Preparation method and application of preparation for RNA cell-free synthesis

Abstract

The invention provides a preparation method and application of a preparation for RNA cell-free synthesis and belongs to the technical field of molecular biology, the preparation method of the preparation for RNA cell-free synthesis mainly comprises the steps that escherichia coli serves as a chassis strain, a pET-28a carrier is utilized, a recombinant strain for expressing T7RNA polymerase is constructed, and the T7RNA polymerase has enzyme activity at the temperature of 50-60 DEG C; inducing, culturing and collecting thalli, carrying out ultrasonic lysis, and treating a lysis solution to prepare a preparation for RNA cell-free synthesis; the preparation provided by the invention naturally contains various cofactors, and RNA polymerase does not need to be extracted and purified, so that the production cost is reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and particularly relates to a preparation method and application of a preparation for RNA cell-free synthesis. BACKGROUND

[0002] RNA is an important biomolecule and plays a crucial role in various biochemical reactions. In recent years, with the continuous development of technology, RNA vaccines, RNA biological pesticides and other innovative products have been derived. At present, the synthesis of RNA mainly relies on two traditional technologies of chemical synthesis and in vitro transcription. However, the chemical method can only effectively synthesize RNA molecules with a length of not more than 40 nucleotides (nt), and the synthesis process is often accompanied by a large amount of harmful waste and by-products, causing serious environmental pollution. The enzymes required for in vitro transcription are often expensive, and the large-scale level is low and difficult, and the industrial application is insufficient.

[0003] In the prior art, RNA in vitro transcription requires the use of highly purified RNA polymerase, such as T7 RNA polymerase. In addition, a variety of cofactors need to be added, which is cumbersome and high in cost. Therefore, it is necessary to develop a preparation for RNA cell-free synthesis which is convenient to use and low in cost. SUMMARY

[0004] In view of the deficiencies of the prior art, the application provides a preparation method and application of a preparation for RNA cell-free synthesis.

[0005] The application adopts the following technical scheme: A preparation method for RNA cell-free synthesis preparation, comprising the following steps: (1) Using Escherichia coli as a chassis strain, a recombinant strain expressing T7 RNA polymerase is constructed by using a pET-28a vector; The T7 RNA polymerase has enzyme activity at 50-60℃; (2) Inducing the culture of the recombinant strain constructed in step (1) to obtain a strain fermentation broth; (3) Collecting the bacterial cells by solid-liquid separation of the strain fermentation broth obtained in step (2), washing the bacterial cells by using a washing buffer, resuspending the bacterial cells by using a lysis buffer, performing ultrasonic lysis, and obtaining a crude lysis solution; (4) Centrifuging the crude lysis solution obtained in step (3) at 4℃ below at 4500-5000xg for 15-20 minutes to take the supernatant, performing ultrafiltration on the supernatant by using a polyether sulfone (PES) ultrafiltration membrane with a molecular weight cut-off of 3.5KDa to obtain a filtrate and a retentate, and storing the filtrate at 4℃ below for standby; (5) The cut-off liquid is heated at 55-60℃ for 8-10 minutes, centrifuged at 4500-5000xg for 15-20 minutes to obtain the supernatant, and the pH is adjusted to 7.8-8.0, and the supernatant is stored at 4℃ below for standby; (6) The cut-off liquid after step (5) is mixed with the filtrate in step (4), and the pH is adjusted to 7.8-8.0 to obtain a preparation for RNA cell-free synthesis.

[0006] According to the present application, preferably, in step (1), the T7 RNA polymerase has an amino acid sequence as shown in SEQ ID NO. 1, and the gene sequence of the T7 RNA polymerase is as shown in SEQ ID NO. 2. E. coli BL21 (DE3) is used as the chassis strain; In step (2), the ZYM-5052 auto-induction medium is used to induce the culture of the recombinant strain constructed in step (1), and the recombinant strain is cultured at 36-37℃ until the OD 600 is 30-35, the temperature is reduced to 18-25℃, and the culture is continued until the OD 600 is 150-180, and the strain fermentation liquor is obtained; In step (3), the strain fermentation liquor obtained in step (2) is centrifuged at 3000-3500xg for 8-10 minutes below 4℃ to separate the solid and liquid and collect the bacteria, the bacteria are washed 2-3 times with the washing buffer, the bacteria are resuspended with the lysis buffer at a volume ratio of 1: (1-1.5), ultrasonic lysis is performed, and the crude lysis liquor is obtained.

