Reverse transcriptase UCRT vZ mutant with improved thermal stability as well as construction method and application of reverse transcriptase UCRT vZ mutant

Abstract

The invention belongs to the technical field of biology, and particularly provides reverse transcriptase UCRT vZ mutants with improved thermal stability, the reverse transcriptase UCRT vZ mutants comprise four single-point mutants, six double mutants and four three mutants, and compared with wild type reverse transcriptase UCRT vZ, the half-life period of the mutants is longer at 65 DEG C; the double-mutant has a better effect, and the half-life period of the double-mutant is about four times that of the wild reverse transcriptase UCRT vZ. The invention also provides a genetically engineered bacterium containing the mutant. The reverse transcriptase UCRT vZ mutant obtained by the construction method provided by the invention has better thermal stability, shows higher thermal stability when being used for reverse transcription of RNA (Ribonucleic Acid) into cDNA (Complementary Deoxyribose Nucleic Acid) at a higher temperature, and has higher application potential.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a reverse transcriptase UCRT vZ mutant with improved thermal stability, a construction method and an application thereof. Background Art

[0002] Reverse transcriptase UCRT vZ uses RNA as a template, catalyzes dNTP polymerization, and generates DNA-RNA hybrid double strands. It has multiple enzyme activities and high temperature resistance. The optimal enzyme activity temperature is 60-70°C. The efficiency of opening the RNA secondary structure is significantly improved, which improves the transcription efficiency and cDNA integrity. Under this condition, there are fewer competitors for reverse transcriptase, which has a greater advantage than the common reverse transcriptases on the market. During the cDNA synthesis process, reverse transcription at a higher temperature can reduce the secondary structure of RNA and increase the efficiency of reverse transcription. Bioactive enzymes are widely used in industrial production, and improving the thermal stability of enzymes is one of the research focuses of industrial enzymes.

[0003] Protein engineering is based on the structural laws of protein molecules and the relationship between their biological functions. Through chemical, physical and molecular biological means, gene modification or gene synthesis is performed to transform existing proteins or create a new protein to meet human needs for production and life. Rational design is the most commonly used method in protein engineering. Computer-assisted molecular models are combined with site-directed mutagenesis to achieve protein function optimization, such as improving catalytic activity, thermal stability, acid and alkali resistance, etc. In order to effectively optimize the thermal stability of proteins, Markus Wyss et al. proposed the Consensus Concept theory in 2001. Unlike conventional protein rational design methods based on the precise structure-function relationship of proteins, the Consensus Concept theory is based on the amino acid sequence information of homologous proteins, and analyzes information that can improve the thermal stability of enzymes from an evolutionary perspective. The present invention takes the Consensus Concept theory as the guiding ideology, integrates and analyzes the reverse transcriptase UCRT vZ family sequence, and combines bioinformatics and crystallographic methods to obtain a new reverse transcriptase UCRT vZ mutant with high stability. Summary of the invention

[0004] The purpose of the invention is to improve the thermal stability of the existing reverse transcriptase UCRT vZ.

[0005] To this end, the present invention provides a reverse transcriptase UCRT vZ mutant with improved thermal stability, wherein the reverse transcriptase UCRT vZ mutant is as follows (a1) or (a2):

[0006] (a1) a derivative protein in which one or more amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO.2 and which has the same function as the amino acid sequence shown in SEQ ID NO.2;

[0007] (a2) A derivative protein in which one or more amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO. 2 and which has at least 90% homology with the amino acid sequence shown in SEQ ID NO. 2.

[0008] Specifically, the amino acid sequence of the above-mentioned reverse transcriptase UCRT vZ mutant is configured as the amino acid sequence after one or more combinations of mutations at the mutation sites Q319G, N606Y, V188F, and K238W on SEQ ID NO.2 are mutated.

[0009] Specifically, the above mutation sites are Q319G, N606Y, V188F, K238W, Q319G / N606Y, Q319G / V188F, Q319G / K238W, N606Y / V188F, N606Y / K238W, V188F / K238W, Q319G / N606Y / V188F, Q319G / N606Y / K238W, N606Y / V188F / K238W, Q319G / V188F / K238W or Q319G / N606Y / V188F / K238W.

