal stability test showed that the T7 RNA polymerase mutant can synthesize mRNA using modified nucleotides at high reaction temperature and perform co-transcription capping, and the efficiency of the mutant in transcribed mRNA is greatly improved compared with the wild-type T7 RNA polymerase. The expression verification experiment of the transcription product in the cell showed that the mRNA transcribed and synthesized by the mutant can be translated into the corresponding protein in the cell, and the protein expression level is significantly improved.

[0040] Specifically, the dual expression frame single plasmid screening system utilizes a reporter gene downstream of a specific T7 promoter and a mutant library downstream of a constitutive promoter, which are respectively constructed into the dual expression frames of a single plasmid and transferred into host cells without endogenous T7 RNA polymerase. The expression level of the resulting cell reporter gene is proportional to the enzyme activity of the T7 RNA polymerase variant.

[0041] As a preferred method, pRSF-Duet-1 is used as the screening plasmid backbone, GFP is used as the reporter gene, and DH5α is used as the host cell without endogenous T7 RNA polymerase.

[0042] The mutant sequence obtained by the screening system was inserted into the modified pET-28a(+) vector and transferred into the prokaryotic expression cell Escherichia coli BL21(DE3)pLysS. After induced expression, in vitro transcription was performed to verify the transcription performance of the mutant. After in vitro transcription verification, it has high transcription activity at 37°C, and has a significant increase in enzyme activity compared with the wild-type enzyme. In vitro transcription verification performance was also performed at 33°C, 37°C, 41°C, 45°C, and 49°C. The results showed that under 41°C, the mutant had higher enzyme activity than under 37°C, and had a significant increase in enzyme activity compared with a commercial enzyme. In addition, in the enzyme activity half-life experiment at 50°C, the mutant strain also showed a longer half-life, showing its performance advantages of high enzyme activity and high stability.

[0043] The present invention provides a method for preparing mRNA by using the mutant strain. The method comprises using the mutant T7 RNA polymerase to take modified ribonucleoside triphosphates, cap analogs and a DNA template corresponding to a target RNA with a poly-A sequence as raw materials, and adopting an in vitro co-transcription method to efficiently synthesize an mRNA fragment that can be expressed exogenously.

[0044] A sixth object of the present invention is to provide a method for preserving a T7 RNA polymerase mutant, comprising the following steps:

[0045] (B1) concentrating the enzyme solution containing the T7 RNA polymerase mutant to obtain a concentrated enzyme solution;

[0046] (B2) mixing the concentrated enzyme solution prepared in step (B1) with lactose, mannitol, BSA and glycine to obtain a mixed solution;

[0047] (B3) pre-freezing the mixed solution prepared in step (B2) and then freeze-drying it to obtain a freeze-dried powder, which is then stored at low temperature.

[0048] In one embodiment of the present invention, in step (B1), the concentration of the T7 RNA polymerase mutant in the concentrated enzyme solution is 1 to 3 mg / mL;

[0049] In step (B2), the mixed solution contains 200 μL of concentrated enzyme solution, 1-2 (w / v)% lactose, 1-1.5 (w / v)% mannitol, 0.75-1.25 (w / v)% BSA and 0.5-0.75 (w / v)% glycine;

[0050] In step (B3), during the pre-freezing process, the temperature is -30 to -20°C and the time is 2 to 6 hours;

[0051] During the freeze-drying process, the temperature is -60 to -40°C, the pressure is 20 to 100 Pa, and the time is 12 to 16 hours;

[0052] During the low-temperature preservation process, the temperature is 0-4°C and the time is 0-12 months.

[0053] The seventh object of the present invention is to provide a use of a T7 RNA polymerase mutant in in vitro transcription synthesis of mRNA, wherein the temperature during in vitro transcription is 41°C.