[0007] Further preferably, in step (1), the amino acid sequence of the T7 RNA polymerase is as shown in SEQ ID NO. 1, and the gene sequence of the T7 RNA polymerase is as shown in SEQ ID NO. 2. The gene of the T7 RNA polymerase is amplified by PCR using primers, and enzyme cutting sites are introduced at both ends of the fragment, the PCR-amplified T7 RNA polymerase gene fragment is inserted between the NdeI and XhoI enzyme cutting sites of pET-28a, and a recombinant plasmid is obtained; the recombinant plasmid is transformed into E. coli BL21 (DE3) competent cells, and positive clones are screened to obtain a recombinant strain; In step (2), the ZYM-5052 auto-induction medium is composed of the following components per liter: 8-10 g of tryptone, 4.5-5 g of yeast extract, 6.7-7 g of Na2HPO4, 2.5-3.0 g of KH2PO4, 0.8-1.0 g of NH4Cl, 0.45-0.5 g of Na2SO4, 4.5-5 mL of glycerol, 0.45-0.5 g of glucose, 1.8-2 g of lactose, and 0.8-1 mL of 1M MgSO4 solution; In step (3), the washing buffer has a formula of 50mM pH7.5 Tris-HCl, 14mM MgCl2, and 60mM NaCl; The lysis buffer formula is: 50 mM pH 7.5 Tris-HCl, 14 mM MgCl2, 60 mM KCl, 1 mM DTT (dithiothreitol) and 0.1% Triton X-100; The ultrasonic conditions are pulse treatment at 4 2 C under the condition of ultrasonic power density of 300 W / cm o with pulse time of 5 seconds and intermittent time of 10 seconds.

[0008] More preferably, in step (1), the sequence of the primer is as shown in SEQ ID NO. 4 for the forward primer and as shown in SEQ ID NO. 5 for the reverse primer.

[0009] A preparation for RNA cell-free synthesis prepared according to the above method.

[0010] The preparation for RNA cell-free synthesis prepared by the above method is used in the synthesis of RNA.

[0011] A method for RNA cell-free synthesis, comprising the following steps: i. NTPs, MgCl2, potassium glutamate (K-Glu) and DTT (dithiothreitol) are prepared into a reaction premix solution using Tris-HCl buffer, with final concentrations of NTPs not higher than 10 mM, MgCl2 14 mM, potassium glutamate 60 mM, DTT 1 mM, 50 mM pH 8.0 Tris-HCl; ii. The preparation for RNA cell-free synthesis prepared by the above method is mixed with the reaction premix solution prepared in step i. according to a volume ratio of 1:1, and a DNA template of the target RNA is added, and after reaction, the product mixture containing RNA is obtained after processing.

[0012] According to the present application, preferably in step i., the concentrations of ATP, UTP, CTP and GTP in NTPs are the same.

[0013] According to the present application, preferably in step ii., the final concentration of the DNA template is adjusted to 50 ng / μL, and the reaction condition is to react at 37℃ for 30 minutes. The method for processing after reaction is to remove impurities by centrifugation at 2800~3000×g to obtain the product mixture.

[0014] Further preferably, the product mixture is further processed by anion exchange chromatography or silica gel column purification technology to finally obtain the RNA product.

[0015] A T7 RNA polymerase, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0016] The gene sequence of the above-mentioned T7 RNA polymerase is shown in SEQ ID NO.2.

[0017] A vector containing the gene sequence of the T7 RNA polymerase is shown in SEQ ID NO.2.

[0018] A recombinant bacterium, comprising the gene sequence of the T7 RNA polymerase as shown in SEQ ID NO.2.

[0019] The use of the above-mentioned T7 RNA polymerase in synthesizing RNA.

[0020] The beneficial effects of the present invention include at least the following: 1. The preparation for cell-free RNA synthesis provided by the present invention naturally contains multiple cofactors, which do not require additional addition during the RNA synthesis process. The reaction environment is closer to the native intracellular environment and has higher RNA synthesis efficiency.