[0010] Specifically, the amino acid sequence of the single point mutant corresponding to the Q319G is SEQ ID NO.3;

[0011] The amino acid sequence of the single point mutant corresponding to N606Y is SEQ ID NO.4;

[0012] The amino acid sequence of the single point mutant corresponding to V188F is SEQ ID NO.6;

[0013] The amino acid sequence of the single point mutant corresponding to K238W is SEQ ID NO.6;

[0014] The amino acid sequence of the combined mutant corresponding to Q319G / N606Y is SEQ ID NO.7;

[0015] The amino acid sequence of the combined mutant corresponding to Q319G / V188F is SEQ ID NO.8;

[0016] The amino acid sequence of the combined mutant corresponding to Q319G / K238W is SEQ ID NO.9;

[0017] The amino acid sequence of the combined mutant corresponding to N606Y / V188F is SEQ ID NO.10;

[0018] The amino acid sequence of the combined mutant corresponding to N606Y / K238W is SEQ ID NO.11;

[0019] The amino acid sequence of the combined mutant corresponding to V188F / K238W is SEQ ID NO. 12;

[0020] The amino acid sequence of the combined mutant corresponding to Q319G / N606Y / V188F is SEQ ID NO. 13;

[0021] The amino acid sequence of the combined mutant corresponding to Q319G / N606Y / K238W is SEQ ID NO.14;

[0022] The amino acid sequence of the combined mutant corresponding to N606Y / V188F / K238W is SEQ ID NO. 16;

[0023] The amino acid sequence of the combined mutant corresponding to Q319G / V188F / K238W is SEQ ID NO. 16;

[0024] The amino acid sequence of the combined mutant corresponding to Q319G / N606Y / V188F / K238W is SEQ ID NO.17.

[0025] The present invention also provides a method for constructing the above-mentioned reverse transcriptase UCRT vZ mutant with improved thermal stability, comprising the following steps:

[0026] Search and select amino acid sequences with a consistency greater than 60% with the amino acid sequence shown in SEQ ID NO. 2 in the database, then perform multiple sequence alignment, and generate a consensus sequence that can be edited later using software;

[0027] The protein three-dimensional structure of SEQ ID NO.2 was predicted, and the stability-related mutation sites were screened out: Q319G, N606Y, V188F, and K238W.

[0028] Specifically, the amplification primer sequences of the mutation site Q319G are SEQ ID NO.20 and SEQ ID NO.21;

[0029] The amplification primer sequences for the mutation site N606Y are SEQ ID NO.22 and SEQ ID NO.23;

[0030] The amplification primer sequences for the mutation site V188F are SEQ ID NO.24 and SEQ ID NO.26;

[0031] The amplification primer sequences for the mutation site K238W are SEQ ID NO.26 and SEQ ID NO.27.

[0032] The present invention also provides the gene of the reverse transcriptase UCRT vZ mutant with improved thermal stability.

[0033] The invention also provides a recombinant plasmid comprising the gene.

[0034] The present invention also provides a soluble protein, an immobilized enzyme or an engineered bacterium comprising the reverse transcriptase UCRT vZ mutant with improved thermal stability.

[0035] The reverse transcriptase UCRT vZ mutant with improved thermal stability provided by the present invention can be used to reverse transcribe RNA into cDNA.

[0036] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0037] 1. The reverse transcriptase UCRT vZ mutant with improved thermal stability provided by the present invention includes single point mutants and combined mutants. Compared with the wild-type reverse transcriptase UCRT vZ, the single point mutants and combined mutants have longer half-lives at 66°C; in particular, the combined mutants show the superposition effect of the thermal stability of the single point mutants, and their half-life is about 4 times that of the wild type. The UCRT vZ mutant has excellent catalytic activity and has good application prospects.