[0054] In the present invention, after the above freeze-dried storage, the T7 RNA polymerase mutant still has more than 90% residual enzyme activity, which greatly improves the storage capacity of the T7 RNA polymerase mutant and reduces the storage cost.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] (1) The T7 RNA polymerase mutant provided by the present invention is expressed by the modified pET-28a(+) vector, which can be expressed in large quantities in a soluble form while preventing the influence of the purification tag on the performance of the variant enzyme to the greatest extent.

[0057] (2) The T7 RNA polymerase mutant provided by the present invention has the performance advantages of high transcription activity and high expression rate of transcription products while having high stability.

[0058] (3) The present invention provides a freeze-dried storage method for T7 RNA polymerase mutants. Compared with the traditional cryopreservation protection solution, it does not require low temperature and greatly reduces the storage cost.

[0059] (4) The T7 RNA polymerase mutant provided by the present invention has the advantages of high transcription efficiency, high purity, and good mRNA expression effect in the application of in vitro co-transcription synthesis of mRNA compared with the wild type and a commercial enzyme. It can achieve the purpose of accurate, simple, low-cost and efficient synthesis of mRNA, and has important application value in the research of technologies such as mRNA vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Schematic diagram of the T7 RNA polymerase mutant screening system;

[0061] Figure 2 This is a schematic diagram of the results of fluorescence screening of T7 RNA polymerase mutants;

[0062] Figure 3 Schematic diagram of the T7 RNA polymerase mutant expression system;

[0063] Figure 4 Schematic diagram of the transcription template PVAX-EGFP (UTR: IHNV);

[0064] Figure 5The agarose gel electrophoresis diagram of the transcription products of T7 RNA polymerase mutant, wild type and a commercial enzyme after heat treatment for different time periods; A is the commercial enzyme, B is the T7 RNA polymerase mutant; C is the wild type; lanes 1, 2, 3, 4, 5, and 6 correspond to treatment times of 0 min, 10 min, 20 min, 30 min, 40 min, and 50 min, respectively;

[0065] Figure 6 This is a graph showing the transcription yield (enzyme activity) of T7 RNA polymerase mutant, wild type and a commercial enzyme after heat treatment for different time periods;

[0066] Figure 7 The agarose gel electrophoresis diagrams of the transcription products of T7 RNA polymerase mutant, wild type and a commercial enzyme at different temperatures; A is the commercial enzyme, B is the T7 RNA polymerase mutant; C is the wild type; lanes 1, 2, 3, 4, and 5 correspond to treatment times of 33°C, 37°C, 41°C, 45°C, and 49°C, respectively;

[0067] Figure 8 This is a graph showing the transcription yield (enzyme activity) of T7 RNA polymerase mutant, wild type and a commercial enzyme at different temperatures;

[0068] Fig. 9 The transfection effect diagram of the transcription products of T7 RNA polymerase mutant, wild type and a commercial enzyme (fluorescence microscope); A is the T7 RNA polymerase mutant, B is the commercial enzyme, and C is the wild type;

[0069] Fig.10 The figure shows the quantitative results of transfection flow cytometry of the transcription products of T7 RNA polymerase mutant, wild type and a commercial enzyme; ***P<0.001. DETAILED DESCRIPTION

[0070] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0071] In the following examples, unless otherwise specified, all reagents used are commercially available reagents, and all detection means and methods used are conventional detection means and methods in the art.

[0072] For ease of presentation, the amino acid mutations are indicated where the numbers refer to the position of the amino acid residue along the reference sequence of the wild-type T7 RNA polymerase given in SEQ ID NO. 1. Amino acid identification uses the amino acid three-letter abbreviation and the single-letter alphabet, as shown in the following table:

[0073] Table 1 Comparison table of amino acid abbreviations

[0074]

[0075]

[0076] The amino acid at a specific position in the amino acid sequence is given by its three-letter abbreviation and number. For example, K333 means that the 333rd amino acid is lysine, and K333R means that the 333rd amino acid is mutated from lysine to arginine, and the same applies to other substitutions.

[0077] Example 1

[0078] This example provides the construction and screening of a single plasmid screening system for T7 RNA polymerase.

