A high-temperature-resistant MMLV reverse transcriptase mutant and its application

Abstract

The present invention discloses a thermostable MMLV reverse transcriptase mutant and its application. The thermostable MMLV reverse transcriptase mutant is based on the amino acid sequence of the wild-type MMLV reverse transcriptase shown in SEQ ID NO:1, and amino acid mutations are performed at one or more of H126E, F199L, T246H, W388R, A404N, and D422E. The thermostable MMLV reverse transcriptase mutant prepared by the present invention has the advantages of both higher enzyme activity and higher thermal stability compared to the wild-type.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a high-temperature-resistant MMLV reverse transcriptase mutant and application thereof. Background Art

[0002] During the COVID-19 pandemic, reverse transcription and polymerase chain reaction (RT-PCR) is an excellent method for detecting SARS-CoV-2. The Moloney murine leukemia virus (MMLV) reverse transcriptase is the most commonly used cDNA synthesizer in molecular biology. The synthesized cDNA can be used as a substrate for downstream PCR and qPCR applications.

[0003] As an indispensable tool for RNA research and detection, MMLV reverse transcriptase has undergone numerous improvements to enhance various properties, the most important of which is thermal stability, which largely determines its performance in practical applications. Higher reaction temperatures, generally above 55°C, unravel the complex secondary structure of the RNA template, enabling the reverse transcriptase to read the sequence and allowing for smooth and continuous cDNA synthesis. Therefore, reverse transcriptases that can tolerate higher temperatures can achieve full-length cDNA synthesis with higher yields, thereby improving the reverse transcription of RNA into cDNA. Therefore, research on reverse transcriptases with improved thermal stability and amplification efficiency is urgently needed. Summary of the Invention

[0004] 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 thermostable MMLV reverse transcriptase mutant.

[0005] The present invention also provides a nucleic acid molecule encoding the above-mentioned high-temperature-resistant MMLV reverse transcriptase mutant.

[0006] The present invention also provides biological materials related to the above nucleic acid molecules.

[0007] The present invention also provides an enzyme preparation.

[0008] The present invention also provides applications of the high-temperature-resistant MMLV reverse transcriptase mutant, nucleic acid molecule and biological material.

[0009] The present invention also provides a product.

[0010] The present invention also provides a method for preparing the high-temperature-resistant MMLV reverse transcriptase mutant.

[0011] According to a first aspect of the present invention, a thermostable MMLV reverse transcriptase mutant is proposed, wherein amino acid mutations are performed on the wild-type MMLV reverse transcriptase amino acid sequence as shown in SEQ ID NO: 1, and the amino acid mutation sites include one or more of H126E, F199L, T246H, W388R, A404N, and D422E.

[0012] In some embodiments of the present invention, the high temperature is represented by a temperature > 55°C.

[0013] In some embodiments of the present invention, the high temperature is 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65°C.

[0014] In some embodiments of the present invention, the thermostable MMLV reverse transcriptase mutant can also achieve better reverse transcription effect than the wild type at ≤55°C.

[0015] In some embodiments of the present invention, the amino acid sequence of the thermostable MMLV reverse transcriptase mutant is shown in any one of SEQ ID NOs: 2-7.

[0016] According to a second aspect of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the above-mentioned thermostable MMLV reverse transcriptase mutant.

[0017] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is:

[0018] (1) the nucleotide sequence shown in any one of SEQ ID NOs: 9-14; or

[0019] (2) A synonymous codon sequence of the thermostable MMLV reverse transcriptase mutant encoded by the nucleotide sequence described in (1).

[0020] 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):

[0021] a1) an expression cassette comprising the above nucleic acid molecule;

[0022] a2) a vector comprising the above nucleic acid molecule;

[0023] a3) a vector comprising the expression cassette described in a1);

[0024] a4) a transgenic cell line comprising the above nucleic acid molecule;

[0025] a5) a transgenic cell line comprising the expression cassette described in a1);

[0026] a6) a transgenic cell line comprising the vector described in a2);

[0027] a7) A transgenic cell line comprising the vector described in a3).

[0028] According to a fourth aspect of the present invention, an enzyme preparation is provided, comprising the above-mentioned thermostable MMLV reverse transcriptase mutant.

[0029] According to a fifth aspect of the present invention, applications of the thermostable MMLV reverse transcriptase mutant, nucleic acid molecule, biomaterial, and enzyme preparation are provided, wherein the applications are applications in preparing products.

[0030] 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.

[0031] 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):

[0032] b1) Detecting RNA samples;

[0033] b2) reverse transcription of RNA into cDNA;

[0034] b3) preparing RT-PCR detection reagents or RT-qPCR detection reagents.

[0035] According to a sixth aspect of the present invention, a product is provided, comprising the above-mentioned thermostable MMLV reverse transcriptase mutant.

[0036] 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.

[0037] 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):

[0038] b1) Detecting RNA samples;

[0039] b2) reverse transcription of RNA into cDNA;

[0040] b3) preparing RT-PCR detection reagents or RT-qPCR detection reagents.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] In some embodiments of the present invention, the cDNA library construction kit further comprises: a linker.

[0046] According to a seventh aspect of the present invention, a method for preparing the above-mentioned thermostable MMLV reverse transcriptase mutant is provided, and the preparation method comprises the following steps: obtaining the mutant by expressing it in the above-mentioned transgenic cell line.

[0047] In some embodiments of the present invention, the method specifically comprises the following steps:

[0048] (1) transferring the recombinant vector containing the nucleic acid molecule into competent cells and culturing the cells, and selecting single clones for screening;

[0049] (2) extracting the expression vector of the screened positive clone, transferring it into host cells, inoculating it into culture medium and culturing it to obtain seed solution;

[0050] (3) inoculating the obtained seed solution into a culture medium for culturing to obtain a bacterial solution;

[0051] (4) Add the inducer IPTG to the resulting bacterial solution to a final concentration of 0.8-1 mmol / L. After induction of expression, the cells are collected by centrifugation.

[0052] (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 through a 0.22-0.5 μm microporous membrane;

[0053] (6) subjecting the filtered liquid phase to nickel ion affinity chromatography, detecting the eluted solution by polyacrylamide electrophoresis, and collecting a sample containing the target protein;

[0054] (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 thermostable MMLV reverse transcriptase mutant.

[0055] In some embodiments of the present invention, the culture medium is LB medium.

[0056] In some embodiments of the present invention, the culture medium in step (2) and step (3) both contain 80-120 μg / mL ampicillin.

[0057] In some embodiments of the present invention, the culture conditions in step (2) are 20-30° C. and 150-200 r / min shaking culture.

