An MMLV reverse transcriptase mutant and its application

Abstract

The present invention discloses an MMLV reverse transcriptase mutant and its application. The MMLV reverse transcriptase mutant is mutated at amino acid sites based on the wild-type MMLV reverse transcriptase amino acid sequence shown in SEQ ID NO: 1. The amino acid mutation sites include one or more of M39L, Q68L, Q68R, L72K, L72I, I74L, Q84L, F155N, C157H, I179T, D200N, D200H, V223I, M289L, C310Q, T330G, T330S, T330P, L435R, L435P, L435K, L435M, V475E, L478I, D524A, and D583N. The MMLV reverse transcriptase mutant prepared by the present invention has the advantages of high yield, high thermal stability, and high sensitivity compared with the wild type.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an MMLV reverse transcriptase mutant and its application. Background Art

[0002] Reverse transcriptase is derived from RNA-containing retroviruses and is a multifunctional enzyme with three enzymatic activities, including RNA- and DNA-dependent DNA polymerase activities, and RNase H activity that catalyzes the cleavage of RNA in RNA-DNA hybrid chains. It can polymerize dNTPs to synthesize cDNA strands and is a standard tool in modern biotechnology and molecular diagnostics, being widely used in many research applications, including cDNA cloning, reverse transcriptase-PCR quantification, microarray analysis, RACE, etc.

[0003] Currently, the most commonly used reverse transcriptase on the market is the MMLV reverse transcriptase from Moloney murine leukemia virus. The MMLV reverse transcriptase is a single-subunit enzyme with a molecular weight of 71 kDa and a length of 671 aa. It is more active in the presence of Mn 2+ than in the presence of Mg 2+ The optimal working temperature of wild-type MMLV reverse transcriptase is 37–42 °C. During reverse transcription, when encountering an RNA template with a complex secondary structure, the reverse transcription process will pause, resulting in truncated reaction products. When the reverse transcription reaction temperature is increased from 37 °C to 55 °C, the hydrogen bonds of the secondary structure of the complex RNA template are opened, becoming linear single-stranded RNA, enabling the reverse transcription reaction to continue. However, the stability of wild-type MMLV reverse transcriptase decreases at 55 °C, thus reducing its reverse transcription activity. Therefore, there is an urgent need for a reverse transcriptase with better thermal stability and amplification efficiency. 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. For this purpose, the present invention provides an MMLV reverse transcriptase mutant.

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

[0006] The present invention also provides a biological material related to the above nucleic acid molecule.

[0007] The present invention also provides the applications of the above MMLV reverse transcriptase mutant, nucleic acid molecule, and biological material.

[0008] The present invention also provides a product.

[0009] The present invention also provides a preparation method of the above MMLV reverse transcriptase mutant.

[0010] According to a first aspect of the present invention, there is provided an MMLV reverse transcriptase mutant, which is mutated at amino acid sites based on the wild-type MMLV reverse transcriptase amino acid sequence shown in SEQ ID NO: 1, and the amino acid mutation sites include one or more of M39L, Q68L, Q68R, L72K, L72I, I74L, Q84L, F155N, C157H, I179T, D200N, D200H, V223I, M289L, C310Q, T330G, T330S, T330P, L435R, L435P, L435K, L435M, V475E, L478I, D524A, and D583N.

[0011] In some embodiments of the present invention, the MMLV reverse transcriptase mutant comprises an amino acid sequence shown in any one of SEQ ID NOs: 2-11.

[0012] According to a second aspect of the present invention, there is provided a nucleic acid molecule encoding the above-mentioned MMLV reverse transcriptase mutant.

[0013] In some embodiments of the present invention, the nucleic acid molecule is as shown in SEQ ID NOs: 17-26.

[0014] According to a third aspect of the present invention, there is provided a biological material related to the above-mentioned nucleic acid molecule, and the biological material comprises at least one of a1) to a7):

[0015] a1) An expression cassette containing the above-mentioned nucleic acid molecule;

[0016] a2) A vector containing the above-mentioned nucleic acid molecule;

[0017] a3) A vector containing the expression cassette described in a1);

[0018] a4) A transgenic cell line containing the above-mentioned nucleic acid molecule;

[0019] a5) A transgenic cell line containing the expression cassette described in a1);

[0020] a6) A transgenic cell line containing the vector described in a2);

[0021] a7) A transgenic cell line containing the vector described in a3).

[0022] According to a fourth aspect of the present invention, there is provided an application of at least one of the above-mentioned MMLV reverse transcriptase mutant, nucleic acid molecule, and biological material, and the application is for use in the preparation of a product.

[0023] In some embodiments of the present invention, the product comprises at least one of a reagent, a test plate, a kit, and a detection chip.

[0024] In some embodiments of the present invention, the reagent, detection plate, detection chip or kit has at least one of the functions of b1) to b3):

[0025] b1) Detecting an RNA sample;

[0026] b2) Reverse transcribing RNA to synthesize cDNA;

[0027] b3) Preparing an RT-PCR detection reagent or an RT-qPCR detection reagent.