[0079] Since the double plasmid screening system based on the transcriptional activity of T7 promoter by T7 RNA polymerase has various defects, it was modified to achieve efficient, high-throughput and high-specificity screening of T7 RNA polymerase mutant strain library. First, the double plasmid screening system was changed to single plasmid double expression frame screening to reduce the impact of the copy number difference of double plasmids between different individuals on the screening results; at the same time, the reporter gene was changed from chloramphenicol resistance gene (CAT) to GFP expression gene to reduce false positive interference caused by the instability of chloramphenicol and other reasons; finally, the screening cells were changed from BL21 (DE3) to DH5α, and the promoter expressing T7 RNA polymerase library was changed to constitutive promoter J23100 to remove the interference of endogenous T7 RNA polymerase in BL21 (DE3). The GFP reporter gene expressed by the T7 promoter and the T7 RNA polymerase library expressed by the J23100 promoter were constructed into the dual expression frame of the pRSF-Duet-1 plasmid, and the original redundant T7 promoter was removed. The plasmid was named T7 RNA polymerase screening plasmid. Its structure is as follows: Figure 1 When the plasmid is transformed into competent cells DH5α, the expression of GFP in the resulting colonies is proportional to the activity of T7 RNA polymerase. The gene coding sequence of the T7 RNA polymerase with the best enzyme activity can be confirmed by sequencing the colony individuals with the highest GFP expression.

[0080] The specific implementation steps are as follows:

[0081] Material:

[0082] The screening vector pRSF-Duet-1 was purchased commercially; PFU high-fidelity enzyme was purchased from Vazyme, and homologous recombination enzyme was purchased from abclonal; other materials such as competent cells DH5α, plasmid mini-extraction kit, random mutagenesis kit, IPTG, kanamycin, LB medium components (yeast extract, tryptone, sodium chloride) and agarose powder were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0083] Construction of wild-type T7 RNA polymerase expression vector, mutant library and screening plasmid

[0084] The wild-type T7 RNA polymerase sequence (amino acid sequence shown in SEQ ID NO. 1) was derived from Escherichia coli BL21 (DE3), and its sequence fragment was obtained by genomic PCR;

[0085] Among them, the specific sequence of SEQ ID NO.1 is as follows:

[0086] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFE

[0087] RQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAV

[0088] AYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKR

[0089] VGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSL

[0090] HRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYW

[0091] ANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITK

[0092] WKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFM

[0093] LEQANKFANKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGY

[0094] YWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCF

[0095] EYAGVQHHGLSYNCSLPLAFDGCSSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIV

[0096] AKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTK

[0097] RSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTV

[0098] VAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQT

[0099] RLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYG

[0100] IESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMP

[0101] ALPAKGNLNLRDILESDFAFA

[0102] The PCR primers are:

[0103] WT-T7-F (SEQ ID NO.2): 5'-ATGAACACGATTAACATCGCTAAGAACG-3'

[0104] WT-T7-R (SEQ ID NO.3): 5'-GGACTTCGCGTTCCGG-3'

[0105] While the pRSF-Duet-1 plasmid and GFP gene sequence were linearized by PCR (the primer pairs were pRSF-F and pRSF-R, GFP-F and GFP-R, respectively), homologous sequences were added as homology arms, and the GFP gene (SEQ ID NO.4) was constructed into the MCS-1 expression frame of pRSF-Duet-1 by homologous recombination using the homology arms. After the transformation and sequencing were verified to be correct, the plasmid was extracted and the next step of construction was carried out;

[0106] The plasmid obtained in the previous step was further constructed using primer pairs T7-J2300-F and T7-J2300-R. The T7 promoter of the MCS-2 expression frame on the plasmid was mutated to the J23100 promoter by circular PCR. The plasmid was named pRSF-GFP-J23100.