[0038] 2. The method for constructing a reverse transcriptase UCRT vZ mutant with improved thermal stability provided by the present invention is different from the rational design based on the precise structure-function relationship of the protein. The present invention is guided by the Consensus Concept theory, analyzes the information that can improve the thermal stability of the enzyme from an evolutionary perspective, integrates and analyzes the reverse transcriptase UCRT vZ family sequence, and combines bioinformatics and crystallography methods to obtain a new reverse transcriptase UCRT vZ mutant with high stability.

[0039] The present invention will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a schematic diagram of the simulated crystal structure of the reverse transcriptase UCRT vZ protein provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0041] The technical scheme in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be limited by the attached claims and their equivalents.

[0042] The present invention provides a reverse transcriptase UCRT vZ mutant with improved thermal stability, wherein the reverse transcriptase UCRTvZ mutant is as follows (a1) or (a2):

[0043] (a1) a derivative protein in which one or more amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO.2 and which has the same function as the amino acid sequence shown in SEQ ID NO.2;

[0044] (a2) A derivative protein in which one or more amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO. 2 and which has at least 90% homology with the amino acid sequence shown in SEQ ID NO. 2.

[0045] The amino acid sequence of the reverse transcriptase UCRT vZ mutant is configured as the amino acid sequence after one or more combinations of mutation sites Q319G, N606Y, V188F, and K238W on SEQ ID NO.2 are mutated.

[0046] The mutation sites are Q319G, N606Y, V188F, K238W, Q319G / N606Y, Q319G / V188F, Q319G / K238W, N606Y / V188F, N606Y / K238W, V188F / K238W, Q319G / N606Y / V188F, Q319G / N606Y / K238W, N606Y / V188F / K238W, Q319G / V188F / K238W or Q319G / N606Y / V188F / K238W.

[0047] Among them, the amino acid sequence of the single point mutant corresponding to Q319G is SEQ ID NO.3;

[0048] The amino acid sequence of the single point mutant corresponding to N606Y is SEQ ID NO.4;

[0049] The amino acid sequence of the single point mutant corresponding to V188F is SEQ ID NO.6;

[0050] The amino acid sequence of the single point mutant corresponding to K238W is SEQ ID NO.6;

[0051] The amino acid sequence of the combined mutant corresponding to Q319G / N606Y is SEQ ID NO.7;

[0052] The amino acid sequence of the combined mutant corresponding to Q319G / V188F is SEQ ID NO.8;

[0053] The amino acid sequence of the combined mutant corresponding to Q319G / K238W is SEQ ID NO.9;

[0054] The amino acid sequence of the combined mutant corresponding to N606Y / V188F is SEQ ID NO.10;

[0055] The amino acid sequence of the combined mutant corresponding to N606Y / K238W is SEQ ID NO.11;

[0056] The amino acid sequence of the combined mutant corresponding to V188F / K238W is SEQ ID NO. 12;

[0057] The amino acid sequence of the combined mutant corresponding to Q319G / N606Y / V188F is SEQ ID NO. 13;

[0058] The amino acid sequence of the combined mutant corresponding to Q319G / N606Y / K238W is SEQ ID NO. 14;

[0059] The amino acid sequence of the combined mutant corresponding to N606Y / V188F / K238W is SEQ ID NO. 16;

[0060] The amino acid sequence of the combined mutant corresponding to Q319G / V188F / K238W is SEQ ID NO. 16;

[0061] The amino acid sequence of the combined mutant corresponding to Q319G / N606Y / V188F / K238W is SEQ ID NO.17.

[0062] The present invention also provides a method for constructing the above-mentioned reverse transcriptase UCRT vZ mutant with improved thermal stability, comprising the following steps:

[0063] By searching the amino acid sequence shown in SEQ ID NO.2 in the Pfam database and the NCBI database, the repeated identical sequences were removed, and the amino acid sequences with a consistency of more than 30% with the amino acid sequence shown in SEQ ID NO.2 were selected. Then, multiple sequence alignment was performed using Clustalx1.83 software, and the remaining amino acid sequences were organized into fasta files and uploaded to the Consensus Maker v2.0.0 server. After modifying the setting parameters as needed, the online software will generate a consensus sequence that can be edited later.

