A MMLV reverse transcriptase mutant and its application

Abstract

The present invention discloses a MMLV reverse transcriptase mutant and its application. The MMLV reverse transcriptase mutant is based on the amino acid sequence of the wild-type MMLV reverse transcriptase shown in SEQ ID NO:1, with amino acid sites modified. The modified amino acid sites include at least one of D200, T246, G358, W388, L435, V444, D524, and D583. Compared to the wild-type, the MMLV reverse transcriptase mutant prepared by the present invention has the advantages of high yield, high thermal stability, and high sensitivity.

Description

Technical Field

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

[0002] Reverse transcriptases (RTs) are enzymes that synthesize DNA using RNA as a template and are widely used in molecular biology and medical research. The discovery and development of RTs has rapidly advanced RNA-related research, making them key tools for cDNA synthesis, gene expression analysis, and 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). These techniques play a vital role in gene function research, disease diagnosis, and new drug development.

[0003] Among the numerous reverse transcriptases, MMLV (murine leukemia virus reverse transcriptase), a single-subunit enzyme with strong activity and stability, has garnered extensive attention over the past few decades. Compared to other retroviral reverse transcriptases, MMLV offers advantages in its relatively simple structure, making it easy to produce and purify. Furthermore, MMLV operates under relatively mild reaction conditions, making it suitable for use in a variety of experimental settings. Despite its promising performance, MMLV's stability at high temperatures remains a limiting factor.

[0004] Mutation strategies are an effective approach to improving the performance of MMLV reverse transcriptase. Researchers have improved the stability of enzyme-template binding by mutating the template-interacting region or fusing it with other DNA-binding protein domains. The resulting thermostable MMLV mutants are capable of reverse transcription at high temperatures, resulting in higher amplification efficiency. This advance not only improves the stability of the RNA template during reverse transcription but also effectively reduces the potential for nonspecific amplification 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. In particular, rapid and accurate cDNA synthesis is crucial for subsequent PCR amplification and analysis in early disease diagnosis and pathogen detection. These mutants allow researchers to conduct experiments across a wider temperature range, ensuring efficient and reliable reactions. Therefore, there is a significant 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-mentioned 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 applications of the MMLV reverse transcriptase mutant, nucleic acid molecule and biological material.

[0010] The present 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 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 present invention, the modified amino acid positions 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 positions 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 positions 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 positions 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 positions 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 positions 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 activity at a temperature not lower than 55°C. For example, it can 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 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, which 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:

[0028] (1) a nucleotide sequence as shown in any one of SEQ ID NOs: 3-8; or

[0029] (2) A synonymous codon sequence of the MMLV reverse transcriptase mutant that is the same as the nucleotide sequence described in (1).

[0030] 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):

[0031] a1) an expression cassette comprising the above nucleic acid molecule;

[0032] a2) a vector comprising the above nucleic acid molecule;

[0033] a3) a vector comprising the expression cassette described in a1);

[0034] a4) a transgenic cell line comprising the above nucleic acid molecule;

[0035] a5) a transgenic cell line comprising the expression cassette described in a1);

[0036] a6) a transgenic cell line comprising the vector described in a2);

[0037] a7) A transgenic cell line comprising the vector described in a3).

[0038] In some embodiments of the present invention, the parent 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, the PET-32a vector.

[0039] In some embodiments of the present invention, the transgenic cell line comprises prokaryotic cells or eukaryotic cells.

[0040] According to a fourth aspect of the present invention, an enzyme preparation is provided, comprising the aforementioned MMLV reverse transcriptase mutant.

[0041] According to a fifth aspect of the present invention, applications of the aforementioned MMLV reverse transcriptase mutants, nucleic acid molecules, biomaterials, and enzyme preparations are provided.

[0042] In some embodiments of the present invention, the application is the application of the aforementioned MMLV reverse transcriptase mutant or enzyme preparation in the field of detecting RNA samples or reverse transcription reactions.

[0043] In some embodiments of the present invention, the application is the application of the aforementioned MMLV reverse transcriptase mutant, nucleic acid molecule, biological material or enzyme preparation in the preparation of a product.

[0044] 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.

[0045] 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):

[0046] b1) Detecting RNA samples;

[0047] b2) reverse transcription of RNA into cDNA;

[0048] b3) preparing RT-PCR detection reagents or RT-qPCR detection reagents.

[0049] According to a sixth aspect of the present invention, a product is provided, comprising the aforementioned MMLV reverse transcriptase mutant.

[0050] 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.

[0051] 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):

[0052] b1) Detecting RNA samples;

[0053] b2) reverse transcription of RNA into cDNA;

[0054] b3) preparing RT-PCR detection reagents or RT-qPCR detection reagents.

