T7 RNA polymerase mutant and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a T7 RNA polymerase mutant and application thereof. The amino acid sequence of the T7 RNA polymerase mutant is as shown in SEQ ID No. 1. The invention provides the T7 RNA polymerase mutant which is high in transcriptional activity and can tolerate a high-salt environment, the enzyme activity and the yield of an mRNA transcriptional product are remarkably improved, the integrity of the transcriptional product is relatively high, the T7 RNA polymerase mutant can be used for in-vitro transcription and large-scale synthesis of high-quality mRNA, the technical problems of high cost, tedious steps and low yield of existing mRNA preparation are favorably solved, and the T7 RNA polymerase mutant has a good application prospect. And the method is more suitable for large-scale industrial production. The T7 RNA polymerase mutant disclosed by the invention has important application values in the aspects of RNA in-vitro transcription, cell-free protein synthesis, pathogen nucleic acid detection, mRNA vaccine and drug research and development and preparation and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a T7 RNA polymerase mutant and its application. Background Art

[0002] In recent years, with the rapid development of biotechnology, in vitro transcription technology of RNA has been widely used in the fields of nucleic acid vaccines, drug development, gene therapy and detection, synthetic biology, etc. The core raw material of in vitro transcription technology of RNA is RNA polymerase. T7 RNA polymerase is a DNA-dependent RNA polymerase derived from bacteriophage T7, which has high synthesis ability, specific recognition of promoters and a simple single-subunit structure, thus becoming the most common choice in in vitro transcription. However, in the face of the growing demand for in vitro transcription technology of RNA, the existing T7 RNA polymerase has limitations in terms of production cost, stability and transcription activity, and the improvement of the in vitro transcription process is not obvious. The fundamental way to improve the efficiency and quality of in vitro transcription to synthesize mRNA lies in the activity modification of T7 RNA polymerase. Therefore, it is particularly important to engineer T7 RNA polymerase.

[0003] Currently, the production cost of T7 RNA polymerase is relatively high, which limits its application in large-scale industrial production. By optimizing the expression system and purification process, the production cost can be significantly reduced, making it more suitable for large-scale production requirements. Secondly, the stability of this enzyme is limited. Especially under long-term reactions or extreme conditions (such as high temperature or high salt environment), its activity is prone to decline, thus affecting the synthesis efficiency and quality of mRNA. By means of protein engineering to enhance its structural stability and environmental adaptability, its performance under complex conditions can be significantly improved. In addition, there is still room for further improvement in the transcription activity of T7 RNA polymerase. Especially when synthesizing ultra-long mRNA or mRNA with complex secondary structures, its efficiency may be insufficient. By directed evolution or rational design to optimize its catalytic efficiency and substrate specificity, the yield and quality of mRNA can be effectively improved. At the same time, with the increasingly wide application of mRNA technology in personalized medicine and precision therapy, higher requirements are put forward for the functional diversity of T7 RNA polymerase. For example, developing a T7 RNA polymerase that can recognize unnatural nucleotides or promoter variants will greatly expand its application potential in synthetic biology and new mRNA therapies.

[0004] In summary, by engineering T7 RNA polymerase, higher-quality mRNA molecules can be obtained, while reducing the production cost and improving the synthesis efficiency. This is not only a direct, economical and effective way to achieve the preparation of high-quality mRNA molecules, but also a key driving force for the further development of mRNA technology. Summary of the Invention

[0005] To solve the defects that the current T7 RNA polymerase has low transcriptional activity and cannot tolerate high salt, based on the wild-type T7 RNA polymerase, the present invention provides a T7 RNA polymerase mutant through a large number of single-point mutations and screening and verification, and its amino acid sequence is shown in SEQ ID No.1.

[0006] This T7 RNA polymerase mutant is obtained by replacing the 772nd amino acid residue from histidine (His) to arginine (Arg) based on the wild-type T7 RNA polymerase (shown in SEQ ID No.2). This T7 RNA polymerase mutant has high transcriptional activity, and the yield and purity of the transcription product are higher than those of the wild-type T7 RNA polymerase, and it has the characteristic of tolerating high salt (NaCl and MgCl 2 ) and under the conditions of 120 mM NaCl and 100 mM MgCl 2 , the wild-type T7 RNA polymerase has almost no transcriptional activity, while this T7 RNA polymerase mutant still maintains high transcriptional activity.