[0064] The three-dimensional structure of the protein shown in SEQ ID NO.2 was predicted by the Swissmodel online tool, and the crystal structure of the protein shown in SEQ ID NO.2 was observed using PyMOL, and the mutation sites related to thermal stability were screened out as follows: Q319G, N606Y, V188F, and K238W.

[0065] Among them, the amplification primer sequences of the mutation site Q319G are SEQ ID NO.20 and SEQ ID NO.21;

[0066] The amplification primer sequences for the mutation site N606Y are SEQ ID NO.22 and SEQ ID NO.23;

[0067] The amplification primer sequences for the mutation site V188F are SEQ ID NO.24 and SEQ ID NO.26;

[0068] The amplification primer sequences for the mutation site K238W are SEQ ID NO.26 and SEQ ID NO.27.

[0069] The effect of the reverse transcriptase UCRT vZ mutant with improved thermal stability of the present invention is studied through specific examples below.

[0070] Embodiment 1:

[0071] This embodiment provides a reverse transcriptase UCRT vZ mutant with improved thermal stability, wherein the reverse transcriptase UCRTvZ is an uncultured virus isolated from the heat-resistant bacteria in Yellowstone Park, the wild-type reverse transcriptase is named UCRTvZ, the nucleic acid sequence encoding the reverse transcriptase UCRT vZ protein is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO.2.

[0072] SEQ ID NO.1

[0073]

[0074] SEQ ID NO.2

[0075] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0076] The reverse transcriptase UCRT vZ mutant with improved thermal stability provided in this embodiment includes: the amino acid sequence shown in SEQ ID NO.2 is substituted, deleted or added with one or more amino acids to form a derivative protein with the same function as the amino acid sequence shown in SEQ ID NO.2 (i.e., the reverse transcriptase UCRT vZ protein), or the amino acid sequence shown in SEQ ID NO.2 is substituted, deleted or added with one or more amino acids to form a derivative protein with at least 90% homology with the amino acid sequence shown in SEQ ID NO.2 (i.e., the reverse transcriptase UCRT vZ protein).

[0077] Specifically, a single point mutation was performed on a certain site in the amino acid sequence shown in SEQ ID NO.2 to obtain four reverse transcriptase UCRT vZ single point mutants, whose mutation sites were: Q319G, N606Y, V188F, and K238W. The activity of the four reverse transcriptase UCRT vZ single point mutants was tested, and their amino acid sequences were SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.6, respectively.

[0078] SEQ ID NO.3

[0079] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKGIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0080] SEQ ID NO.4

[0081] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIAYGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0082] SEQ ID NO.6

[0083] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIFDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0084] SEQ ID NO.6

[0085] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFWNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0086] Alternatively, multiple mutation sites are selected and combined in the amino acid sequence shown in SEQ ID NO.2, such as selecting 2 mutation sites from the above 4 mutation sites for combination, to obtain the following 6 reverse transcriptase UCRTvZ mutants with improved thermal stability, wherein the combined mutation sites are: Q319G / N606Y, Q319G / V188F, Q319G / K238W, N606Y / V188F, N606Y / K238W, V188F / K238W, Q319G / N606Y / V188F, Q319G / N606Y / K238W, N606Y / V188F / K238W, Q319G / V188F / K238W, Q319G / N606Y / V188F / K238W, and the amino acid sequences are SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12.

[0087] SEQ ID NO.7

[0088] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKGIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIAYGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0089] SEQ ID NO.8

[0090] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIFDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKGIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0091] SEQ ID NO.9

[0092] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFWNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKGIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0093] SEQ ID NO.10

[0094] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIFDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIAYGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0095] SEQ ID NO.11

[0096] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFWNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIAYGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0097] SEQ ID NO.12

[0098] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIFDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFWNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0099] If three mutation sites are selected from the above four mutation sites and combined, four reverse transcriptase UCRT vZ mutants with improved thermal stability are obtained, and their combined mutation sites are: Q319G / N606Y / V188F, Q319G / N606Y / K238W, Q319G / V188F / K238W, N606Y / V188F / K238W, and their amino acid sequences are SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.16, and SEQ ID NO.16, respectively.