[0107] The nucleotide sequence of GFP (SEQ ID NO.4) is as follows:

[0108] 5'-ATGGGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAAT -3'

[0109] The primer sequences are:

[0110] pRSF-F (SEQ ID NO.5): 5'-TGAACTATACAAATAACGCCGGTGATGCCGGC CACG-3'

[0111] pRSF-R (SEQ ID NO.6): 5'-CCCCTGTAGAAATAATTTTGTTTAACTTTAATAAGGAGATATACC-3'

[0112] GFP-F (SEQ ID NO.7): 5'-CTTTAATAAGGAGATATACCATGGGCAAAGGAGAAGAACTTTTCAC-3'

[0113] GFP-R (SEQ ID NO.8): 5'-GGATTACACATGGCATGGATGAACTATACAA ATAACGCC-3'

[0114] T7-J2300-F (SEQ ID NO.9): 5'-GTCCTAGGTACAGTGCTAGCGAGCTCAGGAGGACAGCTCCATGGGCAGCAGCCATCA-3'

[0115] T7-J23100-R (SEQ ID NO.10): 5'-CTCGCTAGCACTGTACCTAGGACTGAGCTAGCCGTCAAATCCTCTACGCCGGACGCA-3'

[0116] The wild-type T7 RNA polymerase fragment was randomly mutated, and the homologous arms of MCS-2 of pRSF-Duet-1 were added to both ends of the fragment using primer pairs Smut7-F (SEQ ID NO.11) and Smut7-R (SEQ ID NO.12) (i.e., a mutant library named Smut7). At the same time, the modified pRSF-GFP-J23100 plasmid was linearized using primer pairs PGJ-F (SEQ ID NO.13) and PGJ-R (SEQ ID NO.14), and homologous recombination was performed using the homologous sequences at the ends of the two linear fragments to obtain the pRSF-GFP-J23100-Smut7 screening plasmid (SEQ ID NO.15).

[0117] The primers are:

[0118] Smut7-F (SEQ ID NO.11): 5'-GAGGACAGCTCCATGAACACGATTAACATC GCTAAGAACG-3'

[0119] Smut7-R (SEQ ID NO.12): 5'-GGACTTCGCGTTCGCGTAATTA-3'

[0120] PGJ-F (SEQ ID NO.13): 5'-ACTTCGCGTTCGCGTAATTAACCTAGGCTGCT GCCAC-3'

[0121] PGJ-R (SEQ ID NO.14): 5'-GAGCTCAGGAGGACAGCTCCATGAACAC-3'

[0122] The nucleotide sequence of the pRSF-GFP-J23100-Smut7 screening plasmid (SEQ ID NO.15) is as follows:

[0123]

[0124] Initial screening of T7 RNA polymerase mutants:

[0125] The pRSF-GFP-J23100-Smut7 screening plasmid was transformed into DH5α competent cells, and the transformed E. coli was cultured in LB medium without antibiotics at 37°C and 220rpm for 1 hour, and then 50μg / mL kanamycin was added and cultured at 37°C and 220rpm for 12h. The bacteria were then spread on LB agar plates containing 50μg / mL and 0.1mM IPTG kanamycin and cultured overnight at 37°C. GFP-expressing colonies were picked under blue light, cultured at 37°C and 220rpm for 18h, and detected by an ELISA instrument. The one with the highest GFP fluorescence value was sequenced. After the above three rounds of screening, the mutants and their fluorescence were obtained as shown in Figure 2. Figure 2 shown.

[0126] The amino acid sequence of the T7 RNA polymerase mutant is compared with the amino acid sequence shown in SEQ ID NO.1, and the mutations are as follows:

[0127] St1: K206R, E252D, A304V;

[0128] St2: Q583H, T596A, N601D;

[0129] St3: R395S, G645K;

[0130] St4: A49V, I573V;

[0131] St5: V710I;

[0132] St6:I573V;

[0133] St7: T118A, N485S;

[0134] St8: K333R, H486N;

[0135] St9: S43Y, Q744L, K333R, H486N, P508L;

[0136] St10:W415Y;

[0137] St11:T299R;

[0138] St12:I543L;

[0139] St13: S43Y, Q744L, Q786L;

[0140] St14:G788S;

[0141] St15:D388E;

[0142] St16:A638P.