[0007] Furthermore, the present invention provides a nucleic acid sequence encoding the T7 RNA polymerase mutant.

[0008] Furthermore, the present invention provides a biological material containing the T7 RNA polymerase mutant or the nucleic acid sequence.

[0009] In the specific implementation process, the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or cell.

[0010] Furthermore, the present invention provides a reagent or kit containing the T7 RNA polymerase mutant, the nucleic acid sequence or the biological material.

[0011] In the specific implementation process, the reagent or kit is used for in vitro transcription of RNA.

[0012] In the specific implementation process, the reagent or kit is used for cell-free protein synthesis.

[0013] In the specific implementation process, the reagent or kit is used for pathogen nucleic acid detection.

[0014] Furthermore, the present invention provides the application of the T7 RNA polymerase mutant, the nucleic acid sequence, and the biological material in the preparation of reagents or kits.

[0015] Furthermore, the present invention provides the application of the T7 RNA polymerase mutant, the nucleic acid sequence, and the biological material in at least one of the following aspects: (1) In vitro transcription of RNA; (2) Cell-free protein synthesis; (3) Detection of pathogen nucleic acids (such as detection of pathogen RNA); (4) Development of mRNA vaccines; (5) Preparation of drugs.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a mutant T7 RNA polymerase with high transcriptional activity and the ability to tolerate high-salt environments, which significantly improves enzyme activity and the yield of mRNA transcription products, and the integrity of the transcription products is relatively high. It can be used for in vitro transcription to synthesize high-quality mRNA in large quantities, which is beneficial to solving the technical problems of high cost, cumbersome steps and low yield in the existing preparation of mRNA, and makes it more suitable for large-scale industrial production. The mutant T7 RNA polymerase of the present invention has important application values in aspects such as in vitro transcription of RNA, cell-free protein synthesis, detection of pathogen nucleic acids, development and preparation of mRNA vaccines and drugs. Description of the Drawings

[0017] Figure 1 It is a structural diagram of the mutation sites of 20 mutant T7 RNA polymerases.

[0018] Figure 2 It is a statistical chart of the results of the concentration and integrity of the in vitro transcribed EGFP-mRNA products of 20 mutant T7 RNA polymerases.

[0019] Figure 3 It is the expression result of the in vitro transcribed and synthesized EGFP-mRNA transfected into 293T cells by 20 mutant T7 RNA polymerases and wild-type T7 RNA polymerase; among them, NC is the negative control and PC is the positive control.

[0020] Figure 4 It is a protein purification result diagram of three mutant T7 RNA polymerases and wild-type T7 RNA polymerase.

[0021] Figure 5 It is a test result diagram of the in vitro transcription efficiency of three mutant T7 RNA polymerases and wild-type T7 RNA polymerase; among them, Figure 5 A is a comparative statistical chart of the concentration and integrity of the transcribed EGFP-mRNA products; Figure 5 B is the expression result of the transcribed EGFP-mRNA products transfected into 293T cells.

[0022] Figure 6 It is a test result diagram of the transcription efficiency of the mutant T7 RNA polymerase H772R under high-salt environmental conditions; among them,Figure 6 A is the concentration of the EGFP-mRNA product synthesized by in vitro transcription under different concentrations of MgCl 2 and the analysis result diagram of agarose gel electrophoresis; Figure 6 B is the concentration of the EGFP-mRNA product synthesized by in vitro transcription under different concentrations of NaCl and the analysis result diagram of agarose gel electrophoresis; Figure 6 C is the comparison diagram of the integrity of the in vitro transcription products of wild-type and T7 RNA polymerase mutants under the concentration of 100 mM MgCl 2 ; Figure 6 D is the comparison diagram of the integrity of the in vitro transcription products of wild-type and T7 RNA polymerase mutants under the concentration of 120 mM NaCl.