[0021] 2. The preparation for cell-free RNA synthesis provided by the present invention is used for RNA synthesis without the need for extraction and purification of RNA polymerase, thereby reducing production costs.

[0022] 3. Compared with chemical methods, the technology described in the present invention does not produce toxic and harmful substances and is green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Comparison of the contents of the T7 RNA polymerase mutant of the present invention and wild-type T7 RNA polymerase in the soluble portion of cell lysate; In the figure, A1 is a cell lysate containing the T7 RNA polymerase mutant of the present invention, and A2 is a cell lysate containing wild-type T7 RNA polymerase.

[0024] Figure 2 This is a diagram showing the effect of RNA synthesis using the preparation of the present invention; In the figure, C1 is the RNA synthesis result obtained by a commercial RNA in vitro transcription kit; C2 is the RNA synthesis effect obtained using commercial T7 RNA polymerase and laboratory-prepared in vitro transcription reagents; T1-T6 are the RNA synthesis effects of cell lysates prepared by different methods in this laboratory, and T4 is the RNA synthesis effect obtained using the method of the present invention. DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below through specific examples, but the protection scope of the present invention is not limited thereto.

[0026] The contents not specified in the examples are carried out according to the conventional conditions; the reagents or instruments not specified by the manufacturer are all ordinary commercially available products.

[0027] Example 1 The present application uses a T7 RNA polymerase mutant for the construction of recombinant bacteria, and the mutation sites are C216L and Y312D compared with the wild type T7 RNA polymerase, and the amino acid sequence is shown as SEQ ID NO. 1.

[0028] Step 1: Construction of engineering strain 1. Material preparation (1) Target gene: The T7 RNA polymerase mutant gene sequence used in this example is shown as SEQ ID NO. 2. T7 pol

[0029] The required gene fragment is synthesized by the laboratory itself.

[0030] (2) Vector preparation: using pET-28a as the vector (carrying Kan resistance) for expression T7 pol ; (3) Restriction site design: adding restriction enzyme sites suitable for inserting pET-28a at both ends of T7 pol , this example uses NdeI and XhoI; 2. Construction of vector Vector linearization: pET-28a is treated with NdeI and XhoI double digestion, and the linearized vector is recovered.

[0031] Gene fragment splicing: the gene is amplified by PCR T7 pol , and the restriction sites are introduced at both ends of the fragment, respectively, to obtain T7 pol the fragment. Among them, the forward primer (NdeI) is shown as SEQ ID NO. 4; the reverse primer (XhoI) is shown as SEQ ID NO. 5.

[0032] The PCR reaction system is as follows (25 μL): DNA: 50 ng; Forward primer (10 μM): 0.5 μL; Reverse primer (10 μM): 0.5 μL; dNTP mixture (40 mM): 0.5 μL; 10x Taq Buffer: 2.5 μL; Taq DNA polymerase (5 U / μL): 0.5 μL; ddH2O: supplemented to 25 μL.

[0033] The PCR amplification program is as follows:​ Initial denaturation: 94°C for 3 minutes; Denaturation × 30: 94°C for 30 seconds; Annealing ×30: 55°C for 30 seconds; Extension × 30: 72°C for 2 min; Final extension: 72°C for 5 minutes.

[0034] Gene insertion: T7 pol The fragment was ligated into the linearized pET-28a vector.

[0035] T4 DNA ligase was used to ligate the product. E. coli DH5α competent cells were cultured on LB resistance plates containing 50 μg / mL kanamycin at 37°C overnight, and single colonies were picked for plasmid extraction.

[0036] Verify the vector: Use PCR and enzyme digestion to identify positive clones.

[0037] 3. Construction of engineered strains (1) Plasmid transformation: Transform the verified pET28a- T7 pol Plasmid transformation into E. coli BL21(DE3) competent cells were cultured on LB plates containing 50 µg / mL kanamycin to screen for positive clones.

[0038] (2) Strain verification: Pick a single colony, extract the total DNA of the strain, and perform DNA sequencing verification T7 pol The presence of E. coli BL21(DE3)+pET28a- T7 pol .

[0039] 4. Solubility Verification The T7 RNA polymerase mutant of the present invention and the wild-type T7 RNA polymerase were respectively isolated and cloned using the pET-28a vector. E. coli The cells were expressed in BL21 (DE3), and the cells were collected and ultrasonically disrupted, and the supernatant was taken. The protein distribution of the two proteins was analyzed by protein electrophoresis. The results are as follows Figure 1 As shown, the content of T7 RNA polymerase mutant in the supernatant was significantly higher than that of the wild type.

