T7RNA polymerase mutant as well as preparation method and application thereof

Abstract

The invention discloses a T7RNA polymerase mutant as well as a preparation method and application thereof, and relates to the technical field of biology. The T7RNA polymerase mutant provided by the invention has excellent thermal stability, can be effectively transcribed under a relatively high temperature condition, can be applied to effective synthesis of long fragments, and has the advantages of high transcription efficiency, few byproducts 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 a preparation method and application thereof. Background Art

[0002] T7 RNA polymerase is a DNA-dependent RNA polymerase encoded by T7 bacteriophage gene 1. It is a single-subunit enzyme with a molecular weight of 99 kDa and belongs to the single-subunit DNA-dependent RNA polymerase family (single-subunit DNA-dependent RNAP, sRNAP family). This family includes bacteriophage T3 and SP6 RNAP, mitochondrial and chloroplast RNAP. T7 RNA polymerase catalyzes the formation of mRNA by recognizing the highly specific promoter 5'-TAATACGACTCACTATAGG-3'. T7 RNA polymerase requires magnesium ions as a cofactor. In addition, bovine serum albumin and spermine can promote the enzyme and are not inhibited by rifampicin, which is different from ordinary bacterial RNA polymerases.

[0003] Since its discovery in 1970, T7 RNA polymerase has been maturely applied to efficient in vivo transcription in Gram-negative bacteria Escherichia coli BL21 (DE3), and the plasmid system that matches it is the pET series. The pET plasmid system is a preferred system for overexpressing proteins, but it has the problem of leaky expression and is not suitable for toxic protein expression. However, because T7 lysozyme can bind to T7 RNA polymerase and reduce the background transcription activity of T7 RNA polymerase, leaky expression is usually reduced by expressing T7 lysozyme in the host. At the same time, T7 RNA polymerase can also be used for in vivo transcription in Gram-positive bacteria Corynebacterium glutamicum.

[0004] In terms of in vitro transcription, in addition to its advantages such as high transcription efficiency and strong extension ability, T7 RNA polymerase as an in vitro RNA synthesis tool also has some disadvantages that cannot be ignored. T7 RNA polymerase may produce many byproducts in the process of RNA synthesis, including byproducts such as double-stranded RNA produced during the transcription initiation process and interrupted RNA products caused by termination signals. Therefore, it is urgent to optimize and improve T7 RNA polymerase.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a T7 RNA polymerase mutant and a preparation method and application thereof.

[0007] The present invention is achieved in that:

[0008] In a first aspect, an embodiment of the present invention provides a method for transcribing and synthesizing RNA, the method comprising the following steps:

[0009] (a) adding a DNA template, rNTP raw material and T7 RNA polymerase to a transcription system to produce a reaction mixture, wherein the T7 RNA polymerase comprises an amino acid substitution at position 520 corresponding to SEQ ID NO: 1; and

[0010] (b) incubating the reaction mixture under conditions suitable for transcription of the DNA template to produce an RNA copy of the template.

[0011] In a second aspect, an embodiment of the present invention provides a method for preparing T7 RNA polymerase, the method comprising the following steps:

[0012] (a) selecting a T7 RNA polymerase in which the amino acid at position 520 of SEQ ID NO: 1 is substituted for purification; and

[0013] (b) obtaining the T7 RNA polymerase.

[0014] In a third aspect, an embodiment of the present invention provides a T7 RNA polymerase, wherein the T7 RNA polymerase includes an amino acid substitution at position 520 of SEQ ID NO:1.

[0015] In a fourth aspect, an embodiment of the present invention provides a conjugate comprising the T7 RNA polymerase described in the preceding embodiment and a marker for purification.

[0016] In a fifth aspect, an embodiment of the present invention provides an isolated nucleic acid encoding the T7 RNA polymerase described in the preceding embodiment.

[0017] In a sixth aspect, an embodiment of the present invention provides a vector comprising the isolated nucleic acid described in the preceding embodiment.

