MMLV reverse transcriptase mutant and application thereof

Abstract

The invention discloses an MMLV reverse transcriptase mutant and application thereof, the MMLV reverse transcriptase mutant is subjected to amino acid site modification on the basis of an amino acid sequence of a wild type MMLV reverse transcriptase as shown in SEQ ID NO: 1, and the modified amino acid site comprises at least one of D200, T246, G358, W388, L435, V444, D524 and D583. Compared with a wild type, the MMLV reverse transcriptase mutant prepared by the invention has the advantages of high yield, high thermal stability and high sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an MMLV reverse transcriptase mutant and application thereof, and aims to promote the progress of RNA research and related technologies. Background Art

[0002] Reverse transcriptases (RTs) are a class of enzymes that can synthesize DNA using RNA as a template and are widely used in molecular biology and medical research. The discovery and development of RTs has enabled rapid advancement of RNA-related research and has become a key tool for synthesizing cDNA, performing gene expression analysis, and achieving gene cloning. Reverse transcriptases are widely used in various diagnostic techniques, including cDNA cloning, reverse transcriptase-PCR quantification, microarray analysis, and rapid amplification of cDNA ends (RACE), which have played an important role in gene function research, disease diagnosis, and new drug development.

[0003] Among many reverse transcriptases, MMLV (murine leukemia virus reverse transcriptase) is a single-subunit enzyme with strong activity and stability, so it has received widespread attention in the past few decades. Compared with other retroviral reverse transcriptases, the advantage of MMLV is that its structure is relatively simple and easy to produce and purify. In addition, the reaction conditions of MMLV are relatively mild, which is suitable for use in a variety of experimental environments. Although MMLV has good performance, its stability at high temperature is still a limiting factor.

[0004] Mutation strategy is an effective method to improve the performance of MMLV reverse transcriptase. Researchers improve the binding stability of the enzyme to the template by mutating the template interaction region or fusing it with other DNA binding protein domains. The thermostable MMLV mutants screened in this way can be reverse transcribed under high temperature conditions, thereby achieving higher amplification efficiency. This progress can not only improve the stability of the RNA template during reverse transcription, but also effectively reduce the non-specific amplification phenomenon that may occur during the reaction.

[0005] In practical applications, thermostable MMLV reverse transcriptase mutants can be widely used in gene expression analysis, RNA sequencing, and RNA virus detection. Especially in early disease diagnosis and pathogen detection, fast and accurate cDNA synthesis steps are essential for subsequent PCR amplification and analysis. With the help of these mutants, researchers can conduct experiments over a wider temperature range to ensure the efficiency and reliability of the reaction. Therefore, there is a broad market demand for MMLV reverse transcriptase mutants that are resistant to higher temperatures. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a MMLV reverse transcriptase mutant.

[0007] The present invention also provides a nucleic acid molecule encoding the above MMLV reverse transcriptase mutant.

[0008] The present invention also provides biological materials related to the above nucleic acid molecules.

[0009] The present invention also provides the application of the MMLV reverse transcriptase mutant, nucleic acid molecule and biological material.

[0010] The invention also provides a product.

[0011] The present invention also provides a method for preparing the MMLV reverse transcriptase mutant.

[0012] According to a first aspect of the present invention, a MMLV reverse transcriptase mutant is provided, wherein the amino acid sites are modified based on the amino acid sequence of the wild-type MMLV reverse transcriptase as shown in SEQ ID NO: 1, and the modified amino acid sites include at least one of D200, T246, G358, W388, L435, V444, D524 and D583.

[0013] In some embodiments of the invention, the modified amino acid sites include at least two, at least three, at least four, at least five, at least six, at least seven or at least eight of D200, T246, G358, W388, L435, V444, D524 and D583.

[0014] In some embodiments of the present invention, the modification of the amino acid site comprises amino acid mutation.

[0015] In some embodiments of the present invention, the amino acid mutation of D200 is D200N or D200Y; and / or, the amino acid mutation of T246 is T246H; and / or, the amino acid mutation of G358 is G358A; and / or, the amino acid mutation of W388 is W388T or W388F; and / or, the amino acid mutation of L435 is L435G or L435K; and / or, the amino acid mutation of V444 is V444L or V444I; and / or, the amino acid mutation of D524 is D524A; and / or, the amino acid mutation of D583 is D583N.