[0028] According to the fifth aspect of the present invention, a product is provided, which comprises the above-mentioned MMLV reverse transcriptase mutant.

[0029] In some embodiments of the present invention, the product comprises at least one of a reagent, a detection plate, a kit, and a detection chip.

[0030] In some embodiments of the present invention, the reagent, detection plate, detection chip or kit has at least one of the functions of b1) to b3):

[0031] b1) Detecting an RNA sample;

[0032] b2) Reverse transcribing RNA to synthesize cDNA;

[0033] b3) Preparing an RT-PCR detection reagent or an RT-qPCR detection reagent.

[0034] 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 rapid amplification of cDNA ends kit, and an RNA sequencing kit.

[0035] 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.

[0036] In some embodiments of the present invention, the RT-PCR amplification kit further comprises at least one of PCR water, an RT-PCR amplification buffer, dNTPs, a DNA polymerase, and a PCR amplification primer.

[0037] In some embodiments of the present invention, the RT-qPCR kit further comprises at least one of PCR water, an RT-qPCR amplification buffer, dNTPs, a DNA polymerase, a qPCR amplification primer, and a probe.

[0038] In some embodiments of the present invention, the cDNA library construction kit further comprises: adapters.

[0039] According to the sixth aspect of the present invention, there is provided a method for preparing the above-mentioned MMLV reverse transcriptase mutant, the preparation method comprising the following steps: obtaining by expressing through the above-mentioned transgenic cell line.

[0040] In some embodiments of the present invention, the method specifically comprises the following steps:

[0041] (1) Transfer the recombinant vector containing the above-mentioned nucleic acid molecule to competent cells and culture them, pick monoclonal cells for screening;

[0042] (2) Extract the expression vector of the screened positive clone, transfer it into a host cell, inoculate it into an LB medium for culture to obtain a seed solution;

[0043] (3) Take the obtained seed solution and inoculate it into an LB medium for culture to obtain a bacterial solution;

[0044] (4) Add inducer IPTG to the obtained bacterial solution to a final concentration of 0.8 - 1 mmol / L, after induced expression, centrifuge to collect the bacterial cells;

[0045] (5) Resuspend the obtained bacterial cells with a lysis buffer, disrupt the bacterial cells by high pressure, centrifuge to take the supernatant; and filter with a 0.22 - 0.5 μm microporous filter membrane;

[0046] (6) Perform nickel ion affinity chromatography on the filtered liquid phase, detect the elution solution by polyacrylamide gel electrophoresis, and collect the sample containing the target protein;

[0047] (7) Perform cation exchange chromatography on the sample containing the target protein in step (6), detect the elution solution by polyacrylamide gel electrophoresis, collect the sample containing the target protein and dialyze it into a storage solution to obtain the MMLV reverse transcriptase mutant.

[0048] In some embodiments of the present invention, the LB media in steps (2) and (3) both contain 80 - 120 μg / mL ampicillin.

[0049] In some embodiments of the present invention, the culture conditions in step (2) are 20 - 30 °C, shaking culture at 150 - 200 r / min.

[0050] In some embodiments of the present invention, the culture conditions in step (3) are: 20 - 30 °C, shaking culture at 150 - 200 r / min until OD600 is 0.6 - 0.8.

[0051] In some embodiments of the present invention, the seed liquid described in step (3) is inoculated into the LB medium at a volume ratio of 1:(80 - 120).

[0052] In some embodiments of the present invention, the induction conditions described in step (4) are 20 - 30 °C for 4 - 6 h.

[0053] In some embodiments of the present invention, the centrifugation conditions described in step (4) are: centrifuging at a speed of 4000 - 5000 rpm at 2 - 6 °C for 15 - 25 min.

[0054] In some embodiments of the present invention, the amount of the lysis buffer used in step (5) is 8 - 10 mL added per gram of the bacterial cells;

[0055] In some embodiments of the present invention, the centrifugation conditions described in step (5) are centrifuging at a speed of 10000 - 14000 rpm at 2 - 6 °C for 25 - 35 min;

[0056] In some embodiments of the present invention, the components of the lysis buffer described in step (5) include: 45 - 55 mM Tris - HCl, 280 - 320 mM NaCl, 0.1% - 0.2% Triton X - 100.

[0057] In some embodiments of the present invention, the components of the binding buffer used for nickel - ion affinity chromatography described in step (6) include: 45 - 55 mM Tris - HCl, 280 - 320 mM NaCl, 15 - 25 mM Imidazole, 2% - 6% Glycerol.

[0058] In some embodiments of the present invention, the components of the elution buffer used for nickel - ion affinity chromatography include: 45 - 55 mM Tris - HCl, 280 - 320 mM NaCl, 200 - 300 mM Imidazole, 2% - 6% Glycerol.

[0059] In some embodiments of the present invention, the components of the binding buffer used for cation - exchange chromatography include: 15 - 25 mM Tris - HCl, 190 - 220 mM NaCl, 2% - 6% (V / V) Glycerol, 0.5 - 2 mM DTT, 0.5 - 2 mM EDTA.