[0023] Figure 7 is the test result diagram of the T7 RNA polymerase mutant H772R transcribing the mRNA of different genes; among them, Figure 7 A is G6PT the test result diagram of the concentration and integrity of the mRNA transcription product of the gene; Figure 7 B is CTNNB1 the test result diagram of the concentration and integrity of the mRNA transcription product of the gene.

[0024] Figure 8 is the test result diagram of the T7 RNA polymerase mutant H772R transcribing crRNA; among them, Figure 8 A is the test result diagram of the concentration and integrity of the transcribed crRNA1 product; Figure 8 B is the test result diagram of the concentration and integrity of the transcribed crRNA2 product; Figure 8 C is the test result diagram of the concentration and integrity of the transcribed crRNA3 product. Detailed implementation manners

[0025] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention. In the embodiments provided in this specification, those not specifying specific techniques or conditions shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. Those reagents or instruments not indicating the manufacturer can be obtained as conventional products through regular channels.

[0026] The present invention relates to molecular biology experiments. Unless otherwise specified, reference can be made to the book "Molecular Cloning" (written by J. Sambrook, E.F. Fritsch, and T. Maniatis, published by Science Press in 1994). This book and its subsequent published versions are the most commonly used and guiding reference books for those skilled in the art when conducting experiments related to molecular biology. In addition, depending on different experimental purposes, those skilled in the art can complete corresponding experiments under the guidance of the operation manuals attached to various commercial kits or entrust specialized companies to perform them, such as gene sequencing, plasmid sequencing, and determining molecular weight, etc.

[0027] The reagents used in the following examples are as follows: 1. Non-denaturing lysis buffer: 50 mM NaH 2 PO 4 , 300 mM NaCl (pH 8.0).

[0028] 2. The components of imidazole solutions with different concentrations are as follows: 5 mM imidazole solution: 50 mM NaH 2 PO 4 , 300 mM NaCl, 5 mM imidazole (pH 8.0); 10 mM imidazole solution: 50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole (pH 8.0); 25 mM imidazole solution: 50 mM NaH 2 PO 4 , 300 mM NaCl, 25 mM imidazole (pH 8.0); 50 mM imidazole solution: 50 mM NaH 2 PO 4 , 300 mM NaCl, 50 mM imidazole (pH 8.0); 100 mM imidazole solution: 50 mM NaH 2 PO 4 , 300 mM NaCl, 100 mM imidazole (pH 8.0); 500 mM imidazole solution: 50 mM NaH 2 PO 4 , 300 mM NaCl, 500 mM imidazole (pH 8.0).

[0029] Dialysis buffer: 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM DTT, 0.1% (w / v) Triton X-100.

[0030] Example 1 Design, Preparation and Screening of T7 RNA Polymerase Mutants 1. Design and Preparation of T7 RNA Polymerase Mutants Based on the wild-type T7 RNA polymerase sequence (shown in SEQ ID No.2), site-directed mutations were carried out at single amino acid residue sites respectively to obtain 20 mutation sites as shown in Figure 1 . The specific steps are as follows: According to the nucleotide sequence encoding the wild-type T7 RNA polymerase, corresponding primers were synthesized for the above 20 T7 RNA polymerase mutation sites, as shown in Table 1 and Table 2. According to Table 3, the first round of PCR was carried out. After the first round of PCR, the second round of PCR was carried out according to Table 4. After the two rounds of PCR, the ligation system was prepared according to Table 5, and the PCR amplification products were assembled into the XhoI-BamHI site of the pMB1 / pBR322 vector (Miaoling Biology) by recombinant ligation method respectively. The ligation products were spread on the AMP-resistant plate for resistance screening and gene sequencing to obtain the expression plasmids. The nucleotide sequence of the wild-type T7 RNA polymerase expression plasmid is shown in SEQ ID No.3.