[0058] In some embodiments of the present invention, the culture conditions in step (3) are: 20-30° C., 150-200 r / min shaking culture until OD600 is 0.6-0.8.

[0059] 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).

[0060] 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.

[0061] 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.

[0062] In some embodiments of the present invention, the amount of lysis buffer in step (5) is 8 to 10 mL per gram of bacterial cells.

[0063] In some embodiments of the present invention, the centrifugation condition in step (5) is 2-6° C. and 10,000-14,000 rpm for 25-35 min.

[0064] 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.

[0065] In some embodiments of the present invention, the components of the binding buffer used in 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] In some embodiments of the present invention, the components of the preservation solution include: 15-25 mM Tris-HCl, 550-650 mM NaCl, 0.5-2 mM DTT, 40-60% Glycerol, and 0.5-2 mM EDTA.

[0070] According to some embodiments of the present invention, there are at least the following beneficial effects: the thermostable MMLV reverse transcriptase mutant prepared by the present invention is obtained by using machine learning and rational design to obtain numerous mutation sites, and the mutation sites are combined and screened to obtain the thermostable MMLV reverse transcriptase mutant. Compared with the wild type, the thermostable MMLV reverse transcriptase mutant has the advantages of higher enzyme activity and higher thermal stability, and can be used for reverse transcription of complex RNA templates. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0072] Figure 1 This is an SDS-PAGE image of the purified mutant and wild type in Example 1 of the present invention, wherein M is a marker; 1 is a wild-type MMLV reverse transcriptase, 2 is an MMLV reverse transcriptase mutant Mut1, 3 is an MMLV reverse transcriptase mutant Mut2, 4 is an MMLV reverse transcriptase mutant Mut3, 5 is an MMLV reverse transcriptase mutant Mut4, 6 is an MMLV reverse transcriptase mutant Mut5, and 7 is an MMLV reverse transcriptase mutant Mut6;

[0073] Figure 2This is a statistical graph of the relative enzyme activities of the thermostable MMLV reverse transcriptase mutant and the wild-type MMLV reverse transcriptase in Example 2 of the present invention, wherein 1 is the wild-type MMLV reverse transcriptase, 2 is the MMLV reverse transcriptase mutant Mut1, 3 is the MMLV reverse transcriptase mutant Mut2, 4 is the MMLV reverse transcriptase mutant Mut3, 5 is the MMLV reverse transcriptase mutant Mut4, 6 is the MMLV reverse transcriptase mutant Mut5, and 7 is the MMLV reverse transcriptase mutant Mut6;

[0074] Figure 3 is a curve graph of the Tm values ​​of MMLV reverse transcriptase mutants and wild-type MMLV, wherein 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;

[0075] Figure 4 Figure 3 is a curve showing the changes in the ΔCt values ​​of the high-temperature-resistant MMLV reverse transcriptase mutants compared with the ΔCt values ​​of the high-temperature-treated and untreated MMLV reverse transcriptase wild-type. 1 is the MMLV reverse transcriptase mutant Mut1, 2 is the MMLV reverse transcriptase mutant Mut2, 3 is the MMLV reverse transcriptase mutant Mut3, 4 is the MMLV reverse transcriptase mutant Mut4, 5 is the MMLV reverse transcriptase mutant Mut5, and 6 is the MMLV reverse transcriptase mutant Mut6. DETAILED DESCRIPTION

[0076] 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.

[0077] Example 1 Transformation, expression, purification and identification of thermostable MMLV reverse transcriptase mutants

[0078] 1. Design of thermostable MMLV reverse transcriptase mutants

[0079] Using artificial intelligence machine learning and rational design, numerous mutation sites of MMLV reverse transcriptase mutants were predicted. These mutation sites were combined to screen and identify the thermostable MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6. The mutant sequences were derived based on sites of the wild-type MMLV reverse transcriptase (amino acid sequence shown in SEQ ID NO: 1).

[0080] Wild-type MMLV reverse transcriptase:

[0081] (SEQ ID NO: 1).

[0082] The amino acid sequences of the mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6 are shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively.

[0083] Amino acid sequence of Mut1:

[0084] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDI E PTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTL L DEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGP R RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI(SEQID NO:2).

[0085] Amino acid sequence of Mut2:

[0086] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTP

[0087] VSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLR

[0088] EVNKRVEDI E PTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRD

[0089] PEMGISGQLTWTRLPQGFKNSPTL L DEALHRDLADFRIQHPDLILLQYVDDLLLAATS

[0090] ELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETV

[0091] MGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQ

[0092] EIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGP R RRPVAYLSKKLDPVA

[0093] N GWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTH

[0094] YQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDA

[0095] DHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAE

[0096] GKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI(SEQ ID NO:3)。

[0097] Mut3 amino acid sequence:

[0098] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDI E PTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTL L DEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGP R RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTK E AGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI (SEQ ID NO:4).

[0099] Mut4 amino acid sequence:

[0100] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDI EPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTL L DEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ H LGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGP R RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI(SEQ ID NO:5).

[0101] Amino acid sequence of Mut5:

[0102] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDI E PTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTL LDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGP R RRPVAYLSKKLDPVA N GWPPCLRMVAAIAVLTK E AGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI(SEQ ID NO:6).

[0103] Amino acid sequence of Mut6:

[0104] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDI E PTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTL L DEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQ HLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGP R RRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTK E AGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAK ALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI(SEQID NO:7).

[0105] Among them, the underline “—” indicates the mutation site.

[0106] 2. Synthesis of thermostable MMLV reverse transcriptase mutants

[0107] (1) Recombinant expression vectors (the original vector was PET-32a) containing nucleotide sequences encoding MMLV reverse transcriptase mutants (the sequences of mutants Mut1-6 are shown in SEQ ID NOs: 9-14, respectively) and nucleotide sequences encoding MMLV reverse transcriptase wild-type (shown in SEQ ID NO: 8) were synthesized by whole gene synthesis.