[0060] In some embodiments of the present invention, the components of the gradient elution buffer used for cation - exchange chromatography include: 15 - 25 mM Tris - HCl, 550 - 650 mM NaCl, 2% - 6% (V / V) Glycerol, 0.5 - 2 mM DTT, 0.5 - 2 mM EDTA.

[0061] 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, 0.5 - 2 mM EDTA.

[0062] According to some embodiments of the present invention, it has at least the following beneficial effects: The MMLV reverse transcriptase mutant prepared in the present invention is rationally designed by using software for predicting 3D structures and interactions with templates to obtain numerous mutation sites. By combining the mutation sites and screening, an enhanced MMLV reverse transcriptase mutant is obtained. Compared with the wild type, it has the advantages of high yield, high thermal stability, and high sensitivity, and can be used for reverse transcription of complex RNA templates and detection of trace RNA viruses. It greatly promotes the industrial production and commercial application of MMLV reverse transcriptase. Brief Description of the Drawings

[0063] The following further describes the present invention in conjunction with the drawings and embodiments, where:

[0064] Figure 1 It is the structural diagram of the enhanced MMLV reverse transcriptase mutant sites in Example 1 of the present invention, and the purple part is the mutation sites;

[0065] Figure 2 It is the SDS-PAGE diagram of the purified mutant and wild type in Example 1 of the present invention. Among them, M is the marker; 1 is the wild type MMLV reverse transcriptase, 2 is the enhanced MMLV reverse transcriptase mutant Mut1, 3 is the enhanced MMLV reverse transcriptase mutant Mut2, 4 is the enhanced MMLV reverse transcriptase mutant Mut3, 5 is the enhanced MMLV reverse transcriptase mutant Mut4, 6 is the enhanced MMLV reverse transcriptase mutant Mut5, 7 is the enhanced MMLV reverse transcriptase mutant Mut6, 8 is the enhanced MMLV reverse transcriptase mutant Mut7, 9 is the enhanced MMLV reverse transcriptase mutant Mut8, 10 is the enhanced MMLV reverse transcriptase mutant Mut9, 11 is the enhanced MMLV reverse transcriptase mutant Mut10;

[0066] Figure 3 It is the statistical chart of the yields of the enhanced MMLV reverse transcriptase mutant and the wild type MMLV reverse transcriptase in Example 2 of the present invention;

[0067] Figure 4Electrophoresis result diagram of RT-PCR of enhanced MMLV reverse transcriptase mutants and wild-type MMLV reverse transcriptase under different temperature treatments in Example 3 of the present invention. Among them, M is the marker; 1 is wild-type MMLV reverse transcriptase, 2 is enhanced MMLV reverse transcriptase mutant Mut1, 3 is enhanced MMLV reverse transcriptase mutant Mut2, 4 is enhanced MMLV reverse transcriptase mutant Mut3, 5 is enhanced MMLV reverse transcriptase mutant Mut4, 6 is enhanced MMLV reverse transcriptase mutant Mut5, 7 is enhanced MMLV reverse transcriptase mutant Mut6, 8 is enhanced MMLV reverse transcriptase mutant Mut7, 9 is enhanced MMLV reverse transcriptase mutant Mut8, 10 is enhanced MMLV reverse transcriptase mutant Mut9, 11 is enhanced MMLV reverse transcriptase mutant Mut10;

[0068] Figure 5 Electrophoresis result diagram of RT-PCR of enhanced MMLV reverse transcriptase mutants and wild-type MMLV reverse transcriptase under different template concentrations in Example 4 of the present invention. Among them, M is the marker; 1 is wild-type MMLV reverse transcriptase, 2 is enhanced MMLV reverse transcriptase mutant Mut1, 3 is enhanced MMLV reverse transcriptase mutant Mut2, 4 is enhanced MMLV reverse transcriptase mutant Mut3, 5 is enhanced MMLV reverse transcriptase mutant Mut4, 6 is enhanced MMLV reverse transcriptase mutant Mut5, 7 is enhanced MMLV reverse transcriptase mutant Mut6, 8 is enhanced MMLV reverse transcriptase mutant Mut7, 9 is enhanced MMLV reverse transcriptase mutant Mut8, 10 is enhanced MMLV reverse transcriptase mutant Mut9, 11 is enhanced MMLV reverse transcriptase mutant Mut10;

[0069] Figure 6 Electrophoresis result diagram of RT-PCR of enhanced MMLV reverse transcriptase mutants and wild-type MMLV reverse transcriptase under the template concentration of 10 pg in Example 4 of the present invention. Among them, M is the marker; 1 is wild-type MMLV reverse transcriptase, 2 is enhanced MMLV reverse transcriptase mutant Mut1, 3 is enhanced MMLV reverse transcriptase mutant Mut4, 4 is enhanced MMLV reverse transcriptase mutant Mut5, 5 is enhanced MMLV reverse transcriptase mutant Mut9, 6 is enhanced MMLV reverse transcriptase mutant Mut10. Detailed implementation method