[0143] Example 2

[0144] This example provides protein expression and purification of a T7 RNA polymerase mutant (St9).

[0145] Since the screening system is constructed for enzyme activity, the T7 RNA polymerase mutant obtained through the preliminary screening in Example 1 above is considered to have the potential for transcription with high enzyme activity.

[0146] First, the expression plasmid pET-28a(+) was modified by PCR to remove the original thrombin site sequence and T7 tag sequence and replace it with 8×His tag+(G 4 S) 2 Linker sequence (Petlinker: its nucleotide sequence is shown in SEQID NO.16); the obtained recombinant plasmid is named Petlinker plasmid, in order to facilitate subsequent protein purification and prevent the influence of His tag on enzyme performance. The specific steps are to use primers Petlinker-F and Petlinker-R to perform PCR on plasmid pET-28a(+), insert the His tag and Linker sequence into the plasmid, and remove the thrombin site and T7 tag sequence. The obtained long linear fragment undergoes homologous recombination through the homologous sequence at the end to obtain Petlinker plasmid, and its structure is shown in Figure 3 shown.

[0147] Petlinker Sequence:

[0148] SEQ ID NO.16: 5'-TAATACGACTCACTATAGGGGAATTGTGAGCGGATA

[0149] ACAATTCCCTCTAGAAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGG

[0150] GCAGCAGCCATCATCATCATCATCACCACCACGGCGGAGGTGGCTCTGGCGGTGGC

[0151] GGATCGGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACC

[0152] ACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTT

[0153] GGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG-3'

[0154] Primer sequences:

[0155] Petlinker-F (SEQ ID NO.17): 5'-GAGGTGGCTCTGGCGGTGGCGGATCGGGATCCATGAACACGATTAACA-3'

[0156] Petlinker-R (SEQ ID NO.18): 5'-CACCGCCAGAGCCACCTCCGCCGTGGTGGTGATGATGATGATGATGGCT-3'

[0157] The screened T7 RNA polymerase sequence was homologously recombined with the modified Petlinker plasmid to its expression cassette, and transformed into Escherichia coli BL21 (DE3) pLysS for expansion culture, and the cells were collected after induction. Due to the 8×His tag carried on the Petlinker plasmid, the T7 RNA polymerase mutant protein was purified based on the affinity between the His tag and the Ni-NTA Agarose purification medium. The specific steps are as follows: the cells were collected by centrifugation (9000rpm, 10min), the cells were resuspended in lysis buffer (20mM Tris, pH 8.0, 100mM NaCl), and then ultrasonically broken, and the supernatant was collected by centrifugation (12000rpm, 15 minutes). The supernatant was purified using a Cytiva Ni-NTA Agarose affinity chromatography column. The column was first equilibrated with equilibration buffer (20 mM Tris, pH 8.0, 100 mM NaCl), and then after loading, non-specifically bound impurities were washed away with washing buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 100 mM imidazole), and the target protein was eluted with elution buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 300 mM imidazole).

[0158] Example 3

[0159] This example provides a validation of the stability of a T7 RNA polymerase mutant and compares it with a wild-type T7 RNA polymerase and a commercial enzyme.

[0160] The transcription template PVAX-EGFP (UTR: IHNV) was synthesized by Nanjing GenScript Co., Ltd., and its transcription framework sequence is shown in SEQ ID NO. 19, which contains the UTR sequence derived from the IHNV virus and a 107 bp poly-A tail structure. The transcription template plasmid was transformed into Stbl3 competent cells and then expanded and cultured, and then extracted using a plasmid extraction kit. The resulting plasmid was linearized by XbaI restriction endonuclease and used as a transcription template. Its structure is shown in Figure 4 shown.

[0161] Among them, the sequence of SEQ ID NO.19 is as follows (5'-3'):

[0162]

[0163] The transcription system is shown in the following table, where the transcription buffer composition is: 40mM Tris-HCl, pH=7.9, 6mM MgCl 2 , 1mM DTT, 2mM spermidine. The initial concentration of each nucleotide monomer was 100mM, and inorganic pyrophosphatase and RNaseInhibitor were purchased from Nanjing Novizan Company.