[0100] SEQ ID NO.13

[0101] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIFDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKGIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIAYGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0102] SEQ ID NO.14

[0103] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFWNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKGIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIAYGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0104] SEQ ID NO.16

[0105] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIFDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFWNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIAYGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0106] SEQ ID NO.16

[0107] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIFDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFWNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKGIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0108] For example, by selecting 4 mutation sites from the above 4 mutation sites and combining them, a reverse transcriptase UCRT vZ mutant with improved thermal stability is obtained, and its combined mutation sites are: Q319G / N606Y / V188F / K238W, and its amino acid sequence is SEQ ID NO.17.

[0109] SEQ ID NO.17

[0110] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIFDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFWNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKGIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIAYGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0111] Embodiment 2:

[0112] This embodiment provides a method for constructing a reverse transcriptase UCRT vZ mutant with improved thermal stability, comprising the following steps:

[0113] 1. Cloning of the wild-type reverse transcriptase UCRT vZ gene

[0114] The wild-type reverse transcriptase UCRT vZ gene was codon optimized using Escherichia coli as a host cell to obtain an optimized reverse transcriptase UCRT vZ gene, whose nucleic acid sequence is SEQ ID NO.1 and the expressed amino acid sequence is SEQ ID NO.2; SEQ ID NO.1 was used as the target gene, and the upstream amplification primer SEQ ID NO.18 and the downstream amplification primer SEQ ID NO.19 were used to amplify the target gene;

[0115] The nucleic acid sequence of SEQ ID NO.18 is:

[0116] 6'-ACTGCT CATATG ATGGTAAAAGTTAAGTTTAAGTATAAAG-3' (where the underline indicates the restriction endonuclease NdeI recognition site);

[0117] The nucleic acid sequence of SEQ ID NO.19 is:

[0118] 6'-TCAGCT CTCGAG GGGCTTCTCCCATGTGTTACC-3' (the underline indicates the restriction endonuclease XhoI recognition site).

[0119] The amplification conditions were as follows: amplification at 96°C for 2 min, followed by amplification at 66°C for 20 sec, amplification at 72°C for 90 sec, for a total of 30 cycles, and finally amplification at 72°C for 10 min.

[0120] After the reaction was completed, 1.6% agarose gel electrophoresis was used to detect the PCR amplification product, and a 1.0 kb band was obtained, the length of which was consistent with the expected result. According to the standard operation of the kit, the target fragment was recovered and purified, and the target fragment and the pET28a plasmid were double-digested with restriction endonucleases XhoI and NdeI, and then connected with T4 DNA ligase, and the obtained connection product was transformed into Escherichia coli BL21 (DE3) competent cells, and the transformed cells were spread on LB plates containing 60 μg / ml kanamycin, and the positive clone plasmid was extracted and sequenced. The results showed that the cloned reverse transcriptase UCRT vZ gene sequence was correct and had been correctly connected to the pET28a plasmid to obtain the recombinant plasmid pET28a-Bst;

[0121] Among them, the wild-type reverse transcriptase UCRT vZ is derived from the uncultured virus isolated from the heat-resistant bacteria in Yellowstone Park;

[0122] The reverse transcriptase UCRT vZ gene was provided by Suzhou Genewise Biotechnology Co., Ltd.;

[0123] The PCR amplification enzyme was KOD high-fidelity polymerase provided by Toyobo.

[0124] 2. Expression and Purification of Reverse Transcriptase UCRT vZ Protein

[0125] The engineered bacteria in the glycerol tube were inoculated into a 4mL LB medium test tube containing 100μg / mL Kan at a volume ratio of 1%, and cultured at 37°C and 220rpm for 12h; 4mL of the bacterial solution was transferred to a 1L LB medium shake flask containing 60μg / mL Kan, and cultured at 37°C and 220rpm for 2.6h until the OD600 reached about 0.9, and 0.1mM IPTG inducer was added, and induced culture was carried out at 26°C and 200rpm for 14h. The Escherichia coli cell suspension harvested after fermentation was ultrasonically disrupted, and then subjected to a one-step Ni-NTA affinity chromatography treatment to obtain a reverse transcriptase UCRT vZ protein with a purity of >96%, and the amino acid sequence was SEQ ID NO.2.