[0016] In some embodiments of the present invention, in the MMLV reverse transcriptase mutant, the mutated amino acid sites are D200, T246, W388, L435, V444, D524, and D583, specifically D200Y, T246H, W388T, L435K, V444I, D524A, and D583N.

[0017] In some embodiments of the present invention, in the MMLV reverse transcriptase mutant, the mutated amino acid sites are D200, G358, W388, L435, and V444, specifically D200N, G358A, W388T, L435K, and V444I.

[0018] In some embodiments of the present invention, in the MMLV reverse transcriptase mutant, the mutated amino acid positions are D200, T246, G358, W388, L435, V444, D524, and D583, specifically D200N, T246H, G358A, W388T, L435G, V444L, D524A, and D583N.

[0019] In some embodiments of the present invention, in the MMLV reverse transcriptase mutant, the mutated amino acid sites are D200, T246, W388, L435, V444, and D524, specifically D200Y, T246H, W388F, L435K, V444I, and D524A.

[0020] In some embodiments of the present invention, in the MMLV reverse transcriptase mutant, the mutated amino acid sites are D200, T246, G358, W388, and V444, specifically D200Y, T246H, G358A, W388F, and V444L.

[0021] In some embodiments of the present invention, in the MMLV reverse transcriptase mutant, the mutated amino acid sites are D200, T246, G358, W388, L435, V444, and D524, specifically D200N, T246H, G358A, W388F, L435G, V444L, and D524A.

[0022] In some embodiments of the present invention, the thermal stability and / or specific enzyme activity of the MMLV reverse transcriptase mutant is higher than that of the wild-type MMLV reverse transcriptase.

[0023] In some embodiments of the present invention, the thermal stability refers to the activity at a temperature not lower than 55° C. For example, it may be 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C. or 65° C.

[0024] In some embodiments of the present invention, the thermal stability refers to activity at a temperature not lower than 65°C.

[0025] In some embodiments of the present invention, the MMLV reverse transcriptase mutant can achieve a better reverse transcription effect than the wild-type MMLV reverse transcriptase at ≤65°C.

[0026] According to a second aspect of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the above-mentioned MMLV reverse transcriptase mutant.

[0027] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is: (1) a nucleotide sequence as shown in any one of SEQ ID NOs: 3-8; or, (2) A synonymous codon sequence of the MMLV reverse transcriptase mutant that is the same as the nucleotide sequence described in (1).

[0028] According to a third aspect of the present invention, a biological material related to the nucleic acid molecule is provided, wherein the biological material comprises at least one of a1) to a7): a1) an expression cassette comprising the above nucleic acid molecule; a2) a vector comprising the above nucleic acid molecule; a3) a vector comprising the expression cassette described in a1); a4) a transgenic cell line comprising the above nucleic acid molecule; a5) a transgenic cell line comprising the expression cassette described in a1); a6) a transgenic cell line comprising the vector described in a2); a7) A transgenic cell line comprising the vector described in a3).

[0029] In some embodiments of the present invention, the mother vector used to construct the vector of the present invention is not limited, and any traditional vector used for prokaryotic or eukaryotic transformation can be used, for example, it can be a PET-32a vector.

[0030] In some embodiments of the invention, the transgenic cell line comprises prokaryotic cells or eukaryotic cells.

[0031] According to a fourth aspect of the present invention, an enzyme preparation is provided, comprising the above-mentioned MMLV reverse transcriptase mutant.

[0032] According to a fifth aspect of the present invention, applications of the above-mentioned MMLV reverse transcriptase mutants, nucleic acid molecules, biomaterials and enzyme preparations are provided.

[0033] In some embodiments of the present invention, the application is the application of the above-mentioned MMLV reverse transcriptase mutant or enzyme preparation in the field of detecting RNA samples or reverse transcription reactions.

[0034] In some embodiments of the present invention, the application is the application of the above-mentioned MMLV reverse transcriptase mutant, nucleic acid molecule, biological material or enzyme preparation in the preparation of a product.

[0035] In some embodiments of the present invention, the product comprises at least one of a reagent, a detection plate, a test kit, and a detection chip.

[0036] In some embodiments of the present invention, the reagent, detection plate, detection chip or kit has at least one of the functions b1) to b3): b1) Detect RNA samples; b2) reverse transcription of RNA to synthesize cDNA; b3) preparing RT-PCR detection reagents or RT-qPCR detection reagents.