[0055] In some embodiments of the present invention, the kit is selected from one of a reverse transcription reaction kit, an RT-PCR amplification kit, an RT-qPCR kit, a cDNA library construction kit, a cDNA end rapid amplification kit and an RNA sequencing kit.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] In some embodiments of the present invention, the cDNA library construction kit further comprises: a linker.

[0060] According to a sixth aspect of the present invention, a method for preparing the aforementioned MMLV reverse transcriptase mutant is provided, the method comprising the following steps: obtaining the mutant by expressing it in the aforementioned transgenic cell line.

[0061] In some embodiments of the present invention, the method specifically comprises the following steps:

[0062] (1) Transfecting the recombinant vector containing the above-mentioned nucleic acid molecule into competent cells and culturing them, and picking single clones for screening;

[0063] (2) Extract the expression vector of the screened positive clone, transfer it into the host cell, inoculate it into LB medium for culture, and obtain seed liquid;

[0064] (3) The obtained seed solution was inoculated into LB medium for cultivation to obtain bacterial solution;

[0065] (4) Add the inducer IPTG to the resulting bacterial solution to a final concentration of 0.8-1 mmol / L. After inducing expression, collect the bacteria by centrifugation.

[0066] (5) Add lysis buffer to the obtained bacterial cells for resuspending, crush the bacterial cells under high pressure, centrifuge to obtain the supernatant, and filter using a 0.22-0.5 μm microporous membrane;

[0067] (6) The filtered liquid phase is subjected to nickel ion affinity chromatography, and the eluted solution is detected by polyacrylamide electrophoresis to collect the sample containing the target protein;

[0068] (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.

[0069] In some embodiments of the present invention, the LB culture medium in step (2) and step (3) both contain 80-120 μg / mL ampicillin.

[0070] In some embodiments of the present invention, the culture conditions in step (2) are 20-30° C. and 150-200 rpm shaking culture.

[0071] 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.

[0072] In some embodiments of the present invention, the seed solution in step (3) is inoculated into LB culture medium at a volume ratio of 1:(80-120).

[0073] In some embodiments of the present invention, the induction conditions in step (4) are 20-30° C. and the induction time is 4-6 h.

[0074] 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.

[0075] In some embodiments of the present invention, the amount of lysis buffer in step (5) is 8-10 mL per gram of bacterial cells;

[0076] In some embodiments of the present invention, the centrifugation condition in step (5) is 2-6°C at a speed of 10000-14000 rpm for 25-35 min;

[0077] In some embodiments of the present invention, the components of the lysis buffer in step (5) contain: 45-55 mM Tris-HCl, 280-320 mM NaCl, and 0.1%-0.2% Triton X-100.

[0078] In some embodiments of the present invention, the components of the binding buffer used for 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.

[0079] In some embodiments of the present invention, the elution buffer used in nickel ion affinity chromatography contains: 45-55 mM Tris-HCl, 280-320 mM NaCl, 200-300 mM Imidazole, and 2%-6% Glycerol.

[0080] In some embodiments of the present invention, the binding buffer used in the cation exchange chromatography contains: 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.

[0081] In some embodiments of the present invention, the gradient elution buffer used in cation exchange chromatography contains: 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.

[0082] 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.

[0083] According to some embodiments of the present invention, there are at least the following beneficial effects: the MMLV reverse transcriptase mutants prepared by the present invention are rationally designed using software that predicts 3D structure and interaction with the template, obtaining numerous mutation sites. The MMLV reverse transcriptase mutants obtained by combining the mutation sites and screening have 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

[0084] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0085] Figure 1 is an SDS-PAGE image of the purified mutant and wild type in Example 1 of the present invention;

[0086] Figure 2 Statistical graph of the Tm values of MMLV reverse transcriptase mutants and wild-type MMLV;

[0087] Figure 3 Statistical graph showing the relative enzyme activities of MMLV reverse transcriptase mutants and wild-type MMLV;

[0088] Figure 4This figure shows the electrophoresis results of RT-PCR of MMLV reverse transcriptase mutants and wild-type MMLV reverse transcriptase under different template concentrations and 65°C inversion conditions 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

[0089] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described 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.

[0090] In the description of the present invention, each amino acid substitution is represented by a triplet: letter-number-letter; wherein the number indicates 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 indicates the amino acid used to replace the amino acid before the number.