[0040] Step 2: Preparation of RNA Cell-free Synthesis Preparations 1. Fermentation (1) Seed culture 100 mL of LB medium was added to a 500 mL Erlenmeyer flask and kanamycin (30 μg / mL) was added.

[0041] Inoculate the above recombinant strain E. coliBL21(DE3) + pET28a- T7 pol Single colony or glycerol-preserved recombinant strain E. coli BL21(DE3) + pET28a- T7 pol (Thawed from -80℃), 37℃, 250rpm shaker culture to OD600≈0.8, seed culture obtained.

[0042] Seed culture was inoculated into the fermenter at a volume ratio of 5%.

[0043] (2) Production culture in the fermenter ZYM-5052 auto-induction medium was used, initial temperature of the fermenter was set at 37℃, dissolved oxygen was maintained at >30%, pH was maintained at 7.0-7.2, when OD 600 reached 35, temperature was reduced to 20℃, and culture was continued for 16 hours until OD 600 reached 150, after fermentation, the bacterial solution was cooled to 4℃.

[0044] ZYM-5052 auto-induction medium was composed of 1 liter as shown in Table 1.

[0045] Table 1

[0046] 2. Cell collection The bacterial solution at the end of culture was centrifuged at 3000xg, 4℃ for 10 minutes, continuous centrifuge could be used to further improve efficiency. The bacterial precipitate was collected, and the supernatant was discarded.

[0047] The collected wet bacterial cells were directly used for subsequent processing, or resuspended in 50mM Tris-HCl (pH 7.2, 10% glycerol by volume) and then aliquoted, and stored at -80℃ for later use.

[0048] 3. Cell washing The collected bacterial cells were added with washing buffer at a volume ratio of 1:3 (bacterial cells: washing buffer), the bacterial cells were resuspended and centrifuged at 3000xg, 4℃ for 10 minutes, the supernatant was discarded, and the washing step was repeated twice until the supernatant was clear, and the bacterial cells were collected.

[0049] The washing buffer formula was as follows: 50mM Tris-HCl (pH 7.5); 14mM MgCl2; 60mM NaCl.

[0050] 4. Cell lysis (1) Lysis buffer was prepared with the following formula: 50mM Tris-HCl (pH 7.5); 14 mM MgCl2; 60 mM KCl; 1 mM DTT (dithiothreitol); 0.1% Triton X-100.

[0051] (2) Ultrasonic lysis The collected bacteria were resuspended with lysis buffer at a volume ratio of 1:1, and then subjected to pulse treatment at 4°C with ultrasonic power density of 300 W / cm 2 for 5 seconds and intermittent time of 10 seconds to lyse the cells, obtaining a crude lysis solution.

[0052] 5. Centrifugation The crude lysis solution was centrifuged at 4°C at 5000 x g for 15 minutes, and the supernatant was used to centrifuge a polyether sulfone ultrafiltration membrane tube with a molecular weight cut-off of 3.5 KDa at 4°C at 5000 x g for 20 minutes; the cut-off liquid was heated at 60°C for 10 minutes, and the supernatant was obtained after centrifugation at 5000 x g for 15 minutes, and the pH was adjusted to 8.0, and stored at 4 o C for standby; the filtrate was stored at 4°C for standby.

[0053] 6. Mixing The filtrate obtained in step 5 of centrifugation was mixed with the treated cut-off liquid, and the pH was adjusted to 8.0 to prepare an RNA cell-free synthesis preparation, which was stored at 4°C for standby.

[0054] Third step: construction of reaction template Method 1: construction of template by PCR 1. Primer design Forward primer: contains the first 15-20 bp of the T7 promoter sequence + target RNA sequence; Reverse primer: contains the complementary strand end sequence (15-20 bp) of the target RNA sequence.

[0055] 2. PCR amplification Template DNA: plasmid or synthetic template DNA can be used; PCR conditions Initial denaturation: 95°C for 5 minutes; 30 cycles: 95°C for 30 seconds, annealing temperature (according to primer design) for 30 seconds, extension at 72°C (according to fragment length) for 1 minute, final extension: 72°C for 10 minutes.