[0037] According to a sixth aspect of the present invention, a product is provided, wherein the product comprises the above-mentioned MMLV reverse transcriptase mutant.

[0038] In some embodiments of the present invention, the product comprises at least one of a reagent, a detection plate, a test kit, and a detection chip.

[0039] In some embodiments of the present invention, the reagent, detection plate, detection chip or kit has at least one of the functions b1) to b3): b1) Detect RNA samples; b2) reverse transcription of RNA to synthesize cDNA; b3) preparing RT-PCR detection reagents or RT-qPCR detection reagents.

[0040] In some embodiments of the present invention, the kit is selected from a reverse transcription reaction kit, a RT-PCR amplification kit, a RT-qPCR kit, a cDNA library construction kit, a cDNA end rapid amplification kit and an RNA sequencing kit.

[0041] In some embodiments of the present invention, the reverse transcription reaction kit further comprises: at least one of a reverse transcription reaction buffer, dNTPs, an RNase inhibitor and a reverse transcription reaction primer.

[0042] In some embodiments of the present invention, the RT-PCR amplification kit further comprises: at least one of PCR water, RT-PCR amplification buffer, dNTPs, DNA polymerase and PCR amplification primers.

[0043] In some embodiments of the present invention, the RT-qPCR kit further comprises: at least one of PCR water, RT-qPCR amplification buffer, dNTPs, DNA polymerase, qPCR amplification primers and probes.

[0044] In some embodiments of the present invention, the cDNA library construction kit further comprises: a linker.

[0045] According to a sixth aspect of the present invention, a method for preparing the above-mentioned MMLV reverse transcriptase mutant is provided, and the preparation method comprises the following steps: obtaining it by expressing it through the above-mentioned transgenic cell line.

[0046] In some embodiments of the present invention, the method specifically comprises the following steps: (1) transferring the recombinant vector containing the above-mentioned nucleic acid molecule into competent cells and culturing them, and selecting single clone cells for screening; (2) Extracting the expression vector of the screened positive clone, transferring it into the host cell, inoculating it into LB medium for culture, and obtaining seed solution; (3) taking the obtained seed liquid and inoculating it into LB medium for culturing to obtain bacterial liquid; (4) Add the inducer IPTG to the obtained bacterial solution to a final concentration of 0.8-1 mmol / L. After inducing expression, collect the bacteria by centrifugation. (5) adding lysis buffer to the obtained bacterial cells for resuspending, crushing the bacterial cells under high pressure, centrifuging to obtain the supernatant, and filtering with a 0.22-0.5 μm microporous filter membrane; (6) subjecting the filtered liquid phase to nickel ion affinity chromatography, detecting the eluted solution by polyacrylamide electrophoresis, and collecting samples containing the target protein; (7) The sample containing the target protein in step (6) is subjected to cation exchange chromatography, and the elution solution is detected by polyacrylamide gel electrophoresis. The sample containing the target protein is collected and dialyzed into a preservation solution to obtain the MMLV reverse transcriptase mutant.

[0047] In some embodiments of the present invention, the LB culture medium in step (2) and step (3) both contain 80-120 μg / mL ampicillin.

[0048] In some embodiments of the present invention, the culture conditions in step (2) are 20-30° C. and 150-200 rpm shaking culture.

[0049] In some embodiments of the present invention, the culture conditions in step (3) are: 20-30°C, 150-200 rpm shaking culture until OD600 is 0.6-0.8.

[0050] In some embodiments of the present invention, the seed solution in step (3) is inoculated into LB medium at a volume ratio of 1:(80-120).

[0051] In some embodiments of the present invention, the induction conditions in step (4) are 20-30° C. and the induction time is 4-6 hours.

[0052] In some embodiments of the present invention, the centrifugation conditions in step (4) are: centrifugation at 4000-5000 rpm at 2-6°C for 15-25 min.

[0053] In some embodiments of the present invention, the amount of lysis buffer in step (5) is 8-10 mL per gram of bacterial cells; In some embodiments of the present invention, the centrifugation condition in step (5) is 2-6°C and 10000-14000 rpm for 25-35 min; In some embodiments of the present invention, the components of the lysis buffer in step (5) include: 45-55 mM Tris-HCl, 280-320 mM NaCl, and 0.1%-0.2% Triton X-100.