[0018] In a seventh aspect, an embodiment of the present invention provides a host cell comprising the vector described in the preceding embodiment.

[0019] In an eighth aspect, an embodiment of the present invention provides a kit comprising the T7 RNA polymerase described in the preceding embodiment.

[0020] In a ninth aspect, embodiments of the present invention provide use of the T7 RNA polymerase described in the preceding embodiments in RNA synthesis.

[0021] The present invention has the following beneficial effects:

[0022] The optimized T7 RNA polymerase mutant of the present invention has excellent thermal stability, can be effectively transcribed at 50°C, can be applied to the synthesis of long fragments (15kb), and has the advantages of high transcription efficiency, few by-products, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 This is a standard curve diagram showing the relationship between the enzyme amount and slope of conventional T7 RNA polymerase.

[0025] Figure 2 This is a standard curve diagram showing the relationship between the enzyme amount and slope of the thermostable Hi T7 RNA polymerase. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0027] The inventors of the present application analyzed the function, protein structure and key sites of the T7 RNA polymerase sequence, and designed a large number of mutations for stability modification, and finally obtained the following thermostable T7 RNA polymerase mutant, which can be effectively transcribed at 50°C and can be applied to the synthesis of long fragments (15kb), and has the advantages of high transcription efficiency and few by-products.

[0028] In one aspect, an embodiment of the present invention provides a method for transcribing and synthesizing RNA, the method comprising the following steps:

[0029] (a) adding a DNA template, rNTP raw material and T7 RNA polymerase to a transcription system to produce a reaction mixture, wherein the T7 RNA polymerase comprises an amino acid substitution at position 520 corresponding to SEQ ID NO: 1; and

[0030] (b) incubating the reaction mixture under conditions suitable for transcription of the DNA template to produce an RNA copy of the template.

[0031]

[0032] In some embodiments, the reaction mixture comprises the transcription system, a DNA template, an Rntp raw material and a T7 RNA polymerase.

[0033] In some embodiments, the transcription system comprises Tris-HCl, Mg 2+ 、Na + , spermidine, EDTA, and DTT.

[0034] In some embodiments, the transcription system further comprises a probe.

[0035] In some embodiments, the transcription system further comprises an RNase inhibitor and a pyrophosphatase.

[0036] In some embodiments, the DNA template contains a promoter recognized by a polymerase.

[0037] In some embodiments, the conditions suitable for DNA template transcription include: the reaction temperature can be 45-60°C.

[0038] On the other hand, an embodiment of the present invention provides a method for preparing T7 RNA polymerase, the method comprising the following steps:

[0039] (a) selecting a T7 RNA polymerase in which the amino acid at position 520 of SEQ ID NO: 1 is substituted for purification; and

[0040] (b) obtaining the T7 RNA polymerase.

[0041] In another aspect, an embodiment of the present invention provides a T7 RNA polymerase, wherein the T7 RNA polymerase comprises an amino acid substitution at position 520 of SEQ ID NO:1.

[0042] In some embodiments, the amino acid at position 520 of SEQ ID NO: 1 is substituted with G520Q.

[0043] In some embodiments, the T7 RNA polymerase has an altered transcription efficiency relative to the T7 RNA polymerase shown in SEQ ID NO:1.

[0044] In some embodiments, the T7 RNA polymerase further comprises an amino acid substitution at any one or more of the following positions: positions 108, 205, 206, 312, 333, 362, 388, 446, 534, 567, 584, 724, 832, 843, 849, 863, and 877 of SEQ ID NO:1.

[0045] In some embodiments, the T7 RNA polymerase further comprises a combination of any one or more of the following mutations: E108L, H205S, K206P, Y312D, K333P, M362P, D388E, L446F, L534V, V567P, A584K, A724P, M832F, A843Q, F849V, A863P, and E877R.