[0056] 3. Purification of PCR product PCR fragments were detected by agarose gel electrophoresis; the DNA band of the target size was recovered and extracted using a DNA purification kit.

[0057] Method 2: by synthesizing double-stranded DNA template 1. Gene sequence design Directly design the complete double-stranded DNA sequence of T7 promoter + target RNA sequence + termination signal; 2. Gene synthesis Submit the designed sequence to a gene synthesis company for double-stranded DNA synthesis; 3. Plasmid cloning If the synthesized sequence is long, it can be inserted into a plasmid for storage, which is convenient for amplification and subsequent PCR preparation.

[0058] In this example, the dsRNA target gene fragment of Aspergillus flavus obtained by the laboratory is used to construct the reaction DNA template, and the gene sequence is shown in SEQ ID NO. 3.

[0059] Step 4: RNA synthesis 1. Preparation of reaction mixture Prepare the reaction premix: NTPs: 10mM; Tris-HCl (pH 8.0): 50mM; MgCl2: 14mM; K-Gu (potassium glutamate): 60mM; DTT: 1mM.

[0060] 2. RNA synthesis reaction Mix the above RNA cell-free synthesis reagent and reaction premix according to the volume ratio of 1:1, add the DNA template, and make its final concentration 50ng / μL. Among them, the concentrations of ATP, UTP, CTP and GTP in NTPs are the same.

[0061] React at 37°C for 0.5 hours.

[0062] 3. Purification of RNA product After the reaction is completed, remove the impurities by centrifugation at 3000xg for 10 minutes to obtain the RNA product mixture (marked as T4).

[0063] Step 5: Effect verification The same DNA template is used, and the DNA template shown in SEQ ID NO. 3 is used.

[0064] 1. Use the in vitro transcription kit (Beyotime: R7016M) to synthesize RNA (marked as C1) according to the requirements of the instruction manual.

[0065] 2. RNA synthesis using commercial T7 RNA polymerase, as follows: Prepare the RNA synthesis reaction system (a conventional in vitro transcription reaction system prepared in the laboratory).

[0066] Prepare the reaction solution in an ice bath operating environment without RNase (as an example of a total reaction system of 20 μL): DNA template: 0.5 μg; 10x reaction buffer (Beyotime: 7012M): 2 μL; NTPs mixture (NEB: N0450): 4 μL; RNase inhibitor (Beyotime: R0101): 1 μL; T7 RNA polymerase (NEB: M0251): 1 μL; DEPC water (Solarbio: D8090): make up to 20 μL; Mix the reaction solution.

[0067] Perform RNA synthesis reaction, place the reaction tube in a constant temperature water bath or PCR instrument at 37°C, incubate for 0.5 hours, after incubation, stop the reaction by placing the product mixture on ice, and synthesize RNA (denoted as C2).

[0068] 3. The inventors also conducted research experiments under different conditions during the preparation of the RNA cell-free synthesis preparation for synthesizing RNA, as follows: T1: Different from T4 above, the crude lysate was heated at 45°C for 10 minutes during treatment, and the other conditions were the same, and the RNA product mixture was prepared.

[0069] T2: Different from T4 above, the crude lysate was directly heated at 60°C for 10 minutes, and after centrifugation at 5000 x g for 15 minutes, the supernatant was cooled, the pH was adjusted to 8.0, and it was stored at 4°C for standby, as an RNA cell-free synthesis preparation, and the other conditions were the same, and the RNA product mixture was prepared.

[0070] T3: Different from T4 above, the ultrafiltration membrane material used was different, and an acid cellulose (NC) membrane with a molecular cut-off of 3.5 KDa was used instead of a polyether sulfone ultrafiltration membrane, and the other conditions were the same, and the RNA product mixture was prepared.

[0071] T5: Different from T4 above, an incubation step was added before the ultrafiltration step, the cell crude lysate was incubated at 37°C for 2 hours, and then the subsequent treatment was performed, and the other conditions were the same, and the RNA product mixture was prepared.