[0031] Table 1 Site-directed Mutation Primers for Constructing T7 RNA Polymerase Mutants

[0032] Table 2 Nucleotide Sequences of Site-directed Mutation Primers

[0033] Table 3 Reaction System for the First Round of PCR

[0034] Table 4 Reaction System for the Second Round of PCR

[0035] Table 5 Ligation Reaction System of the Target Fragment and the Vector

[0036] The 20 mutant T7 RNA polymerase plasmids with correct sequencing were transformed into the E.coli BL21 expression strain. After plate culture, single colonies were picked and inoculated into the LB medium containing 100 μg / mL ampicillin. The culture was carried out at 37°C and 220 rpm. When the OD600 value reached 0.6 - 0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) was added with a final concentration of 0.5 mM, and the expression of the target protein was induced at 37°C and 220 rpm for 10 h.

[0037] Subsequently, the bacterial cell pellet was collected by centrifugation at 4°C and 7,800 rpm for 5 min. Then, the bacteria (calculated by adding 4 mL of non-denaturing lysis buffer per 200 mL of bacterial solution) were resuspended in non-denaturing lysis buffer (pH 8.0) containing 1 mg / mL lysozyme and 1 mM protease inhibitor, and lysed on ice for 30 min followed by sonication (sonication for 6 s, pause for 6 s, total sonication for 2 min). After sonication, the protein lysate was centrifuged at 4°C and 7,800 rpm for 30 min. Subsequently, the separated supernatant was filtered through a 0.22 µm pore size filter membrane to remove impurities. The nickel column was pre-equilibrated with twice the volume of non-denaturing lysis buffer, and this was repeated 2 - 3 times. The filtered supernatant was added to the nickel column, and binding was carried out for 1.5 h using a vertical rotator mixer at 4°C. After 1.5 h, it was removed from the vertical rotator mixer at 4°C. After all the protein solution had passed through the nickel column, the non-denaturing lysis buffer was used to wash 20 column volumes, followed by washing with imidazole solutions of different concentration gradients (10 mM - 25 mM - 5 mM) (8 column volumes each) to remove impurities. Finally, elution was carried out using imidazole solutions of different concentration gradients (50 mM - 100 mM - 500 mM) (8 column volumes each), and the eluate was collected and labeled. 20 µL of each eluate was taken for high-temperature denaturation, followed by SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining analysis. The eluate fractions with higher purity were selected and mixed for dialysis. If there was a large amount of the solution to be dialyzed, ultrafiltration concentration could be carried out first. During dialysis, the dialysis buffer was changed every 2 h. After three rounds of dialysis, the protein was collected and quantified using the BCA method. Glycerol (final concentration of 25%) was added to the collected protein solution, and it was stored at -80°C. All the above operations were carried out on ice or at 4°C.

[0038] 2. Screening of T7 RNA polymerase mutants (1) Linearization and purification of the transcription template Prepare the template linearization reaction system according to Table 6. After mixing, react at 50°C for 1 h and verify by agarose gel electrophoresis.

[0039] Table 6 EGFP-mRNA template linearization system

[0040] Preparation of 1% agarose gel: Weigh 0.3 g of agarose and put it into a conical flask. Then add 30 mL of TAE buffer solution and heat it in a microwave oven for 1 min. Take it out and cool it to 40 - 50 °C. Add 1.5 μL of 10000x gel nucleic acid dye, mix well and pour it into the prepared mold. Cool it at room temperature for 20 min and use it after solidification. Take out 1 μL of the enzyme digestion product and add it to a 0.2 mL PCR tube. Add 4 μL of enzyme-free water and 1 μL of 5×DNA / RNA loading Buffer. After centrifugation and mixing, load the sample and perform electrophoresis at 125 V for 30 min, and then carry out gel imaging analysis. Add an equal volume of saturated phenol to the enzyme digestion product, vortex and mix well, centrifuge at 1330 rpm for 5 min, and take the supernatant into a new 1.5 mL centrifuge tube. Add 100 μL of enzyme-free water to the original centrifuge tube, centrifuge at 1330 rpm for 5 min, and take the supernatant and mix it with the supernatant of the previous step. Add an equal volume of CH 3 Cl for extraction, vortex and mix well, centrifuge at 1330 rpm for 5 min, and take the supernatant into a new 1.5 mL centrifuge tube. Add 1 / 10 volume of sodium acetate (pH 5.2), add glycogen at 5 mg / mL to make its final concentration 0.05 mg / mL. After mixing, add 1000 μL of anhydrous ethanol pre-cooled at 4 °C (2.5 times the minimum volume), and place it at -20 °C overnight. Centrifuge at 1330 rpm for 15 min, gently remove the supernatant, slowly add 1 mL of 70% ethanol along the tube wall, centrifuge at 1330 rpm for 15 min, and completely remove the supernatant. Add 10 μL of enzyme-free water for dissolution, dilute 1 μL of the sample by 10 times, and use a ultra-micro spectrophotometer to measure the concentration.