[0046] In some embodiments, the T7 RNA polymerase further comprises the following mutation combinations: E108L, H205S, K206P, Y312D, K333P, M362P, D388E, L446F, L534V, V567P, A584K, A724P, M832F, A843Q, F849V, A863P, and E877R.

[0047] In some embodiments, the reaction temperature of T7 RNA polymerase can be any one of 37°C, 38°C, 40°C, 42°C, 44°C, 45°C, 46°C, 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, 60°C, or any two thereof. Under the same reaction conditions, the T7 RNA polymerase provided by the present invention has increased thermal stability relative to the wild-type RNA polymerase, and has fewer by-products, more uniform target products, and can be applied to the efficient synthesis of long fragments (15kb).

[0048] On the other hand, an embodiment of the present invention provides a conjugate, which contains the T7 RNA polymerase mutant described in any of the above embodiments and a marker for purification.

[0049] In some embodiments, the markers used for purification include: purification tags, including but not limited to His tag (HIS-Tag), glutathione sulfhydryl transferase tag (GST-Tag), maltose binding protein tag (MBP-Tag), transcription termination / anti-termination protein tag (NusA-Tag), and small ubiquitin-related modifier (SUMO).

[0050] In another aspect, an embodiment of the present invention provides an isolated nucleic acid encoding the T7 RNA polymerase described in any of the preceding embodiments.

[0051] Nucleic acids are typically RNA or DNA, and nucleic acid molecules can be single-stranded or double-stranded. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence. DNA nucleic acids are used when they are linked to a vector.

[0052] In another aspect, an embodiment of the present invention provides a vector comprising the isolated nucleic acid described in any of the preceding embodiments.

[0053] On the other hand, an embodiment of the present invention provides a host cell comprising the vector described in any of the above embodiments.

[0054] Based on the disclosure of the amino acid sequence of the T7 RNA polymerase mutant in the present application, those skilled in the art can easily think of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the T7 RNA polymerase mutant. For example, the T7 RNA polymerase mutant can be separated and purified from the culture product of a recombinant cell that can recombinantly express the T7 RNA polymerase mutant described in any of the above embodiments. This is easy to achieve for those skilled in the art. Based on this, no matter what technology is used to prepare the T7 RNA polymerase mutant disclosed in the present invention, it belongs to the protection scope of the present disclosure.

[0055] On the other hand, an embodiment of the present invention provides a kit, which includes the T7 RNA polymerase described in any of the above embodiments.

[0056] In some embodiments, the kit further comprises: at least one of a buffer solution, a preservative solution, and a diluent.

[0057] In some embodiments, the kit further comprises: ribonucleoside triphosphates, such as any one or more of ATP, UTP, GTP, and CTP.

[0058] In some embodiments, the kit further comprises: a reaction buffer solution for in vitro transcription.

[0059] In some embodiments, the kit further comprises: a PCR reaction mixture.

[0060] In addition, an embodiment of the present invention also provides the use of the T7 RNA polymerase described in any of the above embodiments in RNA synthesis.

[0061] In some embodiments, the RNA synthesis comprises: synthesizing RNA by in vitro transcription.

[0062] In some embodiments, the RNA synthesis comprises: mixing T7 RNA polymerase, rNTP and template DNA molecules to transcribe and synthesize the target RNA. The reaction temperature can be 45-60° C. The template DNA molecule contains a promoter recognized by the polymerase.

[0063] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0064] Example 1 G520Q single point mutation experiment

[0065] 1.1 Molecular cloning protocol

[0066] 1.1.1 Gene synthesis

[0067] The wild-type T7 RNA polymerase sequence (GenBank: M38308.1, SEQ ID NO: 1) was obtained from NCBI, and after codon optimization based on Escherichia coli, it was sent to Universal Bio for gene synthesis, and a 6His tag was added to N to obtain a T7 RNA polymerase amplification template.