[0070] The concept of the present invention and the resulting technical effects will be clearly and completely described below in conjunction with embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0071] Example 1 Transformation, Expression, and Purification and Identification of Enhanced MMLV Reverse Transcriptase Mutants

[0072] 1. Design of Enhanced MMLV Reverse Transcriptase Mutants

[0073] Rational design was carried out according to the software mutcompute for predicting the 3D structure and interactions with templates. Numerous mutation sites of the MMLV reverse transcriptase mutants were predicted. By combining the mutation sites, enhanced MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, Mut6, Mut7, Mut8, Mut9, and Mut10 were screened. The mutant sequences were obtained based on the sites of the wild-type MMLV reverse transcriptase mutant (the amino acid sequences are shown in SEQ ID NO.1 respectively), and the site structure diagram is as Figure 1 shown.

[0074] Wild-type MMLV reverse transcriptase mutant:

[0075] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI(SEQ ID NO.1).

[0076] The amino acid sequences of the mutants Mut1, Mut2, Mut3, Mut4, Mut5, Mut6, Mut7, Mut8, Mut9, and Mut10 are shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11, respectively.

[0077] Amino acid sequence of Mut1:

[0078] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGL GLAVRQAPLIIPLKATSTPVSIKQYPMS

[0079] L EARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYN

[0080] LLSGLPPSHQWYTVLDLKDA N F H LRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFD

[0081] EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQ

[0082] VKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKT

[0083] GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLS

[0084] KKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHY

[0085] QALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS

[0086] SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATA

[0087] HIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI(SEQ ID NO.2).

[0088] Amino acid sequence of Mut2:

[0089] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMS

[0090] QEAR K GIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPY

[0091] NLLSGLPPSHQWYTVLDLKDA N FCLRLHPTSQPLFAFEWRDPEMG T SGQLTWTRLPQGFKNSPTLF

[0092] N EALHRDLADFRIQHPDLILLQY I DDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQ

[0093] VKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKT

[0094] GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLS

[0095] KKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHY

[0096] QALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS

[0097] SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATA

[0098] HIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI(SEQ ID NO.3)。

[0099] Mut3 Amino Acid Sequence:

[0100] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMS

[0101] QEAR I GIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYN

[0102] LLSGLPPSHQWYTVLDLKDAFF H LRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFD

[0103] EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQ

[0104] VKYLGYLLKEGQRWLTEARKETV L GQPTPKTPRQLREFLGTAGF Q RLWIPGFAEMAAPLYPLTKTG

[0105] TLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSK

[0106] KLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQ

[0107] ALLLDTDRVQFGP E VALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSS

[0108] LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHI

[0109] HGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI (SEQ ID NO.4).

[0110] Amino acid sequence of Mut4:

[0111] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMS

[0112] QEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPY

[0113] NLLSGLPPSHQWYTVLDLKDAFF H LRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF

[0114] N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQK

[0115] QVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK

[0116] S GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL

[0117] SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVI R APHAVEALVKQPPDRWLSNARMTHY

[0118] QALLLDTDRVQFGPVVA I NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS

[0119] SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYT N SRYAFATA

[0120] HIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI (SEQ ID NO.5).

[0121] Amino acid sequence of Mut5:

[0122] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMS

[0123] QEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPY

[0124] NLLSGLPPSHQWYTVLDLKDA N FCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF

[0125] N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQK

[0126] QVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK

[0127] P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL

[0128] SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVI P APHAVEALVKQPPDRWLSNARMTHY

[0129] QALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS

[0130] SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYT N SRYAFATA

[0131] HIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI (SEQ ID NO.6).

[0132] Mut6 amino acid sequence:

[0133] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGG L GLAVRQAPLIIPLKATSTPVSIKQYPMS

[0134] QEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPY

[0135] NLLSGLPPSHQWYTVLDLKDA N FCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF

[0136] N EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQK

[0137] QVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK

[0138] P GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYL

[0139] SKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVI KAPHAVEALVKQPPDRWLSNARMTH

[0140] YQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYT A G

[0141] SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYT N SRYAFATA

[0142] HIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI(SEQ ID NO.7).

[0143] Amino acid sequence of Mut7:

[0144] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGG L GLAVRQAPLIIPLKATSTPVSIKQYPMS

[0145] R EARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPY

[0146] NLLSGLPPSHQWYTVLDLKDAFF H LRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF

[0147] H EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQK

[0148] QVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK

[0149] TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY

[0150] LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTH

[0151] YQALLLDTDRVQFGP E VALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDG

[0152] SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATA

[0153] HIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI(SEQ ID NO.8).

[0154] Amino acid sequence of Mut8:

[0155] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGG L GLAVRQAPLIIPLKATSTPVSIKQYPMS

[0156] QEAR K GIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPY

[0157] NLLSGLPPSHQWYTVLDLKDA N FCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLF

[0158] DEALHRDLADFRIQHPDLILLQY I DDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQ

[0159] VKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGF Q RLWIPGFAEMAAPLYPLTKT

[0160] GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLS

[0161] KKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHY

[0162] QALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYT A GS

[0163] SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATA

[0164] HIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI (SEQ ID NO.9).