[0126] 3. Multiple sequence alignment and consensus analysis of reverse transcriptase UCRT vZ homologous proteins

[0127] 3.1. Enter the Pfam database homepage (http: / / pfam.xfam.org / ), enter the amino acid sequence of reverse transcriptase UCRT vZ in the SEQUENCE SEARCH tool for search, the server will directly feedback the comparison results of the amino acid sequence of the entire protein family, and display the abundance of various amino acids at each mutation site in the form of a bar graph. The website can also automatically generate a consensus sequence for the protein family;

[0128] 3.2. Input the amino acid sequence shown in SEQ ID NO.2 into the NCBI protein database and the Pfam database, and use the Blast tool to find all protein sequences with a consistency greater than 30% with the amino acid sequence of the reverse transcriptase UCRT vZ protein (SEQ ID NO.2), delete the repeated identical sequences, and organize the remaining amino acid sequences into fasta. format, input Clustalx1.83 software for multiple sequence alignment, and output the alignment results in aln., dnd. and fasta. formats, where the dnd. file is a file for constructing an evolutionary tree, and the aln. and fasta. files are sequence files in different formats;

[0129] Upload the above fasta. file to the Consensus Maker v2.0.0 (http: / / www.hiv.lanl.gov / content / sequence / CONSENSUS / consensus.html) server. After modifying the setting parameters as needed, the online software will generate a consensus sequence that can be edited later.

[0130] 3.3. The amino acid sequence of the reverse transcriptase UCRT vZ protein (SEQ ID NO. 2) was compared with the consensus sequence of the family and the amino acid abundance map of each site.

[0131] 4. Simulation of the three-dimensional structure of the reverse transcriptase UCRT vZ protein and selection of mutation hotspots

[0132] 4.1. The three-dimensional structure prediction of the reverse transcriptase UCRT vZ protein (amino acid sequence SEQID NO.2) was obtained using the Swissmodel online tool;

[0133] 4.2. The crystal structure of the reverse transcriptase UCRT vZ protein (amino acid sequence SEQ ID NO. 2) was observed using PyMOL. The candidate mutation sites and mutation forms were reviewed based on the structural information to screen out the mutant sites that are most likely to improve the thermal stability of the reverse transcriptase UCRT vZ protein. The screening conditions are as follows:

[0134] (1) The criteria for determining a site as a candidate site are:

[0135] ① Most proteins in this family have a high overall abundance of amino acids at this site;

[0136] ②The amino acid at this position is conservative;

[0137] ③ The amino acids with higher frequency at this site have significant differences in physical and chemical properties with the amino acids at this site of the reverse transcriptase UCRT vZ protein, such as charge difference, polarity, and steric hindrance.

[0138] (2) Remove the area near the active center, i.e., the area away from the catalytic residue ( The amino acid residues within the range are removed, except for those in buried or semi-buried states.

[0139] After the above two steps of screening, there are 10 differential sites left, most of which are located on the surface of the reverse transcriptase UCRT vZ protein molecule, such as Figure 1 As shown, the arrow points to the mutation site.

[0140] (3) Based on the crystal structure of the reverse transcriptase UCRT vZ protein, the above 10 mutation forms were analyzed in detail one by one to screen out mutants that may improve the thermal stability of the reverse transcriptase UCRT vZ protein.

[0141] The main judgment criteria are: ① Mutations should eliminate the original forms of forces that are not conducive to thermal stability, such as electrostatic repulsion, charge aggregation, etc.; ② Mutations should not destroy existing forms of forces that are conducive to thermal stability and stable protein structures; ③ Mutations should introduce new forms of forces that are conducive to thermal stability, such as hydrogen bonds, salt bridges, hydrophobic interactions, etc.