[0091] Example 1 Expression, purification and identification of MMLV reverse transcriptase mutants

[0092] 1. Design of MMLV reverse transcriptase mutants: Using artificial intelligence machine learning and rational design, numerous mutation sites of MMLV reverse transcriptase mutants were predicted. These mutation sites were combined and screened to obtain the MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6. The mutant sequences were based on sites of the wild-type MMLV reverse transcriptase (amino acid sequence shown in SEQ ID NO: 1).

[0093] Mutants Mut1, Mut2, Mut3, Mut4, Mut5, Mut6, and Mut7 were obtained by mutation of the wild-type MMLV reverse transcriptase with the amino acid sequence shown in SEQ ID NO: 1. The mutations are as follows:

[0094] Mutant Mut1: D200Y, T246H, W388T, L435K, V444I, D524A, D583N;

[0095] Mutant Mut2: D200N, G358A, W388T, L435K, V444I;

[0096] Mutant Mut3: D200N, T246H, G358A, W388T, L435G, V444L, D524A, D583N;

[0097] Mutant Mut4: D200Y, T246H, W388F, L435K, V444I, D524A;

[0098] Mutant Mut5: D200Y, T246H, G358A, W388F, V444L;

[0099] Mutant Mut6: D200N, T246H, G358A, W388F, L435G, V444L, D524A;

[0100] Mutants Mut7: D200W, W388E.

[0101] 2. Synthesis of MMLV reverse transcriptase mutants

[0102] (1) Recombinant expression vectors (the original vector is PET-32a) containing the nucleotide sequence encoding the wild-type MMLV reverse transcriptase (as shown in SEQ ID NO: 2) and the nucleotide sequence encoding the mutant MMLV reverse transcriptase (the sequences of the mutants Mut1-Mut7 are shown in SEQ ID NO: 3-9, respectively) were synthesized by whole gene synthesis.

[0103] (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 0 D 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 4200 rpm at 4°C. Lysis buffer was added and the cells were ultrasonically disrupted. The expression of the target protein was detected by SDS-PAGE electrophoresis. Figure 1 The results showed that the target protein could be expressed efficiently, and the sequence was verified to be correct.

[0104] (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 shaking incubator at 25°C for overnight shaking culture to obtain seed liquid.

[0105] (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 shaking incubator at 25°C until the OD 600The pH value was 0.6-0.8; IPTG was added to a final concentration of 1.0 mmol / L, and the induction was continued at 25°C for 5 h; the induced bacteria were collected by centrifugation and weighed, the wet weight of the bacteria was recorded, and the bacteria were stored at -20°C.

[0106] (5) After induction of expression, cells frozen at -20°C were taken. Based on the recorded wet weight of cells, 10 mL of lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 0.15% Triton X-100, pH 7.8) was added per gram of cells to resuspend the cells. The cells were lysed using a high-pressure disruptor. The lysed cells were centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was centrifuged at 12,000 rpm for 30 min at 4°C, filtered through a 0.22 μm microporous membrane, and transferred to a 200 mL sterile beaker for purification using a Ni affinity chromatography column.

[0107] (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 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) from 0% to 100%. The eluted fractions were analyzed by SDS-PAGE protein electrophoresis. The eluate was collected based on 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.

[0108] (7) Purification by cation exchange chromatography: The dialyzate sample obtained by purification on the Ni affinity chromatography column was loaded onto a chromatography column equilibrated with buffer C. After loading, the column was first rinsed with equilibrated buffer C, and then gradient eluted with buffer D (20 mM Tris-HCl, 600 mM NaCl, 1 mM DTT, 5% (V / V) Glycerol, 1 mM EDTA, pH 7.8) from 0% to 100%. The eluted fractions were subjected to SDS-PAGE protein electrophoresis. The eluate was collected according to the test results, dialyzed using a 10 kDa dialysis bag, and then stored in a storage solution (20 mM Tris-HCl, 600 mM NaCl, 1 mM DTT, 50% (V / V) Glycerol, 1 mM EDTA, pH 7.8) for later use. The concentration of the purified mutant was determined using the Coomassie Brilliant Blue method.

[0109] Based on the measured concentrations, the yields of MMLV reverse transcriptase WT and MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6 were calculated. The volume of the purified MMLV reverse transcriptase was multiplied by the concentration to obtain the total yield of the MMLV reverse transcriptase.

[0110] Statistical results showed that the total yield of wild-type MMLV reverse transcriptase was approximately 4.67 mg, and the total yields 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.