[0054] In some embodiments of the present invention, the components of the binding buffer used for the nickel ion affinity chromatography in step (6) contain: 45-55 mM Tris-HCl, 280-320 mM NaCl, 15-25 mM Imidazole, and 2%-6% Glycerol.

[0055] In some embodiments of the present invention, the components of the elution buffer used for nickel ion affinity chromatography contain: 45-55 mM Tris-HCl, 280-320 mM NaCl, 200-300 mM Imidazole, and 2%-6% Glycerol.

[0056] In some embodiments of the present invention, the components of the binding buffer used in the cation exchange chromatography contain: 15-25 mM Tris-HCl, 190-220 mM NaCl, 2%-6% (V / V) Glycerol, 0.5-2 mM DTT, and 0.5-2 mM EDTA.

[0057] In some embodiments of the present invention, the components of the gradient elution buffer used in cation exchange chromatography contain: 15-25 mM Tris-HCl, 550-650 mM NaCl, 2%-6% (V / V) Glycerol, 0.5-2 mM DTT, and 0.5-2 mM EDTA.

[0058] In some embodiments of the present invention, the components of the preservation solution contain: 15-25 mM Tris-HCl, 550-650 mM NaCl, 0.5-2 mM DTT, 40-60% Glycerol, and 0.5-2 mM EDTA.

[0059] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the MMLV reverse transcriptase mutant prepared by the present invention is rationally designed by using software that predicts 3D structure and interaction with templates to obtain numerous mutation sites, and the mutation sites are combined and screened to obtain the MMLV reverse transcriptase mutant, which has the advantages of high yield and high thermal stability compared to the wild type, and can be used for reverse transcription on complex RNA templates. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The SDS-PAGE image of the purified mutant and wild type in Example 1 of the present invention; Figure 2 It is a statistical graph of the Tm values ​​of MMLV reverse transcriptase mutants and wild-type MMLV; Figure 3 It is a statistical graph of the enzyme activity of MMLV reverse transcriptase mutants and wild-type MMLV; Figure 4 The figure is the electrophoresis result of RT-PCR of MMLV reverse transcriptase mutant and wild-type MMLV reverse transcriptase under different template concentrations and 65°C condition inversion in Example 3 of the present invention, wherein M is marker GL 10000; 1 is wild-type MMLV reverse transcriptase, 2 is MMLV reverse transcriptase mutant Mut1, 3 is MMLV reverse transcriptase mutant Mut2, 4 is MMLV reverse transcriptase mutant Mut3, 5 is MMLV reverse transcriptase mutant Mut4, 6 is MMLV reverse transcriptase mutant Mut5, and 7 is MMLV reverse transcriptase mutant Mut6. DETAILED DESCRIPTION

[0061] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0062] In the description of the present invention, each amino acid substitution is represented by a triplet: letter-number-letter; wherein the number represents the position of the mutated amino acid, the letter before the number corresponds to the amino acid involved in the mutation, and the letter after the number represents the amino acid used to replace the amino acid before the number.

[0063] Example 1 Expression, purification and identification of MMLV reverse transcriptase mutants 1. Design of MMLV reverse transcriptase mutants: Based on artificial intelligence machine learning and rational design, many mutation sites of MMLV reverse transcriptase mutants were predicted. The mutation sites were combined and screened to obtain MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6. The mutant sequences were obtained based on the sites of the wild-type MMLV reverse transcriptase (amino acid sequence as shown in SEQ ID NO: 1).

[0064] Mutants Mut1, Mut2, Mut3, Mut4, Mut5, Mut6, and Mut7 are all obtained by mutation of the wild-type MMLV reverse transcriptase having an amino acid sequence as shown in SEQ ID NO: 1. The mutations are as follows: Mutant Mut1: D200Y, T246H, W388T, L435K, V444I, D524A, D583N; Mutant Mut2: D200N, G358A, W388T, L435K, V444I; Mutant Mut3: D200N, T246H, G358A, W388T, L435G, V444L, D524A, D583N; Mutant Mut4: D200Y, T246H, W388F, L435K, V444I, D524A; Mutant Mut5: D200Y, T246H, G358A, W388F, V444L; Mutant Mut6: D200N, T246H, G358A, W388F, L435G, V444L, D524A; Mutants Mut7: D200W, W388E.