[0072] T6: The difference from T4 is that the crude lysis solution is treated by ultrafiltration centrifugation, the filtrate is stored, and the storage temperature of the treated retentate is room temperature. Specifically, the crude lysis solution is centrifuged at 5000xg for 15 minutes at room temperature, and the supernatant is used to centrifuge a polyether sulfone ultrafiltration membrane tube with a molecular weight cut-off of 3.5KDa at 5000xg for 20 minutes at room temperature; the retentate is heated at 60°C for 10 minutes, centrifuged at 5000xg for 15 minutes, and the supernatant is taken, the pH is adjusted to 8.0, and stored at room temperature for standby; the filtrate is stored at room temperature for standby. The other conditions are the same, and the RNA product mixture is prepared.

[0073] The RNA synthesized by using the in vitro transcription kit (C1), the RNA synthesized by using the commercial T7 RNA polymerase (C2), the RNA product mixture prepared by the system of the present application (T4), and the RNA product mixtures prepared under different conditions of T1, T2, T3, T5, and T6 are subjected to nucleic acid electrophoresis, and the process is as follows: Preparation of agarose gel 1g of agarose is weighed, 85mL of 1xTAE buffer is added, and heated to completely dissolve. When the solution cools to 60°C, 5µL of nucleic acid dye is added and mixed. Pour the mixed solution into the gel mold, insert the comb, and stand for 30 minutes until it solidifies.

[0074] Remove the comb and place the gel in the electrophoresis tank. Add 1xTAE buffer until the gel is covered.

[0075] Sample loading and electrophoresis Mix the RNA sample with the RNA loading buffer at a volume ratio of 4:1, take 5-10µL of the mixed sample, and load it into the gel sample well. Use 1xTAE buffer as the electrophoresis liquid in the electrophoresis tank, set the voltage to 120V, and run for 30-45 minutes.

[0076] Result observation and analysis Observe the gel fluorescence band under the ultraviolet lamp, use the gel imaging system to take pictures and save the electrophoresis results.

[0077] The results are shown in Figure 2 The electrophoresis results show that the RNA product mixture prepared by the system of the present application (T4) has similar RNA synthesis effect as the other two technologies (RNA synthesized by using the in vitro transcription kit, RNA synthesized by using the commercial T7 RNA polymerase combined with the conventional in vitro transcription reaction system of the prior art), but the electrophoresis results of the RNA product mixtures prepared under different conditions of T1, T2, T3, T5, and T6 show that the RNA synthesis effect is not good.

[0078] The preparation for RNA cell-free synthesis prepared by the present invention can effectively synthesize RNA, does not require the addition of additional cofactors during the RNA synthesis process, and does not require the extraction and purification of RNA polymerase, thereby reducing production costs.

Claims

1. A method for preparing a cell-free RNA synthesis preparation, characterized in that: The steps include: (1) Using Escherichia coli as the base strain and the pET-28a vector, a recombinant strain expressing T7 RNA polymerase was constructed; The T7 RNA polymerase has enzymatic activity at 50-60°C; (2) Inducing the recombinant strain constructed in step (1) to obtain a fermentation broth; (3) collecting the bacterial cells by solid-liquid separation of the fermentation liquid of the strain obtained in step (2), washing the bacterial cells with a washing buffer, and then resuspending the bacterial cells with a lysis buffer, and performing ultrasonic lysis to obtain a crude lysate; (4) The crude lysate obtained in step (3) was centrifuged at 4500-5000 × g for 15-20 minutes at below 4°C to obtain the supernatant, and the supernatant was ultrafiltered using a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 3.5 kDa to obtain a filtrate and a retentate, and the filtrate was stored at below 4°C for later use; (5) Heat the retentate at 55-60°C for 8-10 minutes, centrifuge at 4500-5000 × g for 15-20 minutes, collect the supernatant, adjust the pH to 7.8-8.0, and store below 4°C for later use; (6) The retentate after treatment in step (5) is mixed with the filtrate in step (4), and the pH is adjusted to 7.8-8.0 to obtain a preparation for cell-free RNA synthesis.

2. The preparation method according to claim 1, wherein In step (1), E. coli BL21(DE3) was used as the chassis strain; In step (2), the recombinant strain constructed in step (1) was induced and cultured using ZYM-5052 autoinduction medium, and the recombinant strain was cultured at 36-37°C until the OD 600 When the temperature reaches 30-35°C, reduce the temperature to 18-25°C and continue culturing until the OD 600 When the concentration reaches 150-180, the fermentation liquid of the strain is obtained; In step (3), the fermentation broth of the strain obtained in step (2) is centrifuged at 3000-3500×g for 8-10 minutes at below 4°C to perform solid-liquid separation to collect the bacteria, and the bacteria are washed 2-3 times with a washing buffer. The collected bacteria are resuspended with a lysis buffer at a volume ratio of 1:(1-1.5), and ultrasonically lysed to obtain a crude lysate.

3. The preparation method according to claim 2, wherein In step (1), the amino acid sequence of T7 RNA polymerase is shown in SEQ ID NO.1, and the gene sequence of T7 RNA polymerase is shown in SEQ ID NO.

2. The gene of T7 RNA polymerase is amplified by PCR using primers, and restriction sites are introduced at both ends of the fragment. The gene fragment of T7 RNA polymerase amplified by PCR is inserted between the NdeI and XhoI restriction sites of pET-28a to obtain a recombinant plasmid; the recombinant plasmid is transformed into E. coli BL21 (DE3) competent cells were used to screen positive clones and obtain recombinant strains; In step (2), the composition of the ZYM-5052 autoinduction medium per liter includes: 8-10 g of tryptone, 4.5-5 g of yeast extract, 6.7-7 g of Na2HPO4, 2.5-3.0 g of KH2PO4, 0.8-1.0 g of NH4Cl, 0.45-0.5 g of Na2SO4, 4.5-5 mL of glycerol, 0.45-0.5 g of glucose, 1.8-2 g of lactose, and 0.8-1 mL of 1 M MgSO4 solution; In step (3), the washing buffer formula is: 50mM pH7.5 Tris-HCl, 14mM MgCl2 and 60mM NaCl; The lysis buffer formula is: 50mM pH7.5 Tris-HCl, 14mM MgCl2, 60mM KCl, 1mM DTT and 0.1% Triton X-100; The ultrasonic power density was 300 W / cm 2 Ultrasound at 4 o C, with a pulse time of 5 seconds and a rest time of 10 seconds, and ultrasonic lysis was performed.

4. The preparation method according to claim 3, wherein In step (1), the sequences of the primers are as shown in SEQ ID NO. 4 for the forward primer and SEQ ID NO. 5 for the reverse primer.

5. A preparation for cell-free RNA synthesis prepared according to the method according to any one of claims 1 to 4.

6. Use of the preparation for cell-free RNA synthesis prepared by the method according to any one of claims 1 to 4 in synthesizing RNA.

7. A method for cell-free RNA synthesis, characterized in that: The steps include: i. Prepare a reaction premix of NTPs, MgCl2, potassium glutamate, and DTT using Tris-HCl buffer to a final concentration of no more than 10 mM NTPs, 14 mM MgCl2, 60 mM potassium glutamate, 1 mM DTT, and 50 mM Tris-HCl, pH 8.

0. ii. The preparation for cell-free RNA synthesis prepared by the method according to any one of claims 1 to 4 is mixed with the reaction premix prepared in step i at a volume ratio of 1:1, a DNA template of the target RNA is added, and the reaction is followed by treatment to obtain a product mixture containing RNA.

8. The method according to claim 7, wherein In step i, the concentrations of ATP, UTP, CTP, and GTP in the NTPs are the same.

9. The method according to claim 7, wherein In step ii, the final concentration of the DNA template was adjusted to 50 ng / μL, and the reaction was carried out at 37°C for 30 minutes; The post-reaction treatment method is to remove impurities by centrifugation at 2800-3000×g to obtain a product mixture; Preferably, in step ii, the product mixture containing RNA is further treated by anion exchange chromatography or silica gel column purification technology to finally obtain the RNA product.

10. A T7 RNA polymerase, characterized in that The amino acid sequence of the T7 RNA polymerase is shown in SEQ ID NO.1; Preferably, the gene sequence of the T7 RNA polymerase is shown as SEQ ID NO.2; Preferably, a vector containing the gene sequence of the T7 RNA polymerase is shown in SEQ ID NO.2; Preferably, a recombinant bacterium containing the gene sequence of the T7 RNA polymerase is shown in SEQ ID NO.2; Preferably, the T7 RNA polymerase is used in synthesizing RNA.

Images