[0041] (2) Comparison of the transcription efficiency of different T7 RNA polymerase mutants Prepare the transcription reaction system according to Table 7. After mixing, incubate it in a PCR instrument at 37 °C for 2 h. Add 2 μL of DNase I (Novizan #GMP4104PC) and continue to incubate at 37 °C for 15 min to remove the template DNA. Purify the transcription product with magnetic beads (Novizan #N412-02), dilute it 10 times and then use a ultra-micro spectrophotometer to measure the concentration. The results are as Figure 2 shown.

[0042] Figure 2 The left vertical coordinate is the concentration of the transcription product. The mutants with higher transcription efficiency than wild-type T7 RNA polymerase (WT) are V687K, F55R, H772R, T312K, Y312D, G618K, T566K, V185K, M369K. The mutants with the highest transcription product concentration are V687K, F55R and H772R, which are 4.20 times, 3.06 times and 2.88 times that of wild-type T7 RNA polymerase respectively.

[0043] Table 7 In vitro transcription reaction system

[0044] (3) Analysis of Transcript Integrity The RNA sample was diluted to 25 ng / µL, treated at 70 °C for 2 min, and immediately cooled on ice. Capillary electrophoresis analysis of the RNA sample was performed using a Qsep-100 device. The results showed that the mutants with a transcript integrity of over 90% were: F55R, Y178K, V185K, M306K, Y312D, Y312K, M369K, T566K, G618K, H772R, and H854P.

[0045] (4) Cell Transfection HEK293T cells were seeded in a 96-well plate at a density of 1×10 4 / well. When the cells grew to about 70% of the bottom area, transfection of the transcript was performed. The mRNA samples of the above-mentioned EGFP gene transcripts were transfected at 200 ng / well, and the expression level of EGFP was observed and recorded under a fluorescence microscope 24 h later. The results were as Figure 3 shown. The protein expression levels of the transcripts of the T566K, G618K, and H772R mutants were the highest.

[0046] In summary, the dominant mutants T566K, G618K, and H772R were screened out.

[0047] Furthermore, based on the screened dominant mutants, multiple expression purifications and in vitro transcription repeatability verifications were performed. The results were as Figure 4 and Figure 5 shown. The purities of the three T7 RNA polymerase mutants after quantitative leveling were consistent, all above 85%. Figure 5 In A, the T7 RNA polymerase mutant H772R not only had the highest transcript concentration but also the highest integrity of the synthesized RNA. Its transcript concentration was 4.08 times that of the WT, and its integrity was 1.32 times that of the WT. From Figure 5 B, there was no significant difference in the transcript expression levels between the T7 RNA polymerase mutant H772R and the other two mutants, and they were all higher than the wild-type expression level. Therefore, the T7 RNA polymerase mutant H772R is the best T7 RNA polymerase mutant.

[0048] Example 2 Comparison of Transcription Activities of T7 RNA Polymerase Mutants in a High-Salt Environment To verify whether the activity of the T7 RNA polymerase mutant is more stable, in a high-salt environment (high concentration of MgCl 2In vitro transcription was carried out under the conditions of (and NaCl), and the product concentration and integrity of wild-type T7 RNA polymerase and mutant transcription of EGFP-mRNA were compared. The steps are as follows: Prepare 10× transcription buffer solutions (10× Buffer1 and 10× Buffer2 components) according to Tables 8 and 9, and filter the prepared buffer solutions through a 0.2 µm filter. Prepare in vitro transcription reaction systems according to Tables 10 and 11. After mixing, centrifuge briefly and incubate at 37 °C in a PCR instrument for 2 h. After the reaction is completed, take out the reaction product, add 4 µL of DNase I (Novizan #GMP4104PC), and continue to incubate at 37 °C for 15 min to remove the template. Purify the RNA product with RNA purification magnetic beads (Novizan #N412-02). After diluting the purified product 10-fold, measure the product concentration with a ultra-micro spectrophotometer. Take equal volumes of the RNA product for 1% agarose gel electrophoresis analysis and analysis of the integrity of the transcription product.