[0065] 2. Synthesis of MMLV reverse transcriptase mutants (1) Recombinant expression vectors (the original vector is PET-32a) containing a nucleotide sequence encoding a wild-type MMLV reverse transcriptase (as shown in SEQ ID NO: 2) and a nucleotide sequence encoding a mutant MMLV reverse transcriptase (the sequences of the mutants Mut1 to Mut7 are shown in SEQ ID NO: 3 to 9, respectively) were synthesized by whole gene synthesis.

[0066] (2) Transform the obtained recombinant expression vector into host cells E. coli After culturing in BL21 (DE3), a single colony was picked and inoculated into LB liquid medium (containing 100 μg / mL ampicillin) and cultured until 0D 600 The concentration of IPTG was 0.6-0.8, and IPTG was added to a final concentration of 1.0 mmol / L. The cells were induced at 25°C for 5 h, and the cells were collected by centrifugation at 4°C and 4200 rpm. Lysis buffer was added and the cells were ultrasonically disrupted. The expression of the target protein was detected by SDS-PAGE electrophoresis. The test results are shown in Figure 1 The results showed that the target protein could be expressed efficiently, and the sequence was verified to be correct.

[0067] (3) The recombinant bacteria that were verified in step (2) to be able to express the MMLV reverse transcriptase mutant were inoculated into 100 mL of LB medium containing 100 μg / mL of ampicillin, and placed in a shaker at 25°C for overnight shaking culture to obtain a seed solution.

[0068] (4) The seed solution obtained from the overnight culture was inoculated into 2 L of LB medium containing 100 μg / mL ampicillin at a volume ratio of 1:100, and cultured in a shaker at 25°C until the OD 600 is 0.6-0.8; add IPTG to a final concentration of 1.0mmol / L, and continue to induce with shaking at 25℃ for 5h; collect the induced bacteria by centrifugation and weigh them, record the wet weight of the bacteria, and store them at -20℃.

[0069] (5) Take the cells frozen at -20℃ after induction of expression, add 10mL lysis buffer (50mM Tris-HCl, 300mM NaCl, 0.15% Triton X-100, pH7.8) per gram of cells according to the recorded wet weight of the cells, and lyse the cells with a high-pressure crusher. Centrifuge the lysed cells at 12000rpm for 30min at 4℃. Centrifuge the supernatant at 12000rpm for 30min at 4℃, filter with a 0.22μm microporous filter, take the supernatant to a 200mL sterile beaker, and purify it using a Ni affinity chromatography column.

[0070] (6) Ni affinity chromatography column purification: The supernatant filtered through a 0.22 μm microporous filter was loaded onto a chromatography column equilibrated with buffer A (50 mM Tris-HCl, 300 mM NaCl, 20 mM Imidazole, 5% Glycerol, pH 7.8). After loading, the column was first rinsed with equilibrated buffer A, and then gradient eluted with buffer B (50 mM Tris-HCl, 300 mM NaCl, 250 mM Imidazole, 5% Glycerol, pH 7.8) at a rate of 0% to 100%. The eluted fractions were tested by SDS-PAGE protein electrophoresis. The eluate was collected according to the test results and dialyzed into buffer C (20 mM Tris-HCl, 200 mM NaCl, 5% (V / V) Glycerol, 1 mM DTT, 1 mM EDTA, pH 7.8) for later use.

[0071] (7) Purification by cation exchange chromatography: The dialysate sample obtained by purification by Ni affinity chromatography column was loaded onto the chromatography column equilibrated with buffer C. After loading, the column was first rinsed with equilibrated solution C, and then gradient eluted with buffer D (20mM Tris-HCl, 600mM NaCl, 1mM DTT, 5% (V / V) Glycerol, 1mM EDTA, pH7.8) from 0% to 100%. The eluted fractions were subjected to SDS-PAGE protein electrophoresis. The eluate was collected according to the test results, dialyzed with a 10 kDa dialysis bag, and then stored in a storage solution (20mM Tris-HCl, 600mM NaCl, 1mM DTT, 50% (V / V) Glycerol, 1mM EDTA, pH7.8) for later use. The concentration of the purified mutant was determined by the Coomassie Brilliant Blue method.

[0072] According to the measured concentration, the yields of MMLV reverse transcriptase WT and MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6 were calculated, and the volume of the purified MMLV reverse transcriptase was multiplied by the concentration to obtain the total yield of the MMLV reverse transcriptase.