[0164] Table 2 In vitro transcription (IVT) system (20 μL)

[0165]

[0166] The wild type, mutant strain and commercially available enzyme were incubated at 50°C for 0 min, 10 min, 20 min, 30 min, 40 min, 50 min and 60 min respectively using the above transcription system. The PVAX-EGFP template was transcribed at 37°C for 2 h 30 min. The obtained transcription product was digested with DNaseI (Vazyme) and then purified by magnetic beads (novoprotein). The purified product was specifically detected for single-stranded RNA content by Equal bit RNA BR Assay Kit (Vazyme). The results are shown in Figure 5 , Figure 6 As shown, under the same transcription system and the same amount of DNA template, the half-life of the T7 RNA polymerase mutant at 50°C is higher than that of the commercial enzyme and the wild-type enzyme, indicating that its thermal stability is greatly improved compared with the wild-type and a commercially available enzyme.

[0167] The wild type, mutant and commercially available enzyme were respectively cultured at 33°C, 37°C, 41°C, 45°C and 49°C using the above transcription system.

[0168] The PVAX-EGFP template was transcribed for 2 h 30 min, and the transcription product was digested with enzymes and purified by magnetic beads for RNA quantification. The results are shown in Figure 7 , Figure 8 As shown in the figure, the optimal transcription temperature of the T7 RNA polymerase mutant is higher than that of the wild-type enzyme and a commercial enzyme at 37°C, reaching about 41°C. At 41°C, the transcription activity of the T7 RNA polymerase mutant is significantly higher than that of the wild-type T7 RNA polymerase and a commercial enzyme, further indicating that the stability of the T7 RNA polymerase mutant is greatly improved while maintaining high transcription activity.

[0169] Example 4

[0170] This example provides a T7 RNA polymerase mutant that generates mRNA through in vitro co-transcription, which is verified by transfection expression in HEK-293T and compared with a wild-type T7 RNA polymerase and a commercial enzyme.

[0171] The PVAX-EGFP (UTR: IHNV) in Example 3 was used as the transcription template. The transcription template was co-transcribed in vitro using the transcription system in Example 3 using a T7 RNA polymerase mutant, a wild-type T7 RNA polymerase, and a commercial enzyme. After the template was digested with DNaseI, it was purified by magnetic beads to obtain pure mRNA.

[0172] HEK-293T cells were transfected with the above mRNA at the same concentration, and the transfection reagent was Lipofectamine 3000 (ThermoFisher). Cells were collected 24 hours after transfection and quantitatively detected by flow cytometry. Fig. 9 , Fig.10 As shown, the translation expression positive rate of mRNA transcribed by the T7 RNA polymerase mutant is higher than that of the commercial enzyme and the wild-type enzyme, and the average EGFP fluorescence intensity of the cells is significantly higher than that of a commercial enzyme and the wild-type enzyme, indicating that the mRNA transcription product of the mutant strain has a significantly improved expression rate.

[0173] Example 5

[0174] This example provides freeze-dried storage and residual enzyme activity detection of T7 RNA polymerase mutants.

[0175] Since most T7 RNA polymerases on the market are stored in the form of liquid cryopreservation tubes, the storage cost is relatively high. The present invention provides a freeze-dried storage method for the T7 RNA polymerase mutant, and the specific implementation scheme is as follows:

[0176] The pure enzyme solution of the T7 RNA polymerase mutant obtained in Example 2 was subjected to gradient dialysis to remove NaCl and imidazole in the buffer system, so as to reduce the influence of salt ions on the protein freeze-drying process.