[0068] 1.1.2 Construction of wild-type T7 RNA polymerase

[0069] The target sequence was amplified using Q5 high-fidelity polymerase (NEB, catalog number M0491L), and the amplified fragment and pTrc99A vector were double-digested with EcoR I and BamH I (Takara, catalog numbers 1605 and 1611, respectively). After ligation using T4 DNA ligase (Takara, catalog number 2011A), BL21 (DE3) was transformed and cultured overnight. Appropriate transformants were picked for identification and sequencing verification to obtain pTrc99A-TRP.

[0070] 1.1.3 Construction of the T7 RNA polymerase normal temperature mutant TRPM1 (G520Q single point mutation)

[0071] Primers were designed according to the mutation site, and the target fragment was obtained by overlapping PCR. The construction method was the same as 1.1.2, and pTrc99A-TRPM1 was obtained after sequencing verification.

[0072] 1.2 Expression and purification of mutant TRPM1

[0073] 1.2.1 Recombinant expression

[0074] The recombinant strain was cultured at 37°C, 220 rpm until OD600 was 0.6-0.8, precooled on ice for 30 min, IPTG was added at a final concentration of 1 mM, and the bacteria were harvested after induction at 28°C for 4 hours.

[0075] 1.2.2 Crushing and purification

[0076] The cells were resuspended in lysis buffer, disrupted by ultrasound, purified by butyl, Ni-TED, and CHT II, ​​and placed in a storage solution of 50 mM Tris, 50% glycerol, 100 mM NaCl, 1 mM EDTA, 2 mM DTT, 0.1% tween-20, pH 7.9 and stored at 25°C.

[0077] 1.3 Activity identification of T7 RNA polymerase and its variants

[0078] 1.3.1 Preparation of transcription template

[0079] 1.3.1.1 Preparation of 2× Annealing Buffer

[0080] 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 2 mM EDTA were mixed.

[0081] 1.3.1.2 Template preparation system (T7-DS-2)

[0082] Components Volume (μL) 2×Annealing Buffer 250 T7-min-GGG (50 μM) 100 T7-plus-GGG (50μM) 100 <![CDATA[ddH2O]]> 50 total 500

[0083] The prepared system was divided into eight tubes (50 μL / tube) and annealed using a PCR instrument.

[0084] Template preparation procedure

[0085] 95℃, 5min; 1cycle; 0.1℃ / s;

[0086] 75℃, 10min; 1cycle; 0.1℃ / s;

[0087] 60℃, 10min; 1cycle; 0.1℃ / s;

[0088] 50℃, 10min; 1cycle; 0.1℃ / s;

[0089] 40℃, 10min; 1cycle; 0.1℃ / s;

[0090] 25℃, 30min.

[0091] 1.3.1.3 Combine the annealed templates in the eight tubes into one tube and store it in a -20℃ refrigerator for later use.

[0092] 1.3.2 Dilution of standard and test enzyme

[0093] 1.3.2.1 Preparation of T7 RNA polymerase dilution

[0094] 50mM Tris-HCl, 100mM NaCl, 1mM EDTA, 1mM DTT, 0.1% (w / v) X-100, 50% Glycerol, pH 7.9, placed at 25°C.

[0095] 1.3.2.2 Dilute the enzyme to be tested to 1 mg / mL on ice according to the protein concentration using diluent, and then dilute it 80 times.

[0096] 1.3.2.3 If you need to calibrate the thermostable T7 RNA polymerase (50℃): HR7M1 and HR7, use Hi T7 RNA Polymerase (50U / μL, NEB) as the standard, and there is no need to dilute the standard in advance. If you need to calibrate the conventional T7 RNA polymerase (37℃): TRP and TRPM1, use T7 RNA Polymerase (50U / μL, NEB) as the standard, then you need to dilute the NEB T7 RNA polymerase to 10U / μL (10μL) with diluent.

[0097] 1.3.3 Reaction system preparation

[0098] 1.3.3.1 Prepare the transcription reaction solution in an EP tube on ice according to the table below, mix well and centrifuge.

[0099] 1.3.3.2 Formula for calculating the total number of reactions:

[0100] Total number of reactions = 3 × (n + 1) + 5 + 1 (A1);

[0101] In the formula: n is the number of samples to be tested; 1 is the first "1" indicating the number of blank controls, and the second "1" indicates the number of reserved losses; 3 is 3 replicates for each sample to be tested; 5 is the number of standard curve reactions.

[0102] Transcription reaction system

[0103] Components Single reaction volume (μL) 10× T7 Reaction Buffer(NEB) 2 rNTPs(25mM) 0.4 RNasin (40U / μl) 0.5 T7-MB probe (50μM) 0.25 total 3.15

[0104] 1.3.3.3 Dispense the prepared reaction solution into PCR tubes at a volume of 3.15 μL per well.

[0105] 1.3.4 Enzyme reaction

[0106] 1.3.4.1 Take 1 μL of the diluted enzyme sample to be tested and add it to the reaction solution. Set up 3 replicates for each sample.

[0107] 1.3.4.2 Take 1 μL of T7 RNA polymerase dilution solution (without T7 RNA polymerase) and add it to the reaction solution as a blank control. Set up 3 replicates.

[0108] 1.3.4.3 Conventional T7 RNA polymerase: Take 2.5 / 1.25 / 1 / 0.5 / 0.25 μL of diluted T7 RNA Polymerase (10 U / μL, NEB) and add them to the reaction solution, with one replicate set for each group.

[0109] 1.3.5 Template Addition

[0110] Dilute the T7-DS-2 template 5 times with 1×TE Buffer, add 5ul to each well, cover the PCR tube cap, and centrifuge briefly.

[0111] 1.3.6 On-the-job

[0112] 1.3.6.1 Set up the following program on the Macrostone fully automatic medical PCR analysis system:

[0113]

[0114] 1.3.6.2 Place the PCR tube containing the sample reaction solution in the instrument and start the program.

[0115] 1.3.7 Data Analysis

[0116] 1.3.7.1 Preparation of standard curve

[0117] ①Use the software of Macrostone fully automatic medical PCR analysis system to export Excel file.

[0118] ② Select the fluorescence values ​​of the first 20 cycles of the standard conventional T7 RNA Polymerase and the fluorescence values ​​of the blank control group. First, calculate the average fluorescence value of the three replicates of the blank control group. Subtract the fluorescence value of the conventional T7 RNA Polymerase in each enzyme amount gradient from the average fluorescence value of the blank control group to obtain the net fluorescence value, and calculate the slope of the net fluorescence value increasing with the reaction time, that is, the fluorescence growth rate v. The results of the conventional T7 RNA polymerase enzyme amount and slope are shown in Table 1.

[0119] Table 1

[0120] Enzyme amount (U) 25 12.5 10 5 2.5 Slope (v) 1041.2 464.7 328.7 159.4 55.0

[0121] ③ Conventional T7 RNA polymerase: The amount of T7 RNA Polymerase (25 / 12.5 / 10 / 5 / 2.5U) is the X-axis, and the fluorescence growth rate of each enzyme amount gradient of T7 RNA Polymerase is the Y-axis. The standard curve is as follows: Figure 1 shown.

[0122] ④ Obtain the relationship curve:

[0123] y=Ax+B(R 2 ≥0.99) (A2);

[0124] In the formula: y is the fluorescence growth rate; x is the amount of standard T7 RNA Polymerase, the unit is U.

[0125] Note: If the fluorescence growth rate of an individual enzyme amount deviates from the linearity, one point can be discarded.

[0126] 1.3.7.2 Calculation of enzyme activity

[0127] ① Select the fluorescence values ​​of the first 20 cycles of the enzyme to be tested, subtract the average fluorescence value of the blank control group from the fluorescence value of each enzyme to be tested to obtain the net fluorescence value, calculate the average net fluorescence value of each enzyme to be tested, and its fluorescence growth rate (y1).