[0111] Example 2 Detection of Tm Values of MMLV Reverse Transcriptase Mutants and Wild-Type MMLV

[0112] Differential Scanning Fluorimetry (DSF) is a method for evaluating protein thermal stability by slowly heating a sample in a fluorescent quantitative PCR instrument and measuring the amount of fluorescent dye bound to structurally altered proteins during the heating process. DSF experiments allow for high-throughput screening of MMLV reverse transcriptases with high thermal stability. In this example, the Tm values of MMLV reverse transcriptase mutants and wild-type MMLV reverse transcriptase were determined using differential scanning fluorimetry. The specific method is as follows:

[0113] (1) Dilute Test 1 and Test 2 to 100 μM and mix them in a 1:1 ratio to obtain a primer-template complex;

[0114] Test 1: 5'-TGGAATCAGGTGTCGCACTCTG-3' (SEQ ID NO:10);

[0115] Test 2: 5'-AACAGAGUGCGACACCUGAUUCCAU-3' (SEQ ID NO: 11).

[0116] (2) The MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, Mut6, and Mut7 prepared in Example 1 and the MMLV reverse transcriptase WT were diluted to 500 ng / μL and mixed with the primer-template complex at a ratio of 1:1 to obtain a protein sample;

[0117] (3) The fluorescent dye Sypro Orange was diluted to 20× with 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.

[0118] 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.

[0119] DSF reaction program: 4℃ 2min; then enter the heating program: the temperature change range is generally from 25℃ to 99℃, 0.05℃ / 10s, and the fluorescence intensity is measured once; select X1M3 channel, place it in the real-time fluorescence quantitative PCR instrument, and start the heating reaction.

[0120] The Tm values obtained by software analysis were plotted. The Tm values of the MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6 were 66.22°C, 65.36°C, 64.38°C, 62.70°C, 65.36°C, and 66.55°C, respectively, all significantly higher than those of the MMLV reverse transcriptase WT (58.11°C). This indicates that the thermal stability of the MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6 has been improved. However, the Tm value of the MMLV reverse transcriptase mutant Mut7 was only 57.17°C, which was lower than that of the MMLV reverse transcriptase WT, and was not further tested.

[0121] Example 3 Detection of Enzyme Activity of MMLV Reverse Transcriptase Mutants and Wild-Type MMLV Reverse Transcriptase

[0122] In this example, the enzyme activity of the MMLV reverse transcriptase mutant obtained in Example 1 was detected. The specific method is as follows:

[0123] (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 Bioengineering Co., Ltd.) was used as the control group test sample, and a 5-fold gradient dilution was performed.

[0124] Reverse transcription and quantitative qPCR were then performed using 0.4 μg of MS2 RNA as a template. After the quantitative qPCR was completed, a standard curve was constructed comparing the Ct of each test sample with the logarithm of the enzyme activity concentration. The logarithmic values of the enzyme activity were calculated. After logarithmization, the enzyme activity at different dilutions was calculated and the average value was taken to obtain the reverse transcriptase activity. The specific reverse transcription and quantitative qPCR reactions were performed as follows:

[0125] (2) Use 0.4 μg of MS2 RNA as a template for reverse transcription to obtain cDNA. After the reaction, it can be placed at -20℃ for future use.

[0126] 20 μL reverse transcription system: final concentration of 1×RTase Reaction Buffer Mix II, final concentration of 0.1 μmo1 / L Test 3, final concentration of 1 U / μL RNase Inhibitor, 0.4 μg of MS2 RNA, 5 μL of serially diluted MMLV enzyme, add water to make up to 20 μL.

[0127] Test 3: 5'-TTCAGCGAACTTCTTGTAA-3' (SEQ ID NO:12);

[0128] Test 4: 5'-CATCCGTAGCCTTATTGG-3' (SEQ ID NO: 13).

[0129] The reverse transcription reaction program was 37°C, 30 sec; 85°C, 5 sec; 4°C, hold.

[0130] (3) Perform qPCR reaction using the cDNA synthesized by reverse transcription in step (2) as a template (N=2).

[0131] 25 μL qPCR reaction system: final concentration of 1× SYBR Green Pro Taq HS Premix, final concentration of 0.2 μmo1 / L Test 3, final concentration of 0.2 μmo1 / L Test 4, and the balance is water.

[0132] qPCR reaction program: 95℃ 30 sec; 95℃ 5 sec, 60℃ 30 sec, 40 cycles. Export the Ct value data obtained by the qPCR instrument and create a standard curve of the Ct value of the control and test samples versus the logarithm of the enzyme activity concentration. The six concentration points must be linear 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 multiples 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 multiples after solving the logarithm, and then take the average value to obtain the enzyme activity of the corresponding test sample.

[0133] (4) Calculation of the 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.

[0134] Calculate the statistical enzyme activity results as follows Figure 3 The specific enzyme activities of the 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 higher than those of the wild type (0.54 U / ng).

[0135] Example 4 RT-qPCR performance test of MMLV reverse transcriptase mutants and wild-type MMLV reverse transcriptase at 65°C

[0136] In this example, the thermostable MMLV reverse transcriptase mutant obtained in Example 1 was diluted to 500 ng / μL and then subjected to RT-qPCR at 65°C with different template concentrations (1 μg, 100 ng, and 10 ng). The specific method is as follows:

[0137] The MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6 prepared in Example 1, as well as the MMLV reverse transcriptase WT, were diluted to 500 ng / μL and reverse transcribed at 65°C using 1 μg, 100 ng, and 10 ng of mouse heart RNA, respectively, as templates. The resulting cDNAs were amplified by PCR, and the resulting products were analyzed by electrophoresis on a 1% agarose gel. The specific process is as follows:

[0138] (1) Prepare the RNA template pre-reaction annealing system (dNTP Mix at a final concentration of 1 mmol / L, Oligo dT Primer (from Hunan Aikerui Bioengineering Co., Ltd.) at a final concentration of 0.25 μmol / L, mouse heart RNA (1 μg, 100 ng, or 10 ng), and make up to 10 μL with water). Set the annealing reaction program to 65°C for 5 min and 4°C hold. Place the sample in a PCR instrument for pre-reaction annealing. After the reaction is completed, use the annealing system reaction solution immediately in the subsequent reverse transcription reaction.

[0139] (2) Prepare the reverse transcription system (5× RTase Reaction Buffer, 20 U / μL MMLV reverse transcriptase (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 the reverse transcription reaction in a PCR instrument, and immediately use it as a cDNA template for PCR amplification after the reaction is completed.

[0140] (3) Prepare the PCR amplification system (Exp Taq Master Mix at a final concentration of 1×, Test 5 at a final concentration of 1 μmol / L, Test 6 at a final concentration of 1 μmol / L, 2 μL of cDNA template, and make up to 50 μL with water). Set the PCR reaction program (94°C for 1 min; 94°C for 30 sec; 56°C for 30 sec; 72°C for 4 min, 30 cycles; 72°C for 10 min; 4°C hold). Amplify using a PCR instrument, take 5 μL of the amplified product, and perform electrophoresis on a 1% agarose gel.

[0141] Test 5: 5'-CTCCACTGTTTATGTCTATGTTCG -3' (SEQ ID NO: 14);

[0142] Test 6: 5'-TCCAGAGTTTTCAGCTCTTCTGAT-3' (SEQ ID NO: 15).

[0143] The experimental results are as follows Figure 4The results showed that MMLV reverse transcriptase mutants Mut1-Mut6 could all be reverse transcribed to produce cDNA products at 65°C using 1 μg or 100 ng of template. Among them, MMLV reverse transcriptase mutants Mut1, Mut2, Mut4, and Mut5 could also produce cDNA products at 65°C using 10 ng of template, and the amount of PCR amplification product 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.

[0144] 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 yields, Tm values, and reverse transcription performance at 65°C, thereby enabling reverse transcription of complex RNA templates.

[0145] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a 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, wherein the modification of the amino acid sites is amino acid mutation, and the amino acid mutation sites in the MMLV reverse transcriptase mutant are D200Y, T246H, W388T, L435K, V444I, D524A, D583N; or, D200N, G358A, W388T, L435K, V444I; or, D20 0N, T246H, G358A, W388T, 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.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the MMLV reverse transcriptase mutant according to claim 1.

3. The biomaterial related to the nucleic acid molecule according to claim 2, 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 2; a2) a vector comprising the nucleic acid molecule according to claim 2; a3) a vector comprising the expression cassette described in a1); a4) a transgenic cell line comprising the nucleic acid molecule according to claim 2; 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).

4. An enzyme preparation, characterized in that The invention also comprises the MMLV reverse transcriptase mutant according to claim 1.

5. Use of the MMLV reverse transcriptase mutant according to claim 1 or the enzyme preparation according to claim 4 in the field of detecting RNA samples or reverse transcription reactions.

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

7. The use according to claim 6, characterized in that The product comprises at least one of a reagent, a detection plate, a test kit, and a detection chip.

8. The use according to claim 7, characterized in that The reagent, detection plate, detection chip or kit has at least one of the functions b1) to b3): b1) Detecting RNA samples; b2) reverse transcription of RNA into cDNA; b3) preparing RT-PCR detection reagents or RT-qPCR detection reagents.

9. A method for preparing the MMLV reverse transcriptase mutant according to claim 1, characterized in that: The following steps are involved: Obtained by expression through the transgenic cell line described in claim 3.

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