[0142] A total of four single-point mutants were designed, and their mutation sites were: Q319G, N606Y, V188F, and K238W;

[0143] The activity of the four reverse transcriptase UCRT vZ single point mutants was determined, and four reverse transcriptase UCRT vZ mutants with improved thermal stability were screened out, whose mutation sites were: Q319G, N606Y, V188F, and K238W, and the corresponding amino acid sequences of the single point mutants were SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.6, respectively.

[0144] 6. Construction, expression and purification of mutants

[0145] 6.1. Construction of single-point mutants of reverse transcriptase UCRT vZ protein

[0146] Using the recombinant plasmid pET28a-Bst in step 1 as a template and a pair of complementary oligonucleotides with mutation sites as amplification primers, the whole plasmid PCR amplification was performed using KOD high-fidelity enzyme to obtain a recombinant plasmid with a specific mutation site;

[0147] The amplification primer pairs used were:

[0148] (1) The nucleic acid sequences of the upstream amplification primer SEQ ID NO.20 and the downstream amplification primer SEQ ID NO.21 of the mutation site Q319G are as follows:

[0149] SEQ ID NO.20:

[0150] 6'-AAGGGATCCCCTTTAATTATAACTCCCC-3';

[0151] SEQ ID NO.21:

[0152] 6'-GATGGGTTTTTGCATCTCCGAGCGGA-3';

[0153] (2) The nucleic acid sequences of the upstream amplification primer SEQ ID NO.22 and the downstream amplification primer SEQ ID NO.23 of the mutation site N606Y are as follows:

[0154] SEQ ID NO.22:

[0155] 6'-GCTTATGGAATCAACATGACCGAGGA-3';

[0156] SEQ ID NO.23:

[0157] 6'-TCCATAAGCAATACAATATTCGGCGAAG-3';

[0158] (3) The nucleic acid sequences of the upstream amplification primer SEQ ID NO.24 and the downstream amplification primer SEQ ID NO.26 of the mutation site V188F are as follows:

[0159] SEQ ID NO.24:

[0160] 6'-AAATTTTCGACGTACAATTAGCG-3';

[0161] SEQ ID NO.26:

[0162] 6'-CGTCAAGAATTTTCTTATTTCGGAGTTGT-3';

[0163] (4) The nucleic acid sequences of the upstream amplification primer SEQ ID NO.26 and the downstream amplification primer SEQ ID NO.27 of the mutation site K238W are as follows:

[0164] SEQ ID NO.26:

[0165] 6'-GACATTCTGGAACAACATGTTTCATTCCT-3';

[0166] SEQ ID NO.27:

[0167] 6'-GTTCCAGAATGTCTCACGTATTTTTGTC-3';

[0168] The amplification conditions were as follows: amplification at 96°C for 2 min, then at 66°C for 20 sec, and at 72°C for 90 sec, for a total of 30 cycles, and finally at 72°C for 10 min; the PCR amplification product was recovered by gel, and the gel-recovered product was digested with DpnI enzyme at 37°C for 2 h to degrade the initial template; the digestion product was transformed into Escherichia coli BL21 (DE3) competent cells, spread on LB agar plates containing 60 μg / mL kanamycin, cultured at 37°C overnight, positive clones were screened, and sequencing was performed to obtain recombinant bacteria containing the reverse transcriptase UCRT vZ single-point mutant;

[0169] Among them, the above-mentioned KOD high-fidelity enzyme was provided by TakaRa;

[0170] The above DpnI enzyme was provided by Fermentas.

[0171] 6.2. Construction of reverse transcriptase UCRT vZ protein combination mutants

[0172] Using a construction method similar to that of single point mutants, single point mutants with improved stability are cumulatively combined, and multiple mutation sites are selected in the amino acid sequence shown in SEQ ID NO.2 for combination, such as selecting 2 to 4 mutation sites from the above 4 mutation sites for combination, to obtain different reverse transcriptase UCRT vZ combination mutants:

[0173] (1) Selecting two mutation sites for combination, six reverse transcriