Rat leukemia virus reverse transcriptase mutant and application thereof

Abstract

The invention discloses a mouse leukemia virus reverse transcriptase mutant and application thereof.On the basis of an amino acid sequence SEQ ID NO.1, the mutant is subjected to site mutation including any one or a combination of at least two of S60, K152, Q341, L452, L604 or G608, and the site mutation is used for improving charge distribution of a local region of a mutation site; the mutation of S60 is selected from S60K, S60R or S60H; the mutation of the K152 is selected from K152I, K152Q, K152E or K152D; the mutation of the Q341 is selected from Q341E or Q341D; the mutation of the L452 is selected from L452 C, L452K, L452R or L452H; the mutation of the L604 is selected from L604N, L604D, L604E, L604Y or L604G; and the mutation of the G608 is selected from G608M or G608S. The mutant reverse transcriptase obtained by designing mutations at one or more different sites of amino acids shows improved thermal stability, reactivity at higher temperatures and / or additional beneficial characteristics (improved storage stability, improved transport stability, improved cDNA synthesis ability of a complex structure RNA template or improved specificity).

Description

[0001] This application is a divisional application of the patent application with application number 202310795630.1 (the filing date of the original application is June 30, 2023, and the name of the invention is a murine leukemia virus reverse transcriptase mutant and its application). Technical Field

[0002] The invention belongs to the field of biotechnology and relates to a mouse leukemia virus reverse transcriptase mutant and application thereof. Background Art

[0003] Reverse transcriptase is an enzyme that uses RNA as a template to synthesize DNA. It is widely used in scientific research and various scenarios from pathogen detection to transcriptome analysis, involving RT-PCR, RT-qPCR, RT-LAMP, cDNA library construction and other technologies. Among the known reverse transcriptases, the reverse transcriptase from Moloney murine leukemia virus (MMLV RT) is the most widely used and well-known one. Compared with other reverse transcriptases, MMLV reverse transcriptase has stronger reverse transcription activity, and the expression and purification process is more suitable for commercialization. However, the wild type of MMLV reverse transcriptase has obvious defects. Its optimal reaction temperature is 37-42°C, and it is easily inactivated at high temperatures.

[0004] The RNA used as a template is prone to form secondary structures, which is extremely unfavorable for the reverse transcription process. The secondary structure may cause the reverse transcription process to terminate prematurely, or the synthesized cDNA may be incomplete, have low yields, and have biases. Increasing the reaction temperature of reverse transcription helps to open the secondary structure of RNA and improve the quality of the cDNA product, but the temperature that can open the secondary structure of RNA often inactivates the reverse transcriptase, so it is imperative to improve the thermal stability of MMLV reverse transcriptase. In addition, the improvement of the thermal stability of MMLV reverse transcriptase is often accompanied by an increase in reaction activity and storage stability at high temperatures, making the technology more superior and making transportation and storage more convenient.

[0005] In summary, the existing wild-type MMLV reverse transcriptase has poor thermal stability, cannot withstand high temperatures, and is difficult to apply to a variety of technical scenarios (such as high-temperature reverse transcription reaction, high-temperature RT-qPCR and high-temperature RT-LAMP). How to provide a murine leukemia virus reverse transcriptase mutant with excellent thermal stability and high catalytic activity at high temperatures has become one of the urgent problems to be solved in the field of biotechnology. Summary of the invention

[0006] In view of the deficiencies in the prior art and actual needs, the present invention provides a murine leukemia virus reverse transcriptase mutant and its application, which solves the problems of the existing wild-type MMLV reverse transcriptase, such as poor thermal stability, inability to withstand high temperatures, and difficulty in being suitable for high-temperature reverse transcription reactions, high-temperature RT-qPCR and high-temperature RT-LAMP. The obtained mutant reverse transcriptase exhibits improved thermal stability, reaction activity at higher temperatures and / or additional beneficial properties (improved storage stability, improved transport stability, improved cDNA synthesis ability of complex structure RNA templates or improved specificity).

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a murine leukemia virus reverse transcriptase mutant, wherein the mutant has the following site mutations based on the amino acid sequence SEQ ID NO.1:

[0009] Any one of A46, S60, V88, S92, P100, V101, R121, P127, H143, K152, P166, R211, D216, A229, D339, Q341, T420, L452, M457, T467, A500, G504, A539, ​​L563, A589, L604, T605, S606, G608, E616, Q639 or A646, or a combination of at least two thereof.

[0010] Preferably, the mutant has an amino acid sequence identity of greater than 90% with SEQ ID NO.1; the mutant has the following site mutations based on the amino acid sequence of SEQ ID NO.1:

[0011] A46P, S60K or S60R or S60H, V88K or V88R or V88H, S92C, P100V or P100G or P100S or P100A or P100Y, V101G or V101I or V101C or V101N or V101F or V101K or V101R or V101Y, R121C, P127S, H143C, K152I or K152Q or K152E or K152D, P166C, R211S, D216Q, A229C, D339E, Q341E or Q341D, T420V, L452C or L452K or L452 R or L452H, M457C, T467N, A500P, G504P or G504S, A539C, L563C, A589K or A589R or A589H, L604N or L604D or L604E or L604Y or L604G, T605I or T605S or T605P or T605V or T605D or T605E or T605R or T605K, S606F or S606V or S606H or S606G or S606R or S606K, G608M or G608S, E616C, Q639C or A646C, any one or a combination of at least two thereof.

[0012] Preferably, the mutant has an amino acid sequence identity of greater than 90% with SEQ ID NO.1; the mutant comprises the following mutations based on the amino acid sequence of SEQ ID NO.1:

[0013] Any one of S60, P100, V101, K152, R211, D216, D339, T420, L452, M457, L563, L604, T605, S606, G608, E616 or a combination of at least two thereof.

[0014] Preferably, the mutant has an amino acid sequence identity of greater than 90% with SEQ ID NO.1; the mutant has the following site mutations based on the amino acid sequence of SEQ ID NO.1:

[0015] S60K or S60R or S60H, P100V or P100G or P100S or P100A or P100Y, V101G or V101I or V101C or V101N or V101F or V101K or V101R or V101Y, K152I or K152Q or K152E or K152D, R211S, D216Q, D339E, T420V, L452C or L452K or L452R or l4 52H, M457C, L563C, L604N or L604D or L604E or L604Y or L604G, T605I or T605S or T605P or T605V or T605D or T605E or T605R or T605K, S606F or S606V or S606H or S606G or S606R or S606K, G608M or G608S or E616C, any one or a combination of at least two thereof.

[0016] Preferably, the mutant further undergoes the following mutations based on the amino acid sequence of SEQ ID NO.1:

[0017] Any one of M39, S56, M66, L99, D108, P111, L139, D200, T287, T330, P448, D449, S453, N479, A502, D524, H594, L603, I611, L630, H634, D653, L671, or a combination of at least two of them.

[0018] Preferably, the mutant further undergoes the following site mutations based on the amino acid sequence of SEQ ID NO.1:

[0019] Any one of M39V, S56C, M66L, L99F or L99V or L99Y or L99N or L99G or L99C, D108I or D108L or D108C or D108H, P111L, L139P, D200H or D200Q, T287A, T330P, P448C, D449G, S453K, N479D, A502V, D524A, H594K, L603W, I611K or I611R or I611H, L630P, H634Y, D653T or L671P, or a combination of at least two thereof.

[0020] Preferably, the mutant amino acid sequence SEQ ID NO.1 undergoes any of the following combined site mutations:

[0021] (1) L563C, G608S, E616C, H634Y and D653T;

[0022] (2) S60R, D200Q, A229C, S453K, D524A, L604N, T605D, G608M and I611R;

[0023] (3) D200Q, A229C, T467N and L671P;

[0024] (4) S60R, P127S, D200H, Q341E, S453K and L671P;

[0025] (5) K152I, T382A, G504S and L604D;

[0026] (6) V101F, K152I, R211S, D216Q, T420V, A502V, D524A, T605I, S606K, I611H and A646C;

[0027] (7) M39V, D200H, N479D, L563C and L671P;

[0028] (8) P100A and D339E;

[0029] (9) A46P, P100I, V101G, D200N and G608S;

[0030] (10) K152E, G504S, A539C and S606K;

[0031] (11) S60K, V129R, D200H and A500P;

[0032] (12) S60R, G608S and I611R;

[0033] (13) K152Q, T420V and G608S.

[0034] In some embodiments of the present invention, the amino acid sequence before mutation is not limited to being completely identical to SEQ ID NO.1. For example, an amino acid sequence with an amino acid sequence identity of more than 90%, preferably more than 95%, and further preferably more than 98% with SEQ ID NO.1 is feasible.

[0035] In the present invention, the amino acid sequence shown in SEQ ID NO.1 is as follows:

[0036] TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTP

[0037] VSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN

[0038] KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGIS

[0039] GQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTR

[0040] ALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL

[0041] REFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLP

[0042] DLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVL

[0043] TKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVAL

[0044] NPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGA

[0045] AVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIY

[0046] RRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKA

[0047] AITETPDTSTLL.

[0048] In a second aspect, the present invention provides a nucleic acid molecule comprising a coding sequence of the murine leukemia virus reverse transcriptase mutant described in the first aspect.

[0049] In a third aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule described in the second aspect.

[0050] In a fourth aspect, the present invention provides a recombinant cell, wherein the recombinant cell contains the nucleic acid molecule described in the second aspect and / or the recombinant vector described in the third aspect.

[0051] In a fifth aspect, the present invention provides a kit, wherein the kit contains the murine leukemia virus reverse transcriptase mutant described in the first aspect.

[0052] Preferably, the kit is used to synthesize cDNA using RNA as a template.

[0053] In a sixth aspect, the present invention provides the use of the murine leukemia virus reverse transcriptase mutant described in the first aspect in a reverse transcription reaction.

[0054] Preferably, the reverse transcription reaction includes any one of RT-PCR, RT-qPCR or RT-LAMP.

[0055] Preferably, the mutant retains reverse transcriptase activity.

[0056] Preferably, the mutant also includes improvements in one or at least two of the following properties: thermal stability, optimal reaction temperature, reverse transcriptase activity, storage stability, transport stability, processivity, cDNA synthesis ability of complex structure RNA templates, product specificity, and reverse transcription speed.

[0057] Preferably, the mutant lacks RNase H activity.

[0058] In some embodiments of the present invention, the mutant MMLV reverse transcriptase with excellent thermal stability is obtained by rational design. The rational design strategy used is one or more of generating disulfide bonds, improving substrate binding ability, improving structural flexibility in local regions, optimizing charge distribution in local regions, and reducing Gibbs free energy.

[0059] In some embodiments of the present invention, a mutant MMLV reverse transcriptase with excellent thermal stability is obtained by semi-rational design. The semi-rational design strategy used is to select important amino acid sites for saturation mutation based on rational design analysis.

[0060] In some embodiments of the present invention, a mutant MMLV reverse transcriptase with excellent thermal stability is obtained by directed evolution. The strategy used is to construct a full-length random mutation library of MMLV reverse transcriptase and screen mutants with excellent thermal stability by a high-throughput directed evolution method.

[0061] The present invention obtains mutants that are beneficial to the thermal stability of MMLV reverse transcriptase through rational design, semi-rational design and directed evolution methods. After analysis, the mutation site mainly affects the thermal stability of MMLV reverse transcriptase through one or more of the following pathways.

[0062] In some embodiments of the present invention, the mutant MMLV reverse transcriptase with excellent thermal stability is replaced with cysteine ​​at one or more positions of position 56, position 92, position 121, position 143, position 166, position 229, position 448, position 457, position 563, position 616, position 639, position 646 relative to the wild type, and the thermal stability of the reverse transcriptase is improved by generating disulfide bonds. Disulfide bonds are a commonly used method for improving the thermal stability of proteins, and this method is also effective in MMLV reverse transcriptase. In some embodiments of the present invention, the generation of one or more pairs of disulfide bonds shows better thermal stability than the wild type.

[0063] In some embodiments of the present invention, one or more pairs of disulfide bonds combined with one or more other amino acid substitutions that are beneficial to improving thermal stability also show better thermal stability than the wild type.

[0064] In some embodiments of the present invention, the mutant MMLV reverse transcriptase with excellent thermal stability is replaced with other amino acids at one or more positions 127, 452, 453, 603, 604, 605, 606, 608, and 611 relative to the wild type, which can improve the substrate binding ability. Improving the substrate binding ability helps the protein to be more stable during the catalytic reaction.

[0065] In some embodiments of the present invention, one or more amino acid substitutions that contribute to improving substrate binding ability combined with one or more other amino acid substitutions that are beneficial to improving thermal stability also show better thermal stability than the wild type.

[0066] In some embodiments of the present invention, a mutant MMLV reverse transcriptase having excellent thermal stability is replaced with proline at one or more positions 46, 139, 330, 500, 504, 605, and 671 relative to the wild type, which can improve the structural flexibility of the local region and enhance the thermal stability of the enzyme.

[0067] In some embodiments of the present invention, one or more proline substitutions that help improve structural rigidity combined with one or more other amino acid substitutions that are beneficial to improving thermal stability also show better thermal stability than the wild type.

[0068] In some embodiments of the present invention, the mutant MMLV reverse transcriptase with excellent thermal stability is replaced with other amino acids at one or more positions relative to the wild type at positions 60, 88, 152, 341, 449, 452, 453, 479, 589, 604, 605, 606, 608, and 611, which can improve the local area charge distribution and improve the stability of the enzyme in solution. For example, the 60th amino acid is located on the surface of the protein, and mutating it to K, R, and H basic amino acids can improve the surface charge distribution, which is beneficial to improve the stability in salt solution.

[0069] In some embodiments of the present invention, one or more amino acid substitutions that optimize local charge combined with one or more other amino acid substitutions that are beneficial to improving thermal stability also show better thermal stability than the wild type.

[0070] In some embodiments of the present invention, the mutant MMLV reverse transcriptase with excellent thermal stability is replaced with other amino acids at one or more positions 39, 66, 99, 100, 101, 108, 111, 152, 211, 216, 287, 420, 467, 502, 634, and 653 relative to the wild type, which can reduce the Gibbs free energy of the protein. The protein needs to consume more energy to transform from a normal folded state to a disordered inactivated state, thereby improving the stability of the enzyme.

[0071] In some embodiments of the present invention, one or more amino acid substitutions that reduce Gibbs free energy combined with one or more other amino acid substitutions that are beneficial to improving thermal stability also show better thermal stability than the wild type.

[0072] In some embodiments of the present invention, each of the above-mentioned strategies of generating disulfide bonds, improving substrate binding ability, rigidifying flexible regions, optimizing local charges, and reducing Gibbs free energy is not limited to replacing one or more sites at the same time, nor is it limited to the independent implementation or combination of various strategies. All strategies that are beneficial to improving the thermal stability of reverse transcriptase are applicable.

[0073] In some embodiments of the present invention, the amino acid substitutions disclosed in the present invention that are beneficial to improving thermal stability are combined with reported mutants, which also help to improve the thermal stability of the mutants. For example, after the aspartic acid at position 524 of MMLV is mutated to A, the RNase H activity of MMLV is inactivated, and the thermal stability is improved. Some embodiments of the present invention perform new multi-site mutations on the D524A mutant, further improving the thermal stability of the reverse transcriptase. Similarly, the replacement of the amino acid at position 200 also helps to improve thermal stability. Some embodiments of the present invention perform new multi-site mutations on this basis, and the results also show that thermal stability can be further improved. Therefore, superimposing one or more mutation sites disclosed in the present invention on the known mutant MMLV can further improve the thermal stability of the reverse transcriptase.

[0074] In some embodiments of the present invention, the vector used for the mutant reverse transcriptase is pET21b, and the expression host used is Escherichia coli Rosetta. However, the mutant reverse transcriptase with excellent thermal stability described in the present invention is not limited to the above vectors and hosts.

[0075] In some embodiments of the present invention, the mutant reverse transcriptase with excellent thermal stability is reflected by the melting temperature (Tm). The protein peptide chain folds in water, and its hydrophilic group will wrap the hydrophobic group inside. Under external pressure (high temperature), the protein will gradually expose the hydrophobic group due to denaturation and unfolding. At this time, the hydrophobic fluorescent dye in the system will bind to the hydrophobic group of the protein and emit fluorescence. The Tm of different proteins can be calculated by the change of the fluorescence curve, thereby realizing the screening and comparison of the thermal stability of the mutants.

[0076] The Tm determination of the mutant reverse transcriptase of the present invention is not limited to the above method, and other methods applicable to protein Tm determination may be applicable to the mutant reverse transcriptase of the present invention.

[0077] The optimum reaction temperature of the wild-type MMLV reverse transcriptase is 37-42°C. The mutant reverse transcriptase disclosed in the present invention has better thermal stability and can perform reverse transcription reaction at 50°C. Therefore, when comparing the residual yield of the mutant reverse transcriptase at different temperatures, a reverse transcription temperature of 50°C is used.

[0078] Compared with the prior art, the present invention has the following beneficial effects:

[0079] (1) The present invention can significantly improve the thermal stability of MMLV reverse transcriptase by designing mutations at one or more different amino acid sites, specifically by more than 0.1°C, more than 0.2°C, more than 0.5°C, more than 1°C, more than 2°C, more than 3°C, more than 4°C, and more than 5°C compared with the wild type; the Tm of the MMLV reverse transcriptase with better effects can be increased by more than 6°C. After incubation at 60°C for 15 minutes, the residual yield can still reach more than 50%, and after incubation at 65°C for 15 minutes, the residual yield can still reach more than 15%, and high-temperature RT-LAMP reaction can be performed;

[0080] (2) The mutant reverse transcriptase of the present invention retains the reverse transcriptase activity and has improved one or more of the following characteristics: a. thermal stability; b. optimal reaction temperature; c. reverse transcriptase activity; d. storage stability; e. transport stability; f. continuous synthesis ability; g. cDNA synthesis ability; h. product specificity; i. reverse transcription speed. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 is the ΔTm of the mutant MMLV reverse transcriptase (ΔTm = Tm (Mutation) -Tm (WT) )picture;

[0082] Figure 2 This is the residual yield of mutant MMLV reverse transcriptase after incubation at 60°C for 15 min;

[0083] Figure 3 This is the residual yield of mutant MMLV reverse transcriptase after incubation at 65°C for 15 min;

[0084] Figure 4A This is the result of RT-LAMP reaction of wild-type MMLV reverse transcriptase at 65°C;

[0085] Figure 4B This is the result of RT-LAMP reaction of mutant MMLV reverse transcriptase 356 at 65°C;

[0086] Figure 4C This is the result of RT-LAMP reaction of mutant MMLV reverse transcriptase 215 at 65°C;

[0087] Figure 4D This is the result of RT-LAMP reaction of mutant MMLV reverse transcriptase 306 at 65°C;

[0088] Figure 4E This is the result of RT-LAMP reaction of mutant MMLV reverse transcriptase 347 at 65°C;

[0089] Figure 4FThis is the result of RT-LAMP reaction of mutant MMLV reverse transcriptase 334 at 65°C;

[0090] Figure 4G This is the result of RT-LAMP reaction of mutant MMLV reverse transcriptase 209 at 65°C;

[0091] Figure 4H This is the result of RT-LAMP reaction of mutant MMLV reverse transcriptase 335 at 65°C;

[0092] Fig. 4I This is the result of RT-LAMP reaction of mutant MMLV reverse transcriptase 329 at 65°C;

[0093] Figure 4J This is the result of RT-LAMP reaction of mutant MMLV reverse transcriptase 188 at 65°C;

[0094] Figure 4K This is the result of RT-LAMP reaction of mutant MMLV reverse transcriptase 330 at 65°C;

[0095] Figure 4L This is the result of RT-LAMP reaction of mutant MMLV reverse transcriptase 179 at 65°C;

[0096] Figure 5A This is the result of RT-LAMP reaction of wild-type MMLV reverse transcriptase at 68°C;

[0097] Figure 5B This is the result of RT-LAMP reaction of mutant MMLV reverse transcriptase 215 at 68°C;

[0098] Figure 5C This is the result of RT-LAMP reaction of mutant MMLV reverse transcriptase 209 at 68°C;

[0099] Figure 5D This is the result of RT-LAMP reaction of mutant MMLV reverse transcriptase 306 at 68°C;

[0100] Figure 5E This is the result of RT-LAMP reaction of mutant MMLV reverse transcriptase 188 at 68°C;

[0101] Fig. 5F This is the result of RT-LAMP reaction of mutant MMLV reverse transcriptase 329 at 68°C. DETAILED DESCRIPTION

[0102] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0103] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0104] Example 1

[0105] The thermal stability of MMLV reverse transcriptase was modified by combining rational design, semi-rational design and directed evolution methods. Among them, 160 mutants were rationally designed by site-directed mutation or site-directed combination mutation, 20 site-directed saturation mutation libraries or site-directed combination saturation mutation libraries were semi-rationally designed, and more than 10,000 random mutation libraries were screened by directed evolution, and finally a database of MMLV reverse transcriptase mutants that are beneficial to improving thermal stability was obtained, as shown in Table 1. The amino acid mutations shown in Table 1 are mutations that occur on the basis of the amino acid sequence SEQ ID NO.1.

[0106] Table 1

[0107]

[0108]

[0109]

[0110]

[0111]

[0112] The specific preparation method of the mutant MMLV reverse transcriptase is as follows: the nucleotide sequence encoding the amino acid sequence of the mutant MMLV reverse transcriptase (the corresponding nucleotide sequence encoding the amino acid sequence of the wild-type MMLV reverse transcriptase is SEQ ID NO.2) is connected to the vector pET21b (+) to obtain a recombinant vector, and the recombinant vector is transformed into the expression host Escherichia coli Rosetta to obtain a recombinant strain. The recombinant strain is cultured to induce the expression of the MMLV reverse transcriptase, and the MMLV reverse transcriptase mutant is obtained after purification.

[0113] SEQ ID NO.2:

[0114]

[0115] Example 2

[0116] The Tm value of the murine leukemia virus reverse transcriptase mutant obtained in Example 1 was detected.

[0117] Method: The mouse leukemia virus reverse transcriptase mutant obtained in Example 1 was purified to obtain a pure reverse transcriptase enzyme solution, which was mixed with a hydrophobic fluorescent dye. In a real-time fluorescence quantitative PCR instrument, the temperature was raised to 30°C at a constant rate and kept constant for 20 seconds, then gradually raised to 70°C and kept constant for 1 second. During the heating process, a fluorescent signal was collected every 0.5 seconds, and Tm was calculated based on the collected signal analysis.

[0118] Results: The murine leukemia virus reverse transcriptase mutant obtained in the present invention has a significantly higher melting temperature (Tm) than the wild-type murine leukemia virus reverse transcriptase ( Figure 1 ). Specifically, compared with the wild type, the temperature can be increased by more than 0.1°C, more than 0.2°C, more than 0.5°C, more than 1°C, more than 2°C, more than 3°C, more than 4°C, more than 5°C, more than 6°C for better results, and more than 7°C for the best results. The melting temperature (Tm) reflects the thermal stability of the MMLV reverse transcriptase. The higher the melting temperature (Tm), the better the thermal stability.

[0119] Example 3

[0120] The residual yield of the murine leukemia virus reverse transcriptase mutant obtained in Example 1 at 60° C. and 65° C. was detected. The optimal reaction temperature of the wild-type MMLV reverse transcriptase is 37-42° C. Experiments have shown that the MMLV reverse transcriptase mutant provided by the present invention can perform reverse transcription reaction at 50° C., so when comparing the residual yield of the mutant reverse transcriptase at different temperatures, a reverse transcription temperature of 50° C. was used.

[0121] Methods: The mutant reverse transcriptase was diluted to the same volume activity, incubated at 60℃ for 15min, and then reverse transcribed at 50℃ for 15min. The mutant reverse transcriptase that was not incubated at 60℃ for 15min and directly reverse transcribed at 50℃ for 15min was used as the control group. The MMLV reverse transcriptase mutant with better performance was screened for residual yield test at higher temperature.

[0122] The mutants with higher residual yield at 60°C were screened, and reverse transcription was performed at 50°C for 15 min after incubation at 65°C for 15 min. The mutant reverse transcriptases that were not incubated at 65°C for 15 min and directly reverse transcribed at 50°C for 15 min were used as control groups.

[0123] The configuration of the reverse transcription system is shown in Table 2.

[0124] Table 2

[0125] Components Dosage Oligo dT (50μM) 1μL dNTPs (10 mM each) 0.5μL mRNA 2μg 5×Reaction Buffer 4μL Enzyme 1μL <![CDATA[DEPC-H2O]]> To 20μL

[0126] Results: The residual yield of the murine leukemia virus reverse transcriptase mutant obtained by the present invention after incubation at 60°C for 15 minutes and then reverse transcription reaction was significantly increased compared with the wild type ( Figure 2 ). Specifically, the residual yield of mutants with mutation numbers 188, 329, 330, 331, 334, 335, and 356 can reach more than 50%, the residual yield of mutants with mutation numbers 302, 306, 327, and 341 can reach more than 80%, and the residual yield of mutants with mutation number 342 can reach more than 90%.

[0127] The residual yield of the murine leukemia virus reverse transcriptase mutant obtained by the present invention after incubation at 65°C for 15 minutes and then reverse transcription reaction is significantly increased compared with the wild type ( Figure 3 ). Specifically, the residual yield of mutants with mutation numbers 209 and 215 can reach more than 5%, the residual yield of mutants with mutation numbers 188 and 306 can reach more than 15%, the residual yield of mutants with mutation number 327 can reach more than 20%, and the residual yield of mutant 330 can reach more than 40%.

[0128] Example 4

[0129] The mutant obtained in Example 1 was used to perform RT-LAMP reaction. The effectiveness of the RT-LAMP reaction of the MMLV reverse transcriptase mutant obtained in Example 1 at 60°C, 65°C and 68°C was tested. The experiment proved that the MMLV reverse transcriptase mutant provided by the present invention can effectively peak in the RT-LAMP reaction at 60°C and has repeatability. The MMLV reverse transcriptase mutant with better performance was screened out for RT-LAMP at a higher temperature.

[0130] The RT-LAMP reaction system is shown in Table 3.

[0131] Table 3

[0132]

[0133] Among them, the system without adding template was used as negative control.

[0134] The RT-LAMP reaction procedure at 65°C is shown in Table 4.

[0135] Table 4

[0136] temperature time Cycle times 65℃ 1min (read fluorescence) 60× 95℃ 2min 1×

[0137] Results: The thermal stability of the mutant reverse transcriptases with mutation numbers 356, 215, 306, 347, 334, 209, 335, 329, 188, 330, and 179 was significantly improved compared with the wild-type MMLV reverse transcriptase in the RT-LAMP test. Among them, mutants 209, 215, 188, and 306 could effectively peak in the RT-LAMP reaction at 65°C and had repeatability of at least 7 out of 8 parallel experiments (Figure 4).

[0138] The RT-LAMP reaction procedure at 68°C is shown in Table 5.

[0139] Table 5

[0140] temperature time Cycle times 68℃ 1min (read fluorescence) 60× 95℃ 2min 1×

[0141] Results: The thermal stability of the mutant reverse transcriptases with mutation numbers 215, 209, 306, 188, and 329 was significantly improved in the RT-LAMP test, among which mutants 209, 306, 188, and 329 could effectively peak in the RT-LAMP reaction at 68°C and had repeatability of at least 4 times in 8 parallel experiments (Figure 5).

[0142] In summary, the present invention can significantly improve the thermal stability of MMLV reverse transcriptase by designing mutations at one or more different amino acid sites, the Tm of MMLV reverse transcriptase can be increased by more than 6°C, the residual yield after incubation at 60°C for 15 minutes can still reach more than 90%, and the residual yield after incubation at 65°C for 15 minutes can still reach more than 40%, and the RT-LAMP reaction at 68°C can be performed.

[0143] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A murine leukemia virus reverse transcriptase mutant, characterized in that: The mutant has the following site mutations based on the amino acid sequence SEQ ID NO.1: Any one of S60, K152, Q341, L452, L604 or G608 or a combination of at least two thereof is used to improve the charge distribution in the local area of ​​the mutation site; The mutation of S60 is selected from S60K, S60R or S60H; The mutation of K152 is selected from K152I or K152Q or K152E or K152D; The mutation of Q341 is selected from Q341E or Q341D; The mutation of L452 is selected from L452C or L452K or L452R or L452H; The mutation of L604 is selected from L604N or L604D or L604E or L604Y or L604G; The mutation of G608 is selected from G608M or G608S.

2. The murine leukemia virus reverse transcriptase mutant according to claim 1, characterized in that: The mutant has an amino acid sequence identity of greater than 90% with SEQ ID NO.1; the mutant further has the following mutations based on the amino acid sequence of SEQ ID NO.1: M39, A46, V88, P100, V101, D108, P111, P127, I611, D200, D216, A229, T420, S453, M 457, N479, A500, G504, D524, L563, A589, T605, S606, I611, E616, H634, D653, L671.

3. The murine leukemia virus reverse transcriptase mutant according to claim 1 or 2, characterized in that: The mutant amino acid sequence SEQ ID NO.1 undergoes any of the following combined site mutations: (1)S60R, V101N, M457C, L604Y; (2)S60K, P100I, V101C, D524A; (3) S60R, T420V; (4)S60K, V129R, D200H, A500P; (5)S60R, P127S, D200H, Q341E, S453K, L671P; (6) S60R, G608S, I611R; (7)S60R, K152E, T382A, G504S, D653T; (8)S60K, K152D, T382A, G608M, L604E; (9)S60R, D200Q, A229C, S453K, D524A, L604N, T605D, G608M, I611R; (10) V101I, L604D; (11) G308D, L604G; (12)P100G, D524A, L604D, S606F; (13)A46P, A589K, L604D; (14) V88K, L604E; (15) L604N; (16)D200H, D216Q, S453K, L604D; (17)K152I, T382A, G504S, L604D; (18)P100A, K152Q, T382A, L604D; (19) L604N, T605R; (20) L604G, T605S, S606V; (21)T590I, L604G, T605S, S606G; (22)G308D, L604Y, T605V, S606H; (23)P111L, P127S, L604N, T605I, S606F; (24) D108I, L604G; (25)K152E, T382A, G386S, G504S, D653R; (26)M39V, D200H, N479D, L563C, L671P; (27)L563C, G608S, E616C, H634Y, D653T; (28)K152E.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule contains the coding sequence of the murine leukemia virus reverse transcriptase mutant according to any one of claims 1 to 3.

5. A recombinant vector, characterized in that: The recombinant vector contains the nucleic acid molecule according to claim 4.

6. A recombinant cell, characterized in that The recombinant cell contains the nucleic acid molecule according to claim 4 and / or the recombinant vector according to claim 5.

7. A kit, characterized in that: The kit contains the murine leukemia virus reverse transcriptase mutant described in any one of claims 1-3.

8. The kit according to claim 7, characterized in that The kit is used for synthesizing cDNA using RNA as a template.

9. Use of the murine leukemia virus reverse transcriptase mutant according to any one of claims 1 to 3 in a reverse transcription reaction; Preferably, the reverse transcription reaction includes any one of RT-PCR, RT-qPCR or RT-LAMP.

10. The murine leukemia virus reverse transcriptase mutant according to any one of claims 1 to 3, characterized in that The mutant retains reverse transcriptase activity; Preferably, the mutant further comprises an improvement in one or at least two of the following properties: thermal stability, optimal reaction temperature, reverse transcriptase activity; Preferably, the mutant lacks RNase H activity.

Images