[0165] Amino acid sequence of Mut9:

[0166] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMS

[0167] R EARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPY

[0168] NLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMG T SGQLTWTRLPQGFKNSPTLF

[0169] DEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQK

[0170] QVKYLGYLLKEGQRWLTEARKETV LGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK S

[0171] GTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLS

[0172] KKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVI M APHAVEALVKQPPDRWLSNARMTHY

[0173] QALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGS

[0174] SLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYT N SRYAFATA

[0175] HIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI(SEQ ID NO.10).

[0176] Mut10 amino acid sequence:

[0177] MLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMS

[0178] QEARLG L KPHIQRLLD L GILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPY

[0179] NLLSGLPPSHQWYTVLDLKDA N F H LRLHPTSQPLFAFEWRDPEMG T SGQLTWTRLPQGFKNSPTL

[0180] FDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQ

[0181] KQVKYLGYLLKEGQRWLTEARKETV L GQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTK

[0182] TGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAY

[0183] LSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTH

[0184] YQALLLDTDRVQFGPVVA I NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDG

[0185] SSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATA

[0186] HIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI (SEQ ID NO.11).

[0187] Among them, the underscore “—” indicates the mutation site.

[0188] 2. Synthesis of the enhanced MMLV reverse transcriptase mutant

[0189] (1) Recombinant expression vectors containing nucleotide sequences encoding the enhanced MMLV reverse transcriptase mutant (the sequences of mutants Mut1 - 10 are shown in SEQ ID NOs.17 - 26 in sequence) and the nucleotide sequence encoding the wild - type MMLV reverse transcriptase (shown in SEQ ID NO.16) were respectively synthesized by gene synthesis (the original vector is PET - 32a).

[0190] Nucleotide sequence encoding the wild - type MMLV reverse transcriptase:

[0191]

[0192] Nucleotide sequence encoding MMLV reverse transcriptase mutant Mut 1:

[0193]

[0194] Nucleotide sequence encoding the MMLV reverse transcriptase mutant Mut 2:

[0195]

[0196] Nucleotide sequence encoding MMLV reverse transcriptase mutant Mut 3:

[0197]

[0198] Nucleotide sequence encoding the MMLV reverse transcriptase mutant Mut 4:

[0199]

[0200] Nucleotide sequence encoding the MMLV reverse transcriptase mutant Mut 5:

[0201]

[0202] Nucleotide sequence encoding MMLV reverse transcriptase mutant Mut 6:

[0203]

[0204] Nucleotide sequence encoding MMLV reverse transcriptase mutant Mut 7:

[0205]

[0206] Nucleotide sequence encoding MMLV reverse transcriptase mutant Mut 8:

[0207]

[0208] Nucleotide sequence encoding the MMLV reverse transcriptase mutant Mut 9:

[0209]

[0210] Nucleotide sequence encoding MMLV reverse transcriptase mutant Mut 10:

[0211]

[0212] (2) The obtained recombinant expression vector was transformed into the host cell E. coli BLDE3. After cultivation, single colonies were picked and inoculated into LB (containing 100 μg / mL ampicillin) liquid medium and cultured until the OD600 reached 0.6 - 0.8. IPTG was added to a final concentration of 1.0 mmol / L, and induction was carried out at 25°C for 5 h. The cells were collected by centrifugation at 4°C and 4200 rpm, lysed buffer was added, and ultrasonic disruption was performed. SDS-PAGE electrophoresis was used to detect the expression of the target protein, and the detection results were as Figure 2 shown. It was found that the target protein could be highly expressed, and after sequencing verification, the sequence was correct.

[0213] (3) The recombinant bacteria capable of expressing the enhanced MMLV reverse transcriptase mutant obtained in step (2) were inoculated into 100 mL of LB medium containing 100 μg / mL ampicillin, placed in a shaker at 25°C and cultured overnight to obtain a seed solution.

[0214] (4) The obtained overnight-cultured seed solution 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 reached 0.6 - 0.8; IPTG was added to a final concentration of 1.0 mmol / L, and induction was continued at 25°C with shaking for 5 h; the induced cells were collected by centrifugation and weighed, the wet cell weight was recorded, and stored at -20°C.

[0215] (5) The cells frozen at -20°C after induced expression were taken. According to the recorded wet cell weight, 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, and the cells were lysed with a high-pressure cell disruptor. The lysed cells were centrifuged at 12000 rpm at 4°C for 30 min. The supernatant was centrifuged at 12000 rpm at 4°C for 30 min, filtered through a 0.22 μm microporous filter membrane, and the supernatant was taken into a 200 mL sterilized beaker and purified using a Ni affinity chromatography column.

[0216] (6) Purification using a Ni affinity chromatography column

[0217] 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.

[0218] Experimental method: The supernatant filtered through a 0.22-μm microporous filter membrane was loaded onto a chromatography column that had been equilibrated with buffer A. After loading, the column was first rinsed with equilibration buffer A, and then gradient elution with buffer B from 0% to 100% was performed. The eluted fractions were detected by SDS-PAGE protein electrophoresis. According to the detection results, the eluate was collected and dialyzed into buffer C for standby.

[0219] (7) Purification by cation exchange chromatography

[0220] Experimental reagents: The binding buffer was buffer C: 20 mM Tris-HCl, 200 mM NaCl, 5% (V / V) Glycerol, 1 mM DTT, 1 mM EDTA, pH 7.8; the elution buffer was buffer D: 20 mM Tris-HCl, 600 mM NaCl, 5% (V / V) Glycerol, 1 mM DTT, 1 mM EDTA, pH 7.8; the storage solution: 20 mM Tris-HCl, 600 mM NaCl, 1 mM DTT, 50% Glycerol, 1 mM EDTA, pH 7.8.

[0221] Experimental method: The dialyzed sample obtained by Ni affinity chromatography purification was loaded onto a chromatography column that had been equilibrated with buffer C. After loading, the column was first rinsed with equilibration buffer C, and then gradient elution with buffer D from 0% to 100% was performed. The eluted fractions were detected by SDS-PAGE protein electrophoresis. According to the detection results, the eluate was collected, dialyzed using a dialysis bag with a molecular weight cut-off of 10K, and stored in the storage solution for standby.

[0222] Example 2 Detection of the enzyme activity of the enhanced MMLV reverse transcriptase mutant and the wild type.

[0223] In this example, the enzyme activity of the enhanced MMLV reverse transcriptase mutant obtained in Example 1 was detected. The specific method is as follows:

[0224] The enhanced MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, Mut6, Mut7, Mut8, Mut9, Mut10 and the wild type WT purified in Example 1 were assayed for reverse transcriptase activity according to the following method. The MMLV reverse transcriptase mutants prepared in Example 1 were used as the test samples of the experimental group, and the commercial wild type MMLV reverse transcriptase (purchased from Hunan Aikery Bioengineering Co., Ltd.) was used as the test sample of the control group. They were diluted in a 5-fold gradient, and then reverse transcription and quantitative qPCR were performed using 0.4 μg of MS2 RNA as the template. After the quantitative qPCR was completed, the standard curves of Ct and the logarithm of the enzyme activity concentration of the control and test samples were made respectively. The logarithm of the enzyme activity of the test samples was calculated. After taking the antilogarithm, the enzyme activities at different dilution multiples were calculated, and the average value was taken to obtain the reverse transcriptase activity. The specific operations of the reverse transcription reaction and the quantitative qPCR reaction are as follows:

[0225] Test 1: 5'-TTCAGCGAACTTCTTGTAA-3' (SEQ ID NO.12);

[0226] Test 2: 5'-CATCCGTAGCCTTATTGG-3' (SEQ ID NO.13).

[0227] (1) Using 0.4 μg of MS2 RNA as the template, cDNA was reverse transcribed with the gradient-diluted MMLV enzyme. The specific reverse transcription reaction system is as follows:

[0228] 20 μL reverse transcription system: final concentration of 1×RTase Reaction Buffer Mix II, final concentration of 0.1 μmo1 / L Test 1, final concentration of 1 U / μL RNase Inhibitor, 0.4 μg of MS2 RNA, 5 μL of the diluted MMLV enzyme, and water was added to a final volume of 20 μL.

[0229] The reverse transcription reaction program was 37°C for 30 sec; 85°C for 5 sec; 4°C, hold. It was placed in a PCR instrument for the reverse transcription reaction. After the reaction was completed, it could be stored at -20°C for later use.

[0230] (2) Quantitative qPCR reaction: The cDNA synthesized by reverse transcription in (1) was used as the template for the qPCR reaction (N = 2).

[0231] 25 μL qPCR reaction system: final concentration of 1×SYBR Green Pro Taq HS Premix, final concentration of 0.2 μmol / L Test 1, final concentration of 0.2 μmol / L Test 2, with the balance being water. Set the reaction program: 95°C for 30 sec; then enter the cycling stage: 95°C for 5 sec, 60°C for 30 sec, for 40 cycles, and place it in a quantitative qPCR instrument for reaction.

[0232] (3) Data processing: Export the Ct value data obtained from the qPCR instrument, and respectively prepare standard curves of Ct vs. the logarithm of enzyme activity concentration for the reference substance and the test sample. It is required that the 6 concentration points show a linear relationship and the correlation coefficient R 2 ≥0.99, and the Ct value should be within 13 - 30; the ratio of the slopes of the standard curves of the test sample and the reference sample is 1 ± 0.03. Take 5 dilution multiples of the test sample that conform 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 taking the antilogarithm, and then take the average value to obtain the enzyme activity of the corresponding test sample.