[0073] Calculation and statistical results: The total production of wild-type MMLV reverse transcriptase was about 4.67 mg, and the total production of MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, Mut6, and Mut7 were approximately 7.69 mg, 18.85 mg, 13.50 mg, 12.06 mg, 6.71 mg, 9.89 mg, and 8.26 mg, respectively, which were all higher than those of the wild type.

[0074] Example 2 Detection of Tm values ​​of MMLV reverse transcriptase mutants and wild-type MMLV Differential Scanning Fluorimetry (DSF) is a method for evaluating the thermal stability of proteins by slowly heating the sample on a fluorescent quantitative PCR instrument and detecting the amount of fluorescent dye combined with the protein with a changed structure during the heating process. Through the DSF experiment, MMLV reverse transcriptases with high thermal stability can be screened out in high throughput. In this embodiment, the Tm values ​​of MMLV reverse transcriptase mutants and wild-type MMLV reverse transcriptase are detected by differential scanning fluorescence. The specific method is as follows: (1) Dilute Test 1 and Test 2 to 100 μM and mix them in a 1:1 ratio to obtain a primer-template complex; Test 1: 5'-TGGAATCAGGTGTCGCACTCTG-3' (SEQ ID NO:10); Test 2: 5'-AACAGAGUGCGACACCUGAUUCCAU-3' (SEQ ID NO: 11).

[0075] (2) After diluting the MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, Mut6, Mut7 and MMLV reverse transcriptase WT prepared in Example 1 to 500 ng / μL, the mixture was mixed with the primer template complex at a ratio of 1:1 to obtain a protein sample; (3) The fluorescent dye Sypro Orange was diluted to 20× using dimethyl sulfoxide (DMSO) to prepare the DSF reaction system, which was then placed in a real-time fluorescence quantitative PCR instrument. The excitation and emission wavelengths were adjusted to the corresponding wavelengths of the fluorescent dye, and the temperature was increased to obtain a curve of the change in fluorescence intensity with temperature. The Tm value of the protein sample was calculated using software.

[0076] 20 μL DSF reaction system: 16 μL DSF reaction buffer with a final concentration of 1 ×, 2 μL protein sample with a concentration of 250 ng / μL, and 2 μL 20× fluorescent dye Sypro Orange.

[0077] DSF reaction program: 4℃ 2min; then enter the temperature rising program: the temperature change range is generally from 25℃ to 99℃, 0.05℃ / 10s, and the fluorescence intensity is measured once; select the X1M3 channel, put it into the real-time fluorescence quantitative PCR instrument, and start the temperature rising reaction.

[0078] According to the Tm value obtained by software analysis, the curve was drawn. The Tm values ​​of MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6 were 66.22℃, 65.36℃, 64.38℃, 62.70℃, 65.36℃, and 66.55℃, respectively, which were all improved compared with MMLV reverse transcriptase WT (58.11℃). This shows that the thermal stability of MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6 has been improved. However, the Tm value of MMLV reverse transcriptase mutant Mut7 was only 57.17℃, which was worse than MMLV reverse transcriptase WT, and no subsequent performance test was performed.

[0079] Example 3 Detection of Enzyme Activity of MMLV Reverse Transcriptase Mutants and Wild-type MMLV Reverse Transcriptase In this example, the enzyme activity of the MMLV reverse transcriptase mutant obtained in Example 1 was detected. The specific method is as follows: (1) The purified MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6 were used as the experimental group test samples, and the commercial wild-type MMLV reverse transcriptase (purchased from Hunan Aikerui Biotechnology Co., Ltd.) was used as the control group test sample, and a 5-fold gradient dilution was performed.

[0080] Then, 0.4 μg of MS2 RNA was used as a template for reverse transcription and quantitative qPCR. After the quantitative qPCR was completed, a standard curve of the Ct and the logarithm of the enzyme activity concentration of each test sample was prepared, and the logarithm of the enzyme activity of the test sample was calculated. After solving the logarithm, the enzyme activity at different dilution multiples was calculated, and the average value was taken to obtain the reverse transcriptase activity. The specific operations of the reverse transcription reaction and quantitative qPCR reaction are as follows: (2) Use 0.4 μg of MS2 RNA as a template for reverse transcription to obtain cDNA, which can be placed at -20°C for later use.