[0049] Table 8 Components of 10× Buffer1

[0050] Table 9 Components of 10× Buffer2

[0051] Table 10 In vitro transcription reaction system 1

[0052] Table 11 In vitro transcription reaction system 2

[0053] The results are as Figure 6 shown. The concentration and integrity of the transcription product of H772R under the condition of 100 mM MgCl 2 are 27.99 times and 2 times that of wild-type T7 RNA polymerase, respectively ( Figure 6 A, 6C); under the condition of 120 mM NaCl, the concentration and integrity of the transcription product are 21.47 times and 3 times that of wild-type T7 RNA polymerase, respectively ( Figure 6 B, 6D). In summary, the T7 RNA polymerase mutant H772R still has high transcriptional activity in a high-salt environment, providing a more stable T7 RNA polymerase for the field of RNA applications.

[0054] Example 3 Verification of the universality of the transcriptional activity of the T7 RNA polymerase mutant To verify the universality of the T7 RNA polymerase mutant H772R, two mRNA transcription templates of human genes were designed in this example for verification: G6PT andCTNNB1 The DNA sequences are shown in SEQ ID No. 48 and SEQ ID No. 49 respectively.

[0055] Synthesize G6PT and CTNNB1 The DNA template sequences were respectively ligated to the backbone of the EGFP plasmid template p vector (YaoHai Bio). The inserted restriction enzyme sites were SpeI and XhoI. The ligation products were transformed into DH5α competent cells, and monoclonal colonies were picked for sequencing verification. Monoclonal colonies with correct sequences were inoculated into LB medium for culture, and the template plasmids were extracted using a plasmid large-scale extraction kit (Tiangen #DP120-01). The reaction systems were prepared according to Tables 12 and 13 respectively for plasmid template linearization reactions. The purification steps of the linearized transcription templates were the same as those in Example 1.

[0056] Table 12 G6PT- mRNA (1757nt) template linearization system

[0057] Table 13 CTNNB 1 -mRNA (2832nt) transcription linearization system

[0058] Prepare the 10× transcription buffer solution (10× Buffer 3 components) according to Table 14, and filter the prepared buffer solution through a 0.2 µm filter. Prepare the in vitro transcription reaction system according to Table 15. After mixing, centrifuge briefly and incubate at 37 °C in a PCR instrument for 2 h. After the reaction, take out the reaction product, add 4 µL of DNase I (Novoprotein #GMP4104PC), and continue to incubate at 37 °C for 15 min to remove the template. Purify the RNA product using RNA purification magnetic beads (Novoprotein #N412-02). After purifying the product, dilute it 10-fold and measure the product concentration using a ultra-micro spectrophotometer. Take an equal volume of the RNA product for 1% agarose gel electrophoresis analysis and analysis of the integrity of the transcription product.

[0059] Table 14 10× Buffer 3 components

[0060] Table 15 G6PT -mRNA and CTNNB1 -mRNA transcription reaction system

[0061] The results are as Figure 7 shown. The mutant H772R synthesizes G6PT -mRNA and CTNNB1- The concentration of the mRNA transcript is at least 2.5 times that of the WT. Compared with the WT, the synthesis G6PT - mRNA and CTNNB1 - The integrity of the mRNA has increased by 6.5% and 3.2%.