[0233] (4) Calculation of MMLV reverse transcriptase yield: Multiply the enzyme activities of MMLV reverse transcriptase WT and MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, Mut6, Mut7, Mut8, Mut9, Mut10 calculated in (3) by the volume of MMLV reverse transcriptase purified in Example 1 to calculate the total enzyme activity, and then divide by the weight of the bacteria in Example 1 in g to obtain the enzyme activity yield of MMLV reverse transcriptase per g of weight.

[0234] Calculate the statistical results as Figure 3 shown. It can be seen from the figure that the yields of MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, Mut6, Mut7, Mut8, Mut9, Mut10 are all higher than that of the wild type.

[0235] Example 3 RT-PCR effects of enhanced MMLV reverse transcriptase mutants and wild-type MMLV reverse transcriptase at different reverse transcription temperatures.

[0236] In this example, the enzyme activities of the enhanced MMLV reverse transcriptase mutants obtained in Example 1 were detected at different reverse transcription temperatures. The specific method is as follows:

[0237] The MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, Mut6, Mut7, Mut8, Mut9, Mut10 and MMLV reverse transcriptase WT prepared in Example 1 were diluted to 20 U / μL according to the enzyme activity values determined in Example 2. Using 1 μg of mouse heart RNA as a template, reverse transcription reactions were carried out at four temperatures of 42°C, 50°C, 55°C, and 60°C respectively. The obtained cDNA was subjected to PCR amplification, and the resulting product was electrophoretically detected using 1% agarose gel. The specific procedure is as follows:

[0238] (1) RNA template pre-reaction annealing system: A 10 μL reverse transcription system was composed of dNTP Mix with a final concentration of 1 mmol / L, Oligo dT Primer (from Hunan Aikery Biotechnology Co., Ltd.) with a final concentration of 0.25 μmol / L, 1 μg of mouse heart RNA, and the balance being water.

[0239] The annealing reaction program was 65°C for 5 min; hold at 4°C. It was placed in a PCR instrument for pre-reaction annealing, and immediately used after the reaction ended.

[0240] (2) Reverse transcription system: A 20 μL reverse transcription system was composed of 5×RTase Reaction Buffer, 20 U / μL MMLV enzyme (the MMLV reverse transcriptase mutants Mut1 - 10 and MMLV reverse transcriptase WT prepared in Example 1 respectively), 1 U / μL RNase Inhibitor, and the 10 μL reaction solution of the annealing system obtained in step (1).

[0241] Reverse transcription reaction program: Four reverse transcription temperatures (42°C, 50°C, 55°C, 60°C respectively) were set for 30 min; 70°C for 15 min; hold at 4°C. It was placed in a PCR instrument for reverse transcription reaction, and immediately used after the reaction ended.

[0242] (3) PCR amplification system: Using the cDNA obtained in (2) as a template, with an added amount of 2 μL, PCR amplification was carried out.

[0243] A 50 μL PCR amplification system was composed of 1×Exp Taq Master Mix with a final concentration, Test 3 with a final concentration of 1 μmol / L, Test 4 with a final concentration of 1 μmol / L, 2 μL of cDNA template, and the balance being water.

[0244] PCR reaction program: 94°C for 1 min; then enter the cycling stage: 94°C for 30 sec; 56°C for 30 sec; 72°C for 10 min for 30 cycles; 72°C for 10 min; hold at 4°C.

[0245] The PCR amplification system was amplified using a PCR instrument. 5 μL of the amplification product was taken, and the result was detected by electrophoresis using 1% agarose gel.

[0246] The sequences of Test 3 and Test 4 are as follows:

[0247] Test 3: 5'-CTCCACTGTTTATGTCTATGTTCG-3' (SEQ ID NO.14);

[0248] Test 4: 5'-TCCAGAGTTTTCAGCTCTTCTGAT-3' (SEQ ID NO.15).

[0249] The experimental results are as Figure 4 shown. It can be seen from the figure that the MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, Mut6, Mut7, Mut8, Mut9, Mut10 prepared by the present invention can still reverse transcribe to obtain cDNA products at 60°C for reverse transcription, and then perform PCR amplification, while the wild type cannot reverse transcribe to obtain cDNA products.

[0250] Example 4 RT-PCR effects of enhanced MMLV reverse transcriptase mutants and wild-type MMLV reverse transcriptase under different concentrations of templates

[0251] 1. In this example, the enzyme activity of the enhanced MMLV reverse transcriptase mutants obtained in Example 1 was detected under different concentrations of templates (1 μg, 100 ng, 10 ng). The specific method is as follows:

[0252] The MMLV reverse transcriptase mutants Mut1, Mut2, Mut3, Mut4, Mut5, Mut6, Mut7, Mut8, Mut9, Mut10 and MMLV reverse transcriptase WT prepared and purified in Example 1 were diluted to 20 U / μL according to the enzyme activity values measured in Example 2. Using mouse heart RNA at 1 μg, 100 ng, and 10 ng as templates respectively, reverse transcription reactions were carried out at 50°C. The obtained cDNA was subjected to PCR amplification, and the resulting products were detected by electrophoresis using 1% agarose gel. The specific process is as follows:

[0253] (1) RNA template pre-reaction annealing system: Using mouse heart RNA at 1 μg, 100 ng, and 10 ng as templates respectively, a 10 μL reverse transcription system was used with a final concentration of 1 mmol / L dNTP Mix, a final concentration of 0.25 μmol / L Oligo dT Primer, template RNA (1 μg, 100 ng, 10 ng respectively), and the balance was water.