[0128] ② Substitute the fluorescence growth rate (y1) of each enzyme to be tested into formula (A2) to calculate the enzyme input amount (x1) of the enzyme to be tested.

[0129] ③ Calculate the original enzyme activity according to the following formula:

[0130] Enzyme activity of the original enzyme to be tested = x1 × 80 × C (A3);

[0131] In the formula: x1 is the amount of TRPM1 enzyme to be tested, that is, the unit enzyme activity of the TRPM1 enzyme to be tested (U / ul); 80 is the dilution multiple; C is the concentration of the TRPM1 original enzyme to be tested.

[0132] Note: If the specific enzyme activity of the enzyme to be tested increases or decreases for some reason, the signal is not in the linear range after dilution 80 times. It is necessary to readjust the dilution multiple of the enzyme to be tested and repeat the experiment. At this time, the dilution multiple in the calculation formula should also change accordingly.

[0133] Experimental results: The specific enzyme activity of each protein was calculated based on the experimental data, as shown in Table 2. The specific enzyme activity of the wild-type T7 RNA polymerase (TRP) at room temperature is 931.9 kU / mg, and the specific enzyme activity of the mutant T7 RNA polymerase (TRPM1) at room temperature is 1057.5 kU / mg. TRPM1 has a greater specific enzyme activity and a higher transcription capacity per unit mass of protein. Therefore, the transcription capacity of the T7 RNA polymerase mutant is significantly improved compared with the wild-type T7 RNA polymerase.

[0134] Table 2

[0135]

[0136] 1.3.7.3 Experimental effectiveness analysis

[0137] ①The linear correlation coefficient of at least 5 points of the standard curve is greater than 0.99;

[0138] ② The fluorescence growth rate v of the blank control group in the first 20 cycles is ≤5%;

[0139] ③ The net fluorescence value CV of at least two parallel tubes of the enzyme to be tested is ≤10%;

[0140] ④If the above conditions are not met, repeat the experiment.

[0141] Example 2 Comparative experiment of combined mutations with and without G520Q

[0142] 2.1 Molecular cloning protocol

[0143] 2.1.1 Gene synthesis

[0144] The wild-type T7 RNA polymerase sequence (GenBank: M38308.1) was obtained from NCBI, and after codon optimization based on Escherichia coli, it was sent to Universal Bio for gene synthesis, and a 6His tag was added to obtain a T7 RNA polymerase amplification template.

[0145] 2.1.2 Construction of wild-type T7 RNA polymerase

[0146] The target sequence was amplified using Q5 high-fidelity polymerase (NEB, catalog number M0491L), and the amplified fragment and pTrc99A vector were double-digested with EcoR I and BamH I (Takara, catalog numbers 1605 and 1611, respectively). After ligation using T4 DNA ligase (Takara, catalog number 2011A), BL21 (DE3) was transformed and cultured overnight. Appropriate transformants were picked for identification and sequencing verification to obtain pTrc99A-TRP.

[0147] 2.1.3 Construction of the thermostable mutants HR7M1 and HR7 of T7 RNA polymerase

[0148] 2.1.3.1 Construction of T7 RNA polymerase mutant HR7

[0149] The amino acids E108L, H205S, K206P, Y312D, K333P, M362P, D388E, L446F, G520Q, L534V, V567P, A584K, A724P, M832F, A843Q, F849V, A863P, and E877R were mutated, and primers were designed according to the mutation sites. The construction method was the same as 1.2 to obtain HR7.

[0150] 2.1.3.2 Construction of T7 RNA polymerase mutant HR7M1

[0151] The amino acids E108L, H205S, K206P, Y312D, K333P, M362P, D388E, L446F, L534V, V567P, A584K, A724P, M832F, A843Q, F849V, A863P, and E877R were mutated, and primers were designed according to the mutation sites. The construction method was the same as 1.2 to obtain HR7M1.

[0152] 2.2 Expression and purification of mutant HR7 and HR7M1

[0153] 2.2.1 Recombinant expression

[0154] Same as Example 1.

[0155] 2.2.2 Crushing and purification

[0156] Same as Example 1.

[0157] 2.3 Activity identification of T7 RNA polymerase and its variants

[0158] 2.3.1 Preparation of transcription template

[0159] Same as Example 1.

[0160] 2.3.2 Dilution of standard and test enzyme

[0161] Same as Example 1.

[0162] 2.3.3 Reaction system preparation

[0163] Same as Example 1.

[0164] 2.3.4 Enzyme reaction

[0165] 2.3.4.1 Take 1 μL of the diluted enzyme sample to be tested and add it to the reaction solution. Set up 3 replicates for each sample.

[0166] 2.3.4.2 Take 1 μL of T7 RNA polymerase dilution solution (without T7 RNA polymerase) and add it to the reaction solution as a blank control. Set up 3 replicates.

[0167] 2.3.4.3 Thermoresistant T7 RNA polymerase: Take 1.5 / 1 / 0.75 / 0.5 / 0.25 μL of Hi T7 RNA Polymerase (50 U / μL, NEB) and add them to the reaction solution respectively. Set up one replicate for each group.

[0168] 2.3.5 Template Addition

[0169] Same as Example 1.

[0170] 2.3.6 On-the-job

[0171] 2.3.6.1 Set up the following program on the Macrostone fully automatic medical PCR analysis system:

[0172]

[0173] 2.3.6.2 Place the PCR tube containing the sample reaction solution in the instrument and start the program.

[0174] 2.3 Activity identification of T7 RNA polymerase and its variants

[0175] Same as Example 1.

[0176] 2.3.7 Data Analysis

[0177] 2.3.7.1 Preparation of standard curve

[0178] ①Use the software of Macrostone fully automatic medical PCR analysis system to export Excel file.

[0179] ② Select the fluorescence values ​​of the first 20 cycles of the standard thermostable Hi T7 RNA Polymerase and the fluorescence values ​​of the blank control group. First, calculate the average fluorescence value of the three replicates of the blank control group. Subtract the fluorescence value of the thermostable Hi T7 RNA Polymerase of each enzyme amount gradient from the average fluorescence value of the blank control group to obtain the net fluorescence value, and calculate the slope of the net fluorescence value increasing with the reaction time, that is, the fluorescence growth rate v. The results of the thermostable Hi T7 RNA polymerase enzyme amount and slope are shown in Table 3.

[0180] Table 3

[0181] Enzyme amount (U) 125 100 75 50 37.5 25 12.5 Slope (v) 403.4 321.7 234.4 147.8 107.3 63.5 22.0

[0182] ③ Thermostable T7 RNA polymerase: The amount of Hi T7 RNA Polymerase (75 / 50 / 37.5 / 25 / 12.5U) is the X-axis, and the fluorescence growth rate of each enzyme amount gradient of Hi T7 RNA Polymerase is the Y-axis. The standard curve is as follows: Figure 2 shown.

[0183] ④ Obtain the relationship curve:

[0184] y=Ax+B(R 2 ≥0.99) (A2);

[0185] In the formula: y is the fluorescence growth rate; x is the amount of standard Hi T7 RNA Polymerase, the unit is U.

[0186] Note: If the fluorescence growth rate of an individual enzyme amount deviates from the linearity, one point can be discarded.

[0187] 2.3.7.2 Calculation of enzyme activity

[0188] ① Select the fluorescence values ​​of the first 20 cycles of the enzyme to be tested, subtract the average fluorescence value of the blank control group from the fluorescence value of each enzyme to be tested to obtain the net fluorescence value, calculate the average net fluorescence value of each enzyme to be tested, and its fluorescence growth rate (y1).

[0189] ② Substitute the fluorescence growth rate (y1) of each enzyme to be tested into the formula (A2) of this example to calculate the enzyme input amount (x1) of the enzyme to be tested.

[0190] ③ Calculate the original enzyme activity according to the following formula:

[0191] Enzyme activity of the original enzyme to be tested = x1 × 80 × C (A3);

[0192] In the formula: x1 is the amount of HR7 / HR7M1 enzyme to be tested, that is, the unit enzyme activity of HR7 / HR7M1 enzyme to be tested (U / μl); 80 is the dilution multiple; C is the concentration of HR7 / HR7M1 original enzyme to be tested.

[0193] Note: If the specific enzyme activity of the enzyme to be tested increases or decreases for some reason, the signal is not in the linear range after dilution 80 times. It is necessary to readjust the dilution multiple of the enzyme to be tested and repeat the experiment. At this time, the dilution multiple in the calculation formula should also change accordingly.

[0194] Experimental results: The specific enzyme activity of each protein was calculated based on the experimental data, and the data are shown in Table 4. The specific enzyme activity of the T7 RNA polymerase variant (HR7) containing the G520Q mutation at 50°C is 3837.4 kU / mg, and the specific enzyme activity of the T7 RNA polymerase variant (HR7M1) without the G520Q mutation at 50°C is 3683.3 kU / mg. HR7 has a larger specific enzyme activity and a higher transcription capacity per unit mass of protein. Therefore, the transcription capacity of the T7 RNA polymerase variant containing the G520Q mutation is significantly improved compared with the T7 RNA polymerase variant without the G520Q mutation.

[0195] Table 4

[0196]

[0197] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for transcribing and synthesizing RNA, comprising: (a) adding a DNA template, rNTP raw material and T7 RNA polymerase to a transcription system to produce a reaction mixture, wherein the T7 RNA polymerase comprises an amino acid substitution at position 520 corresponding to SEQ ID NO: 1; and (b) incubating the reaction mixture under conditions suitable for transcription of the DNA template to produce an RNA copy of the template.

2. A method for preparing T7 RNA polymerase, comprising: (a) selecting a T7 RNA polymerase in which the amino acid at position 520 of SEQ ID NO: 1 is substituted for purification; and (b) obtaining the T7 RNA polymerase.

3. A T7 RNA polymerase, characterized in that The T7 RNA polymerase comprises an amino acid substitution at position 520 of SEQ ID NO:

1.

4. The method or T7 RNA polymerase according to any one of claims 1 to 3, characterized in that The amino acid at position 520 of SEQ ID NO: 1 is substituted with G520Q.

5. The method or T7 RNA polymerase according to claim 4, characterized in that The T7 RNA polymerase has an altered transcription efficiency relative to the T7 RNA polymerase shown in SEQ ID NO:

1.

6. The method or T7 RNA polymerase according to claim 4, characterized in that The T7 RNA polymerase further comprises an amino acid substitution at any one or more of the following positions: at positions 108, 205, 206, 312, 333, 362, 388, 446, 534, 567, 584, 724, 832, 843, 849, 863 and 877 of SEQ ID NO: 1; Optionally, the T7 RNA polymerase further comprises a combination of any one or more of the following mutations: E108L, H205S, K206P, Y312D, K333P, M362P, D388E, L446F, L534V, V567P, A584K, A724P, M832F, A843Q, F849V, A863P and E877R; Optionally, the T7 RNA polymerase further comprises the following mutation combinations: E108L, H205S, K206P, Y312D, K333P, M362P, D388E, L446F, L534V, V567P, A584K, A724P, M832F, A843Q, F849V, A863P and E877R.

7. A conjugate, characterized in that: The invention comprises the T7 RNA polymerase according to any one of claims 1 to 6 and a marker for purification.

8. An isolated nucleic acid, vector or host cell, characterized in that The isolated nucleic acid encodes the T7 RNA polymerase according to any one of claims 1 to 6, the vector contains the isolated nucleic acid, and the host cell contains the vector.

9. A kit, characterized in that: It comprises the T7 RNA polymerase according to any one of claims 1 to 6.

10. Use of the T7 RNA polymerase according to any one of claims 1 to 6 in RNA synthesis.

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