[0062] Example 4 Comparison of Transcription of crRNA by Wild-Type T7 RNA Polymerase and Mutants Three crRNA sequences were designed for the hemagglutinin (HA) gene of influenza virus H3N2. The DNA corresponding to each sequence was fused with an antisense sequence complementary to the T7 promoter primer to obtain the corresponding crDNA sequences (shown in Table 16). The specific steps are as follows: (1) Denaturation and annealing: Prepare the annealing reaction system according to Table 17, and denature and anneal the synthesized crDNA with the T7 primer. The prepared reaction system was placed in a PCR instrument, heated at 95 °C for 5 min, and then cooled to 22 °C at a cooling rate of 0.1 °C / s.

[0063] Table 16 Transcription Template Sequences

[0064] Table 17 Annealing Reaction System

[0065] (2) Transcription: Prepare the in vitro transcription system for crRNA according to Table 18 and transcribe at 37 °C for 12 h.

[0066] Table 18 In Vitro Transcription System for crRNA

[0067] (3) Removal of DNA template: To remove the DNA in the system, 3 μL of DNase I (2 U / μL) was added to 10 μL of the transcription product for reaction, and it was placed in a PCR instrument and incubated at 37 °C for 30 min.

[0068] (4) RNA purification and recovery: Add 37 μL of DNase / RNase-free ddH 2O, 50 µL of RNA Binding Buffer and 50 µL of absolute ethanol were mixed well and transferred to a Zymo-Spin™ Column adsorption column, and centrifuged at 12,000 rpm for 1 min; 150 µL of absolute ethanol was added to the filtrate, mixed well, and then all of it was transferred to the adsorption column and centrifuged at 12,000 rpm for 1 min, and the liquid was discarded; 400 µL of RNA Prep Buffer was added to the adsorption column and centrifuged at 12,000 rpm for 1 min, and the liquid was discarded; 700 µL of RNA Wash Buffer was added to the adsorption column and centrifuged at 12,000 rpm for 1 min, and the liquid was discarded; 400 µL of RNA Wash Buffer was added again and centrifuged at 12,000 rpm for 1 min, and the liquid was discarded; the adsorption column was placed in a new 2 mL centrifuge tube and centrifuged at 12,000 rpm for 3 min, and then the adsorption column was transferred to a new 2 mL centrifuge tube, the lid was opened and left standing for 5 min; 10 µL of DNase / RNase-free ddH 2 O was added, left at room temperature for 3 min, and centrifuged at 12,000 rpm for 1 min to collect the RNA; 1 µL of the RNA product was diluted 10-fold, and the concentration of the RNA product was measured using the Qubit RNA BR Assay Kit reagent and the Qubit 4.0 fluorometer, and the integrity of the product was analyzed by capillary electrophoresis.

[0069] The results were as Figure 8 shown. The concentrations of the three different crRNA products synthesized by the T7 RNA polymerase H772R mutant were at least twice that of the wild-type T7 RNA polymerase, and the integrity was higher than that of the wild-type T7 RNA polymerase.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A T7 RNA polymerase mutant, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

2. A nucleic acid sequence encoding the T7 RNA polymerase mutant according to claim 1.

3. A biological material containing the T7 RNA polymerase mutant according to claim 1 or the nucleic acid sequence according to claim 2.

4. The biomaterial according to claim 3, characterized in that The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineered bacteria or cell.

5. A reagent or a kit, characterized in that: It contains the T7 RNA polymerase mutant according to claim 1, the nucleic acid sequence according to claim 2 or the biological material according to claim 3 or 4.

6. The reagent or kit according to claim 5, characterized in that The reagent or kit is used for RNA in vitro transcription.

7. The reagent or kit according to claim 5, characterized in that The reagent or kit is used for cell-free protein synthesis.

8. The reagent or kit according to claim 5, characterized in that The reagent or kit is used for pathogen nucleic acid detection.

9. Use of the T7 RNA polymerase mutant according to claim 1, the nucleic acid sequence according to claim 2, and the biological material according to claim 3 or 4 in the preparation of a reagent or a kit.

10. Use of the T7 RNA polymerase mutant according to claim 1, the nucleic acid sequence according to claim 2, or the biomaterial according to claim 3 or 4 in at least one of the following aspects: (1) RNA in vitro transcription; (2) Cell-free protein synthesis; (3) Pathogen nucleic acid detection; (4) mRNA vaccine research and development; (5) Drug preparation.

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