[0081] 20μL reverse transcription system: final concentration of 1×RTase Reaction Buffer Mix II, final concentration of 0.1μmo1 / L Test 3, final concentration of 1U / μL RNase Inhibitor, 0.4μg MS2 RNA, 5μL gradient diluted MMLV enzyme, add water to make up to 20μL.

[0082] Test 3: 5'-TTCAGCGAACTTCTTGTAA-3' (SEQ ID NO:12); Test 4: 5'-CATCCGTAGCCTTATTGG-3' (SEQ ID NO: 13).

[0083] The reverse transcription reaction program was 37°C, 30 sec; 85°C, 5 sec; 4°C, hold.

[0084] (3) Perform qPCR reaction using the cDNA synthesized by reverse transcription in step (2) as a template (N=2).

[0085] 25 μL qPCR reaction system: final concentration is 1× SYBR Green Pro Taq HS Premix, final concentration is 0.2 μmo1 / L Test 3, final concentration is 0.2 μmo1 / L Test 4, and the balance is water.

[0086] qPCR reaction program: 95℃ 30 sec; 95℃ 5 sec, 60℃ 30sec, 40 cycles. Export the Ct value data obtained by the qPCR instrument, and make standard curves of the Ct of the control and test samples and the logarithm of the enzyme activity concentration. The six concentration points are required to be linearly related and the correlation coefficient R 2 ≥0.99, Ct value must be within 13-30; the slope ratio of the standard curve of the test sample and the control sample is 1±0.03, take 5 dilution times of the test sample that conforms to the standard curve of the reference substance, calculate the logarithm of the enzyme activity of the test sample, calculate the enzyme activity at different dilution times after solving the logarithm, and then take the average value to obtain the enzyme activity of the corresponding test sample.

[0087] (4) Calculation of specific enzyme activity of MMLV reverse transcriptase: The enzyme activities of MMLV reverse transcriptase WT and MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6 calculated in step (3) were divided by the concentration of the MMLV reverse transcriptase purified in Example 1 to calculate the specific enzyme activity of the MMLV reverse transcriptase.

[0088] The results of the statistical enzyme activity were calculated as follows: Figure 3 The specific enzyme activities of MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6 were 2.83 U / ng, 1.40 U / ng, 1.61 U / ng, 3.66 U / ng, 3.78 U / ng, and 1.67 U / ng, respectively, which were significantly increased compared with the wild type (0.54 U / ng).

[0089] Example 4 RT-qPCR effect detection of MMLV reverse transcriptase mutants and wild-type MMLV reverse transcriptase at 65°C In this example, the thermostable MMLV reverse transcriptase mutant obtained in Example 1 was diluted to 500 ng / μL, and RT-qPCR detection was performed at 65°C with different template concentrations (1 μg, 100 ng, 10 ng). The specific method is as follows: The MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, Mut6 and MMLV reverse transcriptase WT prepared in Example 1 were diluted to 500 ng / μL, and 1 μg, 100 ng, and 10 ng of mouse heart RNA were used as templates at 65°C for reverse transcription reaction. The obtained cDNA was amplified by PCR, and the obtained products were detected by electrophoresis using 1% agarose gel. The specific process is as follows: (1) Prepare the RNA template pre-reaction annealing system (dNTP Mix with a final concentration of 1 mmol / L, Oligo dT Primer with a final concentration of 0.25 μmol / L (from Hunan Aikerui Biotechnology Co., Ltd.), mouse heart RNA (1 μg, 100 ng or 10 ng), add water to 10 μL), and set the annealing reaction program to 65℃ for 5 min; 4℃ hold. Place in a PCR instrument for pre-reaction annealing reaction. After the reaction is completed, use it as the annealing system reaction solution for the subsequent reverse transcription reaction.

[0090] (2) Prepare the reverse transcription system (5× RTase Reaction Buffer, 20 U / μL MMLV reverse transcriptase (the MMLV reverse transcriptase mutants Mut1-Mut6 and MMLV reverse transcriptase WT prepared in Example 1, respectively), 1 U / μL RNase Inhibitor, and 10 μL of the annealing system reaction solution obtained in step (1)). Set the reverse transcription reaction program (65°C for 30 min; 70°C for 15 min; 4°C hold), place in a PCR instrument for reverse transcription reaction, and after the reaction is completed, use it as a cDNA template for PCR amplification immediately.