[0108] Nucleotide sequence encoding wild-type MMLV reverse transcriptase:

[0109]

[0110] Nucleotide sequence encoding MMLV reverse transcriptase mutant Mut 1:

[0111] ATGCTGAACATCGAAGACGAACACCGCCTGCACGAAACCTCCAAAGAACCGGATGTCAGCCTGGGTAGCACCTGGCTGTCTGACTTTCCTCAGGCGTGGGCCGAAACCGGCGGTATGGGCCTGGCAGTTCGTCAGGCGCCGCTGATCATTCCGCTGAAGGCGACGTCTACCCCGGTTTCCATCAAACAGTACCCGATGTCCCAGGAAGCCCGCCTGGGCATCAAACCGCACATCCAGCGTCTGCTGGATCAGGGCATCCTGGTGCCGTGCCAAAGCCCATGGAATACTCCACTGCTGCCGGTGAAAAAACCGGGTACCAACGACTATCGTCCTGTCCAGGACCTGCGCGAAGTGAACAAACGTGTGGAAGACATT GAA CCGACCGTTCCGAACCCGTACAATCTGCTGTCCGGCCTGCCGCCGTCCCACCAATGGTATACGGTTCTGGATCTGAAAGACGCGTTTTTTTGTCTGCGTCTGCACCCGACCTCTCAGCCACTGTTCGCGTTTGAGTGGCGCGACCCGGAGATGGGCATCTCTGGTCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTCAAAAACTCCCCGACCCTG CTGGATGAGGCTCTGCATCGCGATCTGGCGGACTTCCGTATCCAGCACCCGGACCTGATCCTGCTGCAGTATGTTGACGACCTGCTGCTGGCGGCAACTTCCGAACTGGATTGCCAACAAGGTACTCGCGCTCTGCTGCAGACCCTGGGCAACCTGGGCTATCGTGCCTCCGCCAAAAAAGCGCAGATTTGTCAGAAACAGGTGAAATACCTGGGCTACCTGCTGAAGGAAGGTCAGCGTTGGCTGACTGAAGCTCGTAAGGAAACCGTTATGGGTCAACCGACTCCGAAAACCCCGCGCCAGCTGCGTGAGTTCCTGGGTACTGCCGGTTTCTGCCGTCTGTGGATCCCAGGCTTCGCTGAAATGGCCGCGCCGCTGTATCCGCTGACTAAAACCGGTACTCTGTTCAACTGGGGTCCGGACCAACAGAAAGCTTATCAGGAAATCAAACAGGCTCTGCTGACTGCGCCGGCTCTGGGTCTGCCGGATCTGACTAAACCGTTCGAACTGTTCGTTGATGAAAAGCAGGGTTACGCAAAAGGCGTACTGACCCAGAAACTGGGTCCG CGTCGTCGTCCGGTCGCTTACCTGTCTAAGAAACTGGACCCAGTAGCAGCTGGTTGGCCGCCGTGCCTGCGTATGGTTGCAGCGATCGCTGTCCTGACCAAAGACGCGGGCAAACTGACCATGGGTCAGCCGCTGGTAATCCTGGCTCCGCATGCTGTGGAAGCTCTGGTAAAACAGCCTCCGGACCGTTGGCTGTCCAACGCACGCATGACCCATTATCAGGCGCTGCTGCTGGACACTGATCGCGTGCAATTCGGTCCTGTTGTTGCCCTGAACCCGGCTACCCTGCTGCCACTGCCGGAGGAGGGCCTGCAACACAACTGTCTGGACATCCTGGCGGAAGCTCACGGCACTCGTCCTGATCTGACTGACCAACCTCTGCCTGACGCGGATCACACCTGGTACACCGACGGTTCTAGCCTGCTGCAAGAAGGTCAACGCAAGGCGGGCGCAGCTGTTACCACTGAAACTGAAGTTATCTGGGCGAAAGCTCTGCCGGCTGGTACCTCTGCTCAGCGTGCTGAACTGATCGCTCTGACTCAGGCGCTGAAAATGGCAGAAGGCAAGAAACTGAACGTGTATACCGATTCTCGTTACGCTTTCGCCACCGCCCACATTCATGGTGAAATTTACCGTCGTCGTGGTCTGCTGACTAGCGAAGGCAAGGAGATTAAGAACAAGGATGAGATCCTGGCGCTGCTGAAAGCTCTGTTCCTGCCGAAACGCCTGTCCATCATCCACTGTCCAGGCCACCAGAAAGGTCACTCCGCCGAAGCACGTGGTAATCGCATGGCGGACCAGGCAGCTCGTAAAGCTGCAATCACTGAAACTCCGGATACCTCTACGCTGCTGATC(SEQ ID NO:9).

[0112] Nucleotide sequence encoding MMLV reverse transcriptase mutant Mut 2:

[0113] ATGCTGAACATCGAAGACGAACACCGCCTGCACGAAACCTCCAAAGAACCGGATGTCAGCCTGGGTAGCACCTGGCTGTCTGACTTTCCTCAGGCGTGGGCCGAAACCGGCGGTATGGGCCTGGCAGTTCGTCAGGCGCCGCTGATCATTCCGCTGAAGGCGACGTCTACCCCGGTTTCCATCAAACAGTACCCGATGTCCCAGGAAGCCCGCCTGGGCATCAAACCGCACATCCAGCGTCTGCTGGATCAGGGCATCCTGGTGCCGTGCCAAAGCCCATGGAATACTCCACTGCTGCCGGTGAAAAAACCGGGTACCAACGACTATCGTCCTGTCCAGGACCTGCGCGAAGTGAACAAACGTGTGGAAGACATT GAA CCGACCGTTCCGAACCCGTACAATCTGCTGTCCGGCCTGCCGCCGTCCCACCAATGGTATACGGTTCTGGATCTGAAAGACGCGTTTTTTTGTCTGCGTCTGCACCCGACCTCTCAGCCACTGTTCGCGTTTGAGTGGCGCGACCCGGAGATGGGCATCTCTGGTCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTCAAAAACTCCCCGACCCTG CTGGATGAGGCTCTGCATCGCGATCTGGCGGACTTCCGTATCCAGCACCCGGACCTGATCCTGCTGCAGTATGTTGACGACCTGCTGCTGGCGGCAACTTCCGAACTGGATTGCCAACAAGGTACTCGCGCTCTGCTGCAGACCCTGGGCAACCTGGGCTATCGTGCCTCCGCCAAAAAAGCGCAGATTTGTCAGAAACAGGTGAAATACCTGGGCTACCTGCTGAAGGAAGGTCAGCGTTGGCTGACTGAAGCTCGTAAGGAAACCGTTATGGGTCAACCGACTCCGAAAACCCCGCGCCAGCTGCGTGAGTTCCTGGGTACTGCCGGTTTCTGCCGTCTGTGGATCCCAGGCTTCGCTGAAATGGCCGCGCCGCTGTATCCGCTGACTAAAACCGGTACTCTGTTCAACTGGGGTCCGGACCAACAGAAAGCTTATCAGGAAATCAAACAGGCTCTGCTGACTGCGCCGGCTCTGGGTCTGCCGGATCTGACTAAACCGTTCGAACTGTTCGTTGATGAAAAGCAGGGTTACGCAAAAGGCGTACTGACCCAGAAACTGGGTCCG CGT CGTCGTCCGGTCGCTTACCTGTCTAAGAAACTGGACCCAGTAGCA AACGGTTGGCCGCCGTGCCTGCGTATGGTTGCAGCGATCGCTGTCCTGACCAAAGACGCGGGCAAACTGACCATGGGTCAGCCGCTGGTAATCCTGGCTCCGCATGCTGTGGAAGCTCTGGTAAAACAGCCTCCGGACCGTTGGCTGTCCAACGCACGCATGACCCATTATCAGGCGCTGCTGCTGGACACTGATCGCGTGCAATTCGGTCCTGTTGTTGCCCTGAACCCGGCTACCCTGCTGCCACTGCCGGAGGAGGGCCTGCAACACAACTGTCTGGACATCCTGGCGGAAGCTCACGGCACTCGTCCTGATCTGACTGACCAACCTCTGCCTGACGCGGATCACACCTGGTACACCGACGGTTCTAGCCTGCTGCAAGAAGGTCAACGCAAGGCGGGCGCAGCTGTTACCACTGAAACTGAAGTTATCTGGGCGAAAGCTCTGCCGGCTGGTACCTCTGCTCAGCGTGCTGAACTGATCGCTCTGACTCAGGCGCTGAAAATGGCAGAAGGCAAGAAACTGAACGTGTATACCGATTCTCGTTACGCTTTCGCCACCGCCCACATTCATGGTGAAATTTACCGTCGTCGTGGTCTGCTGACTAGCGAAGGCAAGGAGATTAAGAACAAGGATGAGATCCTGGCGCTGCTGAAAGCTCTGTTCCTGCCGAAACGCCTGTCCATCATCCACTGTCCAGGCCACCAGAAAGGTCACTCCGCCGAAGCACGTGGTAATCGCATGGCGGACCAGGCAGCTCGTAAAGCTGCAATCACTGAAACTCCGGATACCTCTACGCTGCTGATC(SEQ ID NO:10).

[0114] Nucleotide sequence encoding MMLV reverse transcriptase mutant Mut 3:

[0115] ATGCTGAACATCGAAGACGAACACCGCCTGCACGAAACCTCCAAAGAACCGGATGTCAGCCTGGGTAGCACCTGGCTGTCTGACTTTCCTCAGGCGTGGGCCGAAACCGGCGGTATGGGCCTGGCAGTTCGTCAGGCGCCGCTGATCATTCCGCTGAAGGCGACGTCTACCCCGGTTTCCATCAAACAGTACCCGATGTCCCAGGAAGCCCGCCTGGGCATCAAACCGCACATCCAGCGTCTGCTGGATCAGGGCATCCTGGTGCCGTGCCAAAGCCCATGGAATACTCCACTGCTGCCGGTGAAAAAACCGGGTACCAACGACTATCGTCCTGTCCAGGACCTGCGCGAAGTGAACAAACGTGTGGAAGACATT GAA CCGACCGTTCCGAACCCGTACAATCTGCTGTCCGGCCTGCCGCCGTCCCACCAATGGTATACGGTTCTGGATCTGAAAGACGCGTTTTTTTGTCTGCGTCTGCACCCGACCTCTCAGCCACTGTTCGCGTTTGAGTGGCGCGACCCGGAGATGGGCATCTCTGGTCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTCAAAAACTCCCCGACCCTG CTGGATGAGGCTCTGCATCGCGATCTGGCGGACTTCCGTATCCAGCACCCGGACCTGATCCTGCTGCAGTATGTTGACGACCTGCTGCTGGCGGCAACTTCCGAACTGGATTGCCAACAAGGTACTCGCGCTCTGCTGCAGACCCTGGGCAACCTGGGCTATCGTGCCTCCGCCAAAAAAGCGCAGATTTGTCAGAAACAGGTGAAATACCTGGGCTACCTGCTGAAGGAAGGTCAGCGTTGGCTGACTGAAGCTCGTAAGGAAACCGTTATGGGTCAACCGACTCCGAAAACCCCGCGCCAGCTGCGTGAGTTCCTGGGTACTGCCGGTTTCTGCCGTCTGTGGATCCCAGGCTTCGCTGAAATGGCCGCGCCGCTGTATCCGCTGACTAAAACCGGTACTCTGTTCAACTGGGGTCCGGACCAACAGAAAGCTTATCAGGAAATCAAACAGGCTCTGCTGACTGCGCCGGCTCTGGGTCTGCCGGATCTGACTAAACCGTTCGAACTGTTCGTTGATGAAAAGCAGGGTTACGCAAAAGGCGTACTGACCCAGAAACTGGGTCCG CGT CGTCGTCCGGTCGCTTACCTGTCTAAGAAACTGGACCCAGTAGCAGCTGGTTGGCCGCCGTGCCTGCGTATGGTTGCAGCGATCGCTGTCCTGACCAAA GAAGCGGGCAAACTGACCATGGGTCAGCCGCTGGTAATCCTGGCTCCGCATGCTGTGGAAGCTCTGGTAAAACAGCCTCCGGACCGTTGGCTGTCCAACGCACGCATGACCCATTATCAGGCGCTGCTGCTGGACACTGATCGCGTGCAATTCGGTCCTGTTGTTGCCCTGAACCCGGCTACCCTGCTGCCACTGCCGGAGGAGGGCCTGCAACACAACTGTCTGGACATCCTGGCGGAAGCTCACGGCACTCGTCCTGATCTGACTGACCAACCTCTGCCTGACGCGGATCACACCTGGTACACCGACGGTTCTAGCCTGCTGCAAGAAGGTCAACGCAAGGCGGGCGCAGCTGTTACCACTGAAACTGAAGTTATCTGGGCGAAAGCTCTGCCGGCTGGTACCTCTGCTCAGCGTGCTGAACTGATCGCTCTGACTCAGGCGCTGAAAATGGCAGAAGGCAAGAAACTGAACGTGTATACCGATTCTCGTTACGCTTTCGCCACCGCCCACATTCATGGTGAAATTTACCGTCGTCGTGGTCTGCTGACTAGCGAAGGCAAGGAGATTAAGAACAAGGATGAGATCCTGGCGCTGCTGAAAGCTCTGTTCCTGCCGAAACGCCTGTCCATCATCCACTGTCCAGGCCACCAGAAAGGTCACTCCGCCGAAGCACGTGGTAATCGCATGGCGGACCAGGCAGCTCGTAAAGCTGCAATCACTGAAACTCCGGATACCTCTACGCTGCTGATC(SEQ ID NO:11).