[0177] The dialyzed enzyme solution was ultrafiltered and concentrated (4000rpm, 4°C), and a millipore ultrafiltration tube was used. When the ultrafiltration concentration reached a protein concentration of about 2mg / mL, the solution was packaged in 200uL tubes, and 1.5% lactose, 1.2% mannitol, 1% BSA and 0.6% glycine (W / V, final concentration) were added, and the solution was pre-frozen at -20°C, and then lyophilized at -40°C, 40Pa for 12h after 2h to obtain enzyme lyophilized powder;

[0178] The lyophilized enzyme powder was stored at 4°C, and the enzyme lyophilized powder was reconstituted with nuclease-free water to 200uL after 0, 3, 6, and 12 months, and the difference in enzyme activity before and after lyophilization was verified using the transcription system in Example 3. The results are shown in Table 1. After lyophilization, the residual enzyme activity of the T7 RNA polymerase mutant reached 94.77%, and after 1 year of storage, the residual enzyme activity could still reach more than 90%, which met the actual use requirements and greatly reduced the storage requirements and costs.

[0179] Table 1 Changes in enzyme activity of T7 RNA polymerase mutants after freeze-drying

[0180]

[0181] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the explanation of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A T7 RNA polymerase mutant, characterized in that: The T7 RNA polymerase mutant is St9, Among them, the amino acid sequence of St9 has the following mutations compared with the amino acid sequence shown in SEQ ID NO.1: S43Y, Q744L, K333R, H486N, P508L.

2. A gene, characterized in that Encoding the T7 RNA polymerase mutant according to claim 1.

3. A plasmid, characterized in that Containing the gene according to claim 2.

4. A transformant, characterized in that Containing the plasmid according to claim 3.

5. A method for preparing a T7 RNA polymerase mutant, characterized in that: The following steps are involved: (A1) homologously recombining a nucleotide sequence encoding the T7 RNA polymerase mutant according to claim 1 with a vector to obtain a recombinant expression vector; (A2) transforming the recombinant expression vector prepared in step (A1) into competent Escherichia coli cells, and screening positive transformants; (A3) Cultivation step (A2) The positive transformants obtained are screened and induced to express the T7 RNA polymerase mutant. The bacterial cells are collected and then subjected to disruption treatment and protein purification treatment in sequence to obtain the T7 RNA polymerase mutant.

6. The method for preparing a T7 RNA polymerase mutant according to claim 5, characterized in that: In step (A2), the E. coli competent cells are E. coli BL21 (DE3) pLysS competent cells; In step (A3), 1 mM IPTG is used as an inducing agent, and during the induction process, the temperature is 18 to 37° C. and the time is 6 to 18 hours.

7. A method for preserving the T7 RNA polymerase mutant according to claim 1, characterized in that: The following steps are involved: (B1) concentrating the enzyme solution containing the T7 RNA polymerase mutant to obtain a concentrated enzyme solution; (B2) mixing the concentrated enzyme solution prepared in step (B1) with lactose, mannitol, BSA and glycine to obtain a mixed solution; (B3) pre-freezing the mixed solution prepared in step (B2) and then freeze-drying it to obtain a freeze-dried powder, and storing it at a low temperature; During the low-temperature storage process, the temperature is 0 to 4°C.

8. The method for preserving a T7 RNA polymerase mutant according to claim 7, characterized in that: In step (B1), the concentration of the T7 RNA polymerase mutant in the concentrated enzyme solution is 1 to 3 mg / mL; In step (B2), the mixed solution contains 200 μL of concentrated enzyme solution, 1-2 (w / v)% lactose, 1-1.5 (w / v)% mannitol, 0.75-1.25 (w / v)% BSA and 0.5-0.75 (w / v)% glycine; In step (B3), during the pre-freezing process, the temperature is -30 to -20°C and the time is 2 to 6 hours; During the freeze-drying process, the temperature is -60 to -40°C, the pressure is 20 to 100 Pa, and the time is 12 to 16 hours; During the low-temperature storage process, the time is 0 to 12 months.

9. Use of the T7 RNA polymerase mutant according to claim 1 in in vitro transcription synthesis of mRNA, characterized in that: During in vitro transcription, the temperature was 41 °C.

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