[0254] The annealing reaction program is 65°C for 5 min; 4°C, hold. Put it into a PCR instrument for pre-reaction annealing reaction, and use it immediately after the reaction ends.

[0255] (2) Reverse transcription system: The 20 μL reverse transcription system is 1×RTase Reaction Buffer at the final concentration, MMLV enzyme at the final concentration of 20 U / μL, RNase Inhibitor at the final concentration of 1 U / μL, and 10 μL of the reaction solution of the annealing system obtained in step (1), and the balance is water.

[0256] Set the reaction program as 50°C for 30 min; 70°C for 15 min; 4°C, hold. Put it into a PCR instrument for reverse transcription reaction, and use it immediately after the reaction ends.

[0257] (3) PCR amplification system: Using the cDNA obtained in (2) as the template, the added amount is 2 μL;

[0258] The 50 μL PCR amplification system is 1×Exp Taq Master Mix at the final concentration, Test3 at the final concentration of 1 μmol / L, Test 4 at the final concentration of 1 μmol / L, 2 μL of cDNA template, and the balance is water. After mixing well, set the reaction program. At 94°C for 1 min; then enter the cycling stage: 94°C for 30 sec, 56°C for 30 sec, 72°C for 10 min, 30 cycles; 72°C for 10 min; 4°C, hold. Put it into a PCR instrument for amplification, take 5 μL of the amplification product, and detect the result by electrophoresis using 1% agarose gel.

[0259] The experimental results are as Figure 5 shown. The results show that the MMLV reverse transcriptase mutants Mut1, Mut3, Mut4, Mut5, Mut8, Mut9, Mut10 can be reversed at 50°C, and cDNA products can be reversed from 10 ng of mouse heart RNA as the template amount, and the amount of the product for PCR amplification is significantly higher than that of the wild type.

[0260] 2. This example also performs enzyme activity detection on the enhanced MMLV reverse transcriptase mutant obtained in Example 1 at a template concentration of 10 pg.

[0261] The purified MMLV reverse transcriptase mutants Mut1, Mut4, Mut5, Mut9, Mut10, and MMLV reverse transcriptase WT prepared in Example 1 were diluted to 20 U / μL according to the enzyme activity values measured in Example 2. Using 10 pg of 293T RNA as a template, reverse transcription reactions were carried out at 42°C. The resulting cDNA was subjected to PCR amplification, and the products were electrophoretically detected using 1% agarose gel. The specific procedure was the same as that for the enzyme activity detection of the enhanced MMLV reverse transcriptase mutants in Example 4 under different concentrations of template (1 μg, 100 ng, 10 ng).

[0262] The detection results are as Figure 6 shown. It can be seen from the figure that the mutants Mut1, Mut4, Mut5, Mut9, and Mut10 can achieve detection with a template as low as 10 pg.

[0263] In summary, under the purification conditions in Example 1, the enhanced MMLV reverse transcriptase mutants of the present invention, compared with the wild type, have higher enzyme activity yields per unit gram of bacterial weight for Mut1, Mut2, Mut3, Mut4, Mut5, Mut6, Mut7, Mut8, Mut9, and Mut10; and can still carry out reverse transcription reactions under reverse transcription conditions at 60°C, thus enabling reverse transcription of complex RNA templates; when the template concentration is 10 ng, the reverse transcription cDNA yields of Mut1, Mut3, Mut4, Mut5, Mut8, Mut9, and Mut10 are higher than those of the wild type, further indicating that the sensitivity of the mutants has also been improved. The improvement in these three aspects has great value for the industrial production and commercial application of MMLV reverse transcriptase.

[0264] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the scope of knowledge possessed by those of ordinary skill in the relevant technical field. 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 MMLV reverse transcriptase mutant has an amino acid sequence as shown in SEQ ID NO:

6.

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

3. The biological material 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. Use of at least one of the MMLV reverse transcriptase mutant according to claim 1, the nucleic acid molecule according to claim 2, and the biomaterial according to claim 3 in preparing a product; the product comprises at least one of a reagent, a detection plate, a kit, and a detection chip.

5. The use according to claim 4, 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.

6. The use according to claim 4, characterized in that: The kit is selected from one of 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.

7. The use according to claim 6, characterized in that: 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; And / or, the RT-PCR amplification kit further comprises: at least one of PCR water, RT-PCR amplification buffer, dNTPs, DNA polymerase and PCR amplification primers; And / or, the RT-qPCR kit further comprises: at least one of PCR water, RT-qPCR amplification buffer, dNTPs, DNA polymerase, qPCR amplification primers and probes; And / or, the cDNA library construction kit further comprises: a linker.

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

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