[0116] Nucleotide sequence encoding MMLV reverse transcriptase mutant Mut 4:

[0117] ATGCTGAACATCGAAGACGAACACCGCCTGCACGAAACCTCCAAAGAACCGGATGTCAGCCTGGGTAGCACCTGGCTGTCTGACTTTCCTCAGGCGTGGGCCGAAACCGGCGGTATGGGCCTGGCAGTTCGTCAGGCGCCGCTGATCATTCCGCTGAAGGCGACGTCTACCCCGGTTTCCATCAAACAGTACCCGATGTCCCAGGAAGCCCGCCTGGGCATCAAACCGCACATCCAGCGTCTGCTGGATCAGGGCATCCTGGTGCCGTGCCAAAGCCCATGGAATACTCCACTGCTGCCGGTGAAAAAACCGGGTACCAACGACTATCGTCCTGTCCAGGACCTGCGCGAAGTGAACAAACGTGTGGAAGACATT GAA CCGACCGTTCCGAACCCGTACAATCTGCTGTCCGGCCTGCCGCCGTCCCACCAATGGTATACGGTTCTGGATCTGAAAGACGCGTTTTTTTGTCTGCGTCTGCACCCGACCTCTCAGCCACTGTTCGCGTTTGAGTGGCGCGACCCGGAGATGGGCATCTCTGGTCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTCAAAAACTCCCCGACCCTG CTG GATGAGGCTCTGCATCGCGATCTGGCGGACTTCCGTATCCAGCACCCGGACCTGATCCTGCTGCAGTATGTTGACGACCTGCTGCTGGCGGCAACTTCCGAACTGGATTGCCAACAAGGTACTCGCGCTCTGCTGCAG CATCTGGGCAACCTGGGCTATCGTGCCTCCGCCAAAAAAGCGCAGATTTGTCAGAAACAGGTGAAATACCTGGGCTACCTGCTGAAGGAAGGTCAGCGTTGGCTGACTGAAGCTCGTAAGGAAACCGTTATGGGTCAACCGACTCCGAAAACCCCGCGCCAGCTGCGTGAGTTCCTGGGTACTGCCGGTTTCTGCCGTCTGTGGATCCCAGGCTTCGCTGAAATGGCCGCGCCGCTGTATCCGCTGACTAAAACCGGTACTCTGTTCAACTGGGGTCCGGACCAACAGAAAGCTTATCAGGAAATCAAACAGGCTCTGCTGACTGCGCCGGCTCTGGGTCTGCCGGATCTGACTAAACCGTTCGAACTGTTCGTTGATGAAAAGCAGGGTTACGCAAAAGGCGTACTGACCCAGAAACTGGGTCCG CGTCGTCGTCCGGTCGCTTACCTGTCTAAGAAACTGGACCCAGTAGCAGCTGGTTGGCCGCCGTGCCTGCGTATGGTTGCAGCGATCGCTGTCCTGACCAAAGACGCGGGCAAACTGACCATGGGTCAGCCGCTGGTAATCCTGGCTCCGCATGCTGTGGAAGCTCTGGTAAAACAGCCTCCGGACCGTTGGCTGTCCAACGCACGCATGACCCATTATCAGGCGCTGCTGCTGGACACTGATCGCGTGCAATTCGGTCCTGTTGTTGCCCTGAACCCGGCTACCCTGCTGCCACTGCCGGAGGAGGGCCTGCAACACAACTGTCTGGACATCCTGGCGGAAGCTCACGGCACTCGTCCTGATCTGACTGACCAACCTCTGCCTGACGCGGATCACACCTGGTACACCGACGGTTCTAGCCTGCTGCAAGAAGGTCAACGCAAGGCGGGCGCAGCTGTTACCACTGAAACTGAAGTTATCTGGGCGAAAGCTCTGCCGGCTGGTACCTCTGCTCAGCGTGCTGAACTGATCGCTCTGACTCAGGCGCTGAAAATGGCAGAAGGCAAGAAACTGAACGTGTATACCGATTCTCGTTACGCTTTCGCCACCGCCCACATTCATGGTGAAATTTACCGTCGTCGTGGTCTGCTGACTAGCGAAGGCAAGGAGATTAAGAACAAGGATGAGATCCTGGCGCTGCTGAAAGCTCTGTTCCTGCCGAAACGCCTGTCCATCATCCACTGTCCAGGCCACCAGAAAGGTCACTCCGCCGAAGCACGTGGTAATCGCATGGCGGACCAGGCAGCTCGTAAAGCTGCAATCACTGAAACTCCGGATACCTCTACGCTGCTGATC(SEQ ID NO:12).

[0118] Nucleotide sequence encoding MMLV reverse transcriptase mutant Mut 5:

[0119] ATGCTGAACATCGAAGACGAACACCGCCTGCACGAAACCTCCAAAGAACCGGATGTCAGCCTGGGTAGCACCTGGCTGTCTGACTTTCCTCAGGCGTGGGCCGAAACCGGCGGTATGGGCCTGGCAGTTCGTCAGGCGCCGCTGATCATTCCGCTGAAGGCGACGTCTACCCCGGTTTCCATCAAACAGTACCCGATGTCCCAGGAAGCCCGCCTGGGCATCAAACCGCACATCCAGCGTCTGCTGGATCAGGGCATCCTGGTGCCGTGCCAAAGCCCATGGAATACTCCACTGCTGCCGGTGAAAAAACCGGGTACCAACGACTATCGTCCTGTCCAGGACCTGCGCGAAGTGAACAAACGTGTGGAAGACATT GAA CCGACCGTTCCGAACCCGTACAATCTGCTGTCCGGCCTGCCGCCGTCCCACCAATGGTATACGGTTCTGGATCTGAAAGACGCGTTTTTTTGTCTGCGTCTGCACCCGACCTCTCAGCCACTGTTCGCGTTTGAGTGGCGCGACCCGGAGATGGGCATCTCTGGTCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTCAAAAACTCCCCGACCCTG CTGGATGAGGCTCTGCATCGCGATCTGGCGGACTTCCGTATCCAGCACCCGGACCTGATCCTGCTGCAGTATGTTGACGACCTGCTGCTGGCGGCAACTTCCGAACTGGATTGCCAACAAGGTACTCGCGCTCTGCTGCAGACCCTGGGCAACCTGGGCTATCGTGCCTCCGCCAAAAAAGCGCAGATTTGTCAGAAACAGGTGAAATACCTGGGCTACCTGCTGAAGGAAGGTCAGCGTTGGCTGACTGAAGCTCGTAAGGAAACCGTTATGGGTCAACCGACTCCGAAAACCCCGCGCCAGCTGCGTGAGTTCCTGGGTACTGCCGGTTTCTGCCGTCTGTGGATCCCAGGCTTCGCTGAAATGGCCGCGCCGCTGTATCCGCTGACTAAAACCGGTACTCTGTTCAACTGGGGTCCGGACCAACAGAAAGCTTATCAGGAAATCAAACAGGCTCTGCTGACTGCGCCGGCTCTGGGTCTGCCGGATCTGACTAAACCGTTCGAACTGTTCGTTGATGAAAAGCAGGGTTACGCAAAAGGCGTACTGACCCAGAAACTGGGTCCG CGT CGTCGTCCGGTCGCTTACCTGTCTAAGAAACTGGACCCAGTAGCA AAC GGTTGGCCGCCGTGCCTGCGTATGGTTGCAGCGATCGCTGTCCTGACCAAA GAAGCGGGCAAACTGACCATGGGTCAGCCGCTGGTAATCCTGGCTCCGCATGCTGTGGAAGCTCTGGTAAAACAGCCTCCGGACCGTTGGCTGTCCAACGCACGCATGACCCATTATCAGGCGCTGCTGCTGGACACTGATCGCGTGCAATTCGGTCCTGTTGTTGCCCTGAACCCGGCTACCCTGCTGCCACTGCCGGAGGAGGGCCTGCAACACAACTGTCTGGACATCCTGGCGGAAGCTCACGGCACTCGTCCTGATCTGACTGACCAACCTCTGCCTGACGCGGATCACACCTGGTACACCGACGGTTCTAGCCTGCTGCAAGAAGGTCAACGCAAGGCGGGCGCAGCTGTTACCACTGAAACTGAAGTTATCTGGGCGAAAGCTCTGCCGGCTGGTACCTCTGCTCAGCGTGCTGAACTGATCGCTCTGACTCAGGCGCTGAAAATGGCAGAAGGCAAGAAACTGAACGTGTATACCGATTCTCGTTACGCTTTCGCCACCGCCCACATTCATGGTGAAATTTACCGTCGTCGTGGTCTGCTGACTAGCGAAGGCAAGGAGATTAAGAACAAGGATGAGATCCTGGCGCTGCTGAAAGCTCTGTTCCTGCCGAAACGCCTGTCCATCATCCACTGTCCAGGCCACCAGAAAGGTCACTCCGCCGAAGCACGTGGTAATCGCATGGCGGACCAGGCAGCTCGTAAAGCTGCAATCACTGAAACTCCGGATACCTCTACGCTGCTGATC(SEQ ID NO:13).

[0120] Nucleotide sequence encoding MMLV reverse transcriptase mutant Mut 6:

[0121] ATGCTGAACATCGAAGACGAACACCGCCTGCACGAAACCTCCAAAGAACCGGATGTCAGCCTGGGTAGCACCTGGCTGTCTGACTTTCCTCAGGCGTGGGCCGAAACCGGCGGTATGGGCCTGGCAGTTCGTCAGGCGCCGCTGATCATTCCGCTGAAGGCGACGTCTACCCCGGTTTCCATCAAACAGTACCCGATGTCCCAGGAAGCCCGCCTGGGCATCAAACCGCACATCCAGCGTCTGCTGGATCAGGGCATCCTGGTGCCGTGCCAAAGCCCATGGAATACTCCACTGCTGCCGGTGAAAAAACCGGGTACCAACGACTATCGTCCTGTCCAGGACCTGCGCGAAGTGAACAAACGTGTGGAAGACATT GAA CCGACCGTTCCGAACCCGTACAATCTGCTGTCCGGCCTGCCGCCGTCCCACCAATGGTATACGGTTCTGGATCTGAAAGACGCGTTTTTTTGTCTGCGTCTGCACCCGACCTCTCAGCCACTGTTCGCGTTTGAGTGGCGCGACCCGGAGATGGGCATCTCTGGTCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTCAAAAACTCCCCGACCCTG CTG GATGAGGCTCTGCATCGCGATCTGGCGGACTTCCGTATCCAGCACCCGGACCTGATCCTGCTGCAGTATGTTGACGACCTGCTGCTGGCGGCAACTTCCGAACTGGATTGCCAACAAGGTACTCGCGCTCTGCTGCAG CACCTGGGCAACCTGGGCTATCGTGCCTCCGCCAAAAAAGCGCAGATTTGTCAGAAACAGGTGAAATACCTGGGCTACCTGCTGAAGGAAGGTCAGCGTTGGCTGACTGAAGCTCGTAAGGAAACCGTTATGGGTCAACCGACTCCGAAAACCCCGCGCCAGCTGCGTGAGTTCCTGGGTACTGCCGGTTTCTGCCGTCTGTGGATCCCAGGCTTCGCTGAAATGGCCGCGCCGCTGTATCCGCTGACTAAAACCGGTACTCTGTTCAACTGGGGTCCGGACCAACAGAAAGCTTATCAGGAAATCAAACAGGCTCTGCTGACTGCGCCGGCTCTGGGTCTGCCGGATCTGACTAAACCGTTCGAACTGTTCGTTGATGAAAAGCAGGGTTACGCAAAAGGCGTACTGACCCAGAAACTGGGTCCG CGT CGTCGTCCGGTCGCTTACCTGTCTAAGAAACTGGACCCAGTAGCAGCTGGTTGGCCGCCGTGCCTGCGTATGGTTGCAGCGATCGCTGTCCTGACCAAA GAAGCGGGCAAACTGACCATGGGTCAGCCGCTGGTAATCCTGGCTCCGCATGCTGTGGAAGCTCTGGTAAAACAGCCTCCGGACCGTTGGCTGTCCAACGCACGCATGACCCATTATCAGGCGCTGCTGCTGGACACTGATCGCGTGCAATTCGGTCCTGTTGTTGCCCTGAACCCGGCTACCCTGCTGCCACTGCCGGAGGAGGGCCTGCAACACAACTGTCTGGACATCCTGGCGGAAGCTCACGGCACTCGTCCTGATCTGACTGACCAACCTCTGCCTGACGCGGATCACACCTGGTACACCGACGGTTCTAGCCTGCTGCAAGAAGGTCAACGCAAGGCGGGCGCAGCTGTTACCACTGAAACTGAAGTTATCTGGGCGAAAGCTCTGCCGGCTGGTACCTCTGCTCAGCGTGCTGAACTGATCGCTCTGACTCAGGCGCTGAAAATGGCAGAAGGCAAGAAACTGAACGTGTATACCGATTCTCGTTACGCTTTCGCCACCGCCCACATTCATGGTGAAATTTACCGTCGTCGTGGTCTGCTGACTAGCGAAGGCAAGGAGATTAAGAACAAGGATGAGATCCTGGCGCTGCTGAAAGCTCTGTTCCTGCCGAAACGCCTGTCCATCATCCACTGTCCAGGCCACCAGAAAGGTCACTCCGCCGAAGCACGTGGTAATCGCATGGCGGACCAGGCAGCTCGTAAAGCTGCAATCACTGAAACTCCGGATACCTCTACGCTGCTGATC(SEQ ID NO:14).

[0122] Among them, the underlined “—” indicates the nucleotide sequence of the mutation site.

[0123] (2) The obtained recombinant expression vector was transformed into the host cell E. coli BL21 (DE3). After cultivation, a single colony was picked and inoculated into LB (containing 100 μg / mL ampicillin) liquid medium and cultured until the OD600 was 0.6-0.8. IPTG was added to a final concentration of 1.0 mmol / L and induced at 25°C for 5 h. 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 detection results were as follows: Figure 2 As shown, the results showed that the target protein could be expressed efficiently, and the sequence was verified to be correct by sequencing.

[0124] (3) The recombinant bacteria expressing the MMLV reverse transcriptase mutant obtained in step (2) were inoculated into 100 mL of LB medium containing 100 μg / mL of ampicillin, and the culture was placed in a shaker at 25° C. for overnight shaking to obtain a seed solution.

[0125] (4) The seed liquid 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 OD600 was 0.6-0.8; IPTG was added to a final concentration of 1.0 mmol / L, and the culture was induced by shaking 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.

[0126] (5) After induction of expression, cells were frozen at -20°C and resuspended in 10 mL of lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 0.15% Triton X-100, pH 7.8) per gram of cells, based on the recorded wet weight. The cells were then 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.

[0127] (6) Ni affinity chromatography column purification

[0128] Experimental reagents: The binding buffer was buffer A: 50 mM Tris-HCl, 300 mM NaCl, 20 mM Imidazole, 5% Glycerol, pH 7.8; the elution buffer was buffer B: 50 mM Tris-HCl, 300 mM NaCl, 250 mM Imidazole, 5% Glycerol, pH 7.8.

[0129] Experimental Method: The supernatant filtered through a 0.22 μm microporous membrane was loaded onto a chromatography column equilibrated with Buffer A. After loading, the column was rinsed with equilibration buffer A, followed by gradient elution from 0% to 100% with Buffer B. The eluted fractions were analyzed by SDS-PAGE protein electrophoresis. The eluate was collected based on the test results and dialyzed into Buffer C for later use.

[0130] (7) Cation exchange chromatography purification

[0131] Experimental reagents: Binding buffer is buffer C: 20mM Tris-HCl, 200mM NaCl, 5% (V / V) Glycerol, 1mM DTT, 1mM EDTA, pH7.8; elution buffer is buffer D: 20mM Tris-HCl, 600mM NaCl, 5% (V / V) Glycerol, 1mM DTT, 1mM EDTA, pH7.8; storage solution: 20mM Tris-HCl, 600mM NaCl, 1mM DTT, 50% Glycerol, 1mM EDTA, pH7.8.

[0132] Experimental Methods: The dialysate sample obtained by purification on a Ni affinity chromatography column was loaded onto a chromatography column equilibrated with buffer C. After loading, the column was rinsed with equilibrated buffer C, followed by gradient elution from 0% to 100% with buffer D. The eluted fractions were analyzed by SDS-PAGE protein electrophoresis. Based on the test results, the eluate was collected, dialyzed using a 10 kDa dialysis bag, and stored in storage buffer until further use. The concentration of the purified mutant was determined using the Coomassie Brilliant Blue method.

[0133] Example 2 Enzyme activity detection of thermostable MMLV reverse transcriptase mutants and wild type

[0134] In this example, the enzyme activity of the thermostable MMLV reverse transcriptase mutant obtained in Example 1 was detected. The specific method is as follows:

[0135] The high-temperature resistant MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, Mut6 and wild-type WT purified in Example 1 were assayed for reverse transcriptase activity according to the following method, specifically comprising: using the MMLV reverse transcriptase mutants prepared in Example 1 as the experimental group test sample, and the commercial wild-type MMLV reverse transcriptase (purchased from Hunan Aikerui Bioengineering Co., Ltd.) as the control group test sample, performing a 5-fold gradient dilution, and then performing reverse transcription and quantitative qPCR using 0.4 μg of MS2 RNA as a template. After the quantitative qPCR is completed, a standard curve of the Ct and the logarithm of the enzyme activity concentration of the control and test samples is prepared, the logarithm of the enzyme activity of the test sample is calculated, and the enzyme activity at different dilution multiples is calculated after delogging, and the average value is taken to obtain the reverse transcriptase activity. The specific operations of the reverse transcription reaction and the quantitative qPCR reaction are as follows:

[0136] Test 1: 5'-TTCAGCGAACTTCTTGTAA-3' (SEQ ID NO: 15);

[0137] Test 2: 5'-CATCCGTAGCCTTATTGG-3' (SEQ ID NO: 16).

[0138] (1) Using 0.4 μg of MS2 RNA as a template, reverse transcribe the cDNA using a gradient-diluted MMLV enzyme. The specific reverse transcription reaction system is as follows:

[0139] 20 μL reverse transcription system: final concentration of 1×RTase Reaction Buffer Mix II, final concentration of 0.1 μmo1 / L Test1, final concentration of 1 U / μL RNase Inhibitor, 0.4 μg of MS2 RNA, 5 μL diluted MMLV enzyme, add water to make up to 20 μL.

[0140] The reverse transcription reaction program is 37°C for 30 seconds, 85°C for 5 seconds, and 4°C for hold. Place the tube in a PCR instrument for reverse transcription. After the reaction is complete, store at -20°C until ready for use.

[0141] (2) Quantitative qPCR reaction: The cDNA synthesized by reverse transcription in (1) was used as a template for qPCR reaction (N=2).

[0142] Prepare a 25 μL qPCR reaction system: 1× SYBR Green Pro Taq HS Premix, 0.2 μmol / L Test 1, 0.2 μmol / L Test 2, with the remainder being water. Set the reaction program to 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. Perform the reaction in a quantitative qPCR instrument.

[0143] (3) Data processing: Export the Ct value data obtained by the qPCR instrument, and prepare standard curves of the Ct value 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 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.

[0144] (4) Calculation of MMLV reverse transcriptase specific activity: The enzyme activities of MMLV reverse transcriptase WT and MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6 calculated in (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.

[0145] The statistical results are as follows Figure 2 As shown in the figure, it can be seen that the yield of MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6 is greatly improved compared with the wild type.

[0146] Example 3 Detection of Tm Values ​​of MMLV Reverse Transcriptase Mutants and Wild-Type MMLV

[0147] In this example, the MMLV reverse transcriptase mutants obtained in Example 1 were uniformly diluted to 500 ng / μL. The diluted mutants were then subjected to differential scanning fluorimetry to detect the Tm values ​​of the MMLV reverse transcriptase mutants and wild-type MMLV. The specific method is as follows:

[0148] 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 according to the concentration determined in Example 1. Simultaneously, the fluorescent dye SyproOrange was diluted to 20× using dimethyl sulfoxide (DMSO). The diluted sample was mixed with Sypro Orange and DSF reaction buffer and placed in a real-time fluorescence quantitative PCR instrument. The excitation and emission wavelengths were adjusted to the corresponding wavelengths of the fluorescent dyes, and the temperature was increased to obtain a curve of fluorescence intensity versus temperature. The Tm value of the protein sample was calculated using software. The specific process is as follows:

[0149] (1) DSF reaction system: A 20 μL DSF reaction system consists of 16 μL of 1× DSF reaction buffer, 2 μL of 500 ng / μL protein sample, and 2 μL of 20× fluorescent dye Sypro Orange.

[0150] (2) DSF reaction program: 4℃ for 2 min; 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 the X1M3 channel, place it in the real-time fluorescence quantitative PCR instrument, and start the heating reaction.

[0151] (3) Data processing and analysis: The curve of fluorescence intensity changing with temperature is obtained, and the Tm value and related parameters of the protein sample are calculated on the software.

[0152] According to the Tm value obtained by software analysis, the curve is drawn. The results are as follows Figure 3 As shown in the figure, it can be seen that the Tm values ​​of the MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6 prepared by the present invention are all improved, indicating that the thermal stability of the MMLV reverse transcriptase mutants is improved.

[0153] Example 4 RT-qPCR effect detection of thermostable MMLV reverse transcriptase mutants and wild-type MMLV reverse transcriptase at 60°C

[0154] In this example, the thermostable MMLV reverse transcriptase mutant obtained in Example 1 was diluted to 250 ng / μL, treated at 60°C for 15 minutes, and then subjected to RT-qPCR detection. The specific method is as follows:

[0155] The MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, Mut6 and MMLV reverse transcriptase WT prepared in Example 1 were diluted to 250 ng / μL according to the concentration determined in Example 1, and treated at 60°C for 15 min. The treated and untreated MMLV reverse transcriptase mutants and WT were diluted to final concentrations of 0.5 ng / μL, 0.1 ng / μL, 0.02 ng / μL, and 0.004 ng / μL, respectively; reverse transcription was then performed using 0.4 μg of MS2 RNA as a template; the resulting cDNA was amplified by qPCR to obtain the Ct value of the sample amplification. The specific process is as follows:

[0156] Test 1: 5'-TTCAGCGAACTTCTTGTAA-3' (SEQ ID NO: 15);

[0157] Test 2: 5'-CATCCGTAGCCTTATTGG-3' (SEQ ID NO: 16).

[0158] (1) Using 0.4 μg of MS2 RNA as a template, reverse transcribe the cDNA using diluted MMLV reverse transcriptase. The specific reverse transcription reaction system is as follows:

[0159] 20 μL reverse transcription system: final concentration of 1×RTase Reaction Buffer Mix II, final concentration of 0.1 μmo1 / L Test1, final concentration of 1 U / μL RNase Inhibitor, 0.4 μg of MS2 RNA, 5 μL diluted MMLV enzyme, add water to make up to 20 μL.

[0160] The reverse transcription reaction program is 37°C for 30 seconds, 85°C for 5 seconds, and 4°C for hold. Place the tube in a PCR instrument for reverse transcription. After the reaction is complete, store at -20°C until ready for use.

[0161] (2) Quantitative qPCR reaction: The cDNA synthesized by reverse transcription in (1) was used as a template for qPCR reaction (N=2).

[0162] Prepare a 25 μL qPCR reaction system: 1× SYBR Green Pro Taq HS Premix, 0.2 μmol / L Test 1, 0.2 μmol / L Test 2, with the remainder being water. Set the reaction program to 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. Perform the reaction in a quantitative qPCR instrument.

[0163] Table 1

[0164]

[0165]

[0166] The experimental results obtained the Ct values ​​of the samples after RT-qPCR of the untreated and 60℃ treated samples for 15 minutes. The statistical results are shown in Table 1. The Ct value of the MMLV reverse transcriptase untreated was subtracted from the Ct value of the MMLV reverse transcriptase treated at 60℃ for 15 minutes at the corresponding concentration to obtain the △Ct value of each sample; the △Ct value of the MMLV reverse transcriptase wild type was subtracted from the △Ct value of the MMLV reverse transcriptase mutant, and the curve was plotted. The results are shown in Table 1. Figure 4 As shown in the results, it can be seen that the MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, and Mut6 prepared by the present invention have advanced Ct values ​​after being treated at 60°C for 15 minutes, and Mut3 has the smallest ΔCt, that is, after high-temperature treatment, the RT-qPCR ability is the best.

[0167] Differential Scanning Fluorimetry (DSF) evaluates protein thermal stability by slowly heating a sample in a fluorescence quantitative PCR instrument and measuring the amount of fluorescent dye bound to the structurally altered protein during heating. DSF experiments can be used to screen for MMLV reverse transcriptases with high thermal stability.

[0168] 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 thermostable MMLV reverse transcriptase mutant, characterized in that: The amino acid sequence of the thermostable MMLV reverse transcriptase mutant is shown in any one of SEQ ID NOs: 2-7.

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

5. Use of any one of the thermostable 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; the product comprises at least one of a reagent, a detection plate, a kit, and a detection chip.

6. The use according to claim 5, characterized in that The reagent, detection plate, detection chip or kit has the function of reverse transcription of RNA to synthesize cDNA.

7. The use according to claim 5, characterized in that The kit is selected from one of a reverse transcription reaction kit, an RT-PCR amplification kit, a cDNA library construction kit, a cDNA end rapid amplification kit and an RNA sequencing kit.

8. A product, characterized in that The product comprises the high-temperature-resistant MMLV reverse transcriptase mutant according to claim 1; the product comprises at least one of a reagent, a detection plate, a test kit, and a detection chip.

9. A method for preparing a thermostable 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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