[0091] (3) Prepare the PCR amplification system (Exp Taq Master Mix with a final concentration of 1×, Test 5 with a final concentration of 1μmo1 / L, Test 6 with a final concentration of 1μmo1 / L, 2μL cDNA template, make up to 50μL with water). Set the PCR reaction program (94℃1min; 94℃ 30sec; 56℃ 30sec; 72℃ 4min, 30 cycles; 72℃ 10min; 4℃, hold). Use a PCR instrument for amplification, take 5μL of the amplified product, and perform electrophoresis on 1% agarose gel to detect the results.

[0092] Test 5: 5'-CTCCACTGTTTATGTCTATGTTCG -3' (SEQ ID NO: 14); Test 6: 5'-TCCAGAGTTTTCAGCTCTTCTGAT-3' (SEQ ID NO: 15).

[0093] The experimental results are as follows Figure 4 The results showed that MMLV reverse transcriptase mutants Mut1-Mut6 could be reverse transcribed to obtain cDNA products at 65℃ and template amount of 1μg or 100ng; among them, MMLV reverse transcriptase mutants Mut1, Mut2, Mut4 and Mut5 could also be reverse transcribed to obtain cDNA products at 65℃ and template amount of 10ng, and the amount of PCR amplification products was significantly higher than that of the wild type. The sensitivity of MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5 and Mut6 was also improved.

[0094] In summary, compared with the wild type, the MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6 of the present invention have better protein yield, Tm value, and reverse transcription performance at 65° C., thereby enabling reverse transcription of complex RNA templates.

[0095] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A MMLV reverse transcriptase mutant, characterized in that: The amino acid sites are modified based on the amino acid sequence of the wild-type MMLV reverse transcriptase as shown in SEQ ID NO: 1, and the modified amino acid sites include at least one of D200, T246, G358, W388, L435, V444, D524 and D583.

2. The MMLV reverse transcriptase mutant according to claim 1, characterized in that The modification of the amino acid site is an amino acid mutation, and the sites of the amino acid mutation in the MMLV reverse transcriptase mutant are D200Y, T246H, W388T, L435K, V444I, D524A, D583N; or, D200N, G358A, W388T, L435K, V444I; or, D200N, T246H, G358A, W388 T, L435G, V444L, D524A, D583N; or, D200Y, T246H, W388F, L435K, V444I, D524A; or, D200Y, T246H, G358A, W388F, V444L; or, D200N, T246H, G358A, W388F, L435G, V444L, D524A.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the MMLV reverse transcriptase mutant according to any one of claims 1-2.

4. The biological material related to the nucleic acid molecule according to claim 3, characterized in that: The biological material comprises at least one of a1) to a7): a1) an expression cassette comprising the nucleic acid molecule according to claim 3; a2) a vector comprising the nucleic acid molecule according to claim 3; a3) a vector comprising the expression cassette described in a1); a4) a transgenic cell line comprising the nucleic acid molecule according to claim 3; a5) a transgenic cell line comprising the expression cassette described in a1); a6) a transgenic cell line comprising the vector described in a2); a7) A transgenic cell line comprising the vector described in a3).

5. An enzyme preparation, characterized in that The invention comprises the MMLV reverse transcriptase mutant according to any one of claims 1 to 2.

6. Use of the MMLV reverse transcriptase mutant according to any one of claims 1 to 2 or the enzyme preparation according to claim 5 in the field of detecting RNA samples or reverse transcription reactions.

7. Use of at least one of the MMLV reverse transcriptase mutant according to any one of claims 1 to 2, the nucleic acid molecule according to claim 3, the biomaterial according to claim 4, and the enzyme preparation according to claim 5 in the preparation of a product.

8. The use according to claim 7, characterized in that: The product comprises at least one of a reagent, a detection plate, a test kit, and a detection chip.

9. The use according to claim 8, characterized in that: The reagent, detection plate, detection chip or kit has at least one of the functions b1) to b3): b1) Detect RNA samples; b2) reverse transcription of RNA to synthesize cDNA; b3) preparing RT-PCR detection reagents or RT-qPCR detection reagents.

10. A method for preparing the MMLV reverse transcriptase mutant according to claim 1 or 2, characterized in that: The following steps are involved: Obtained by expression of the transgenic cell line described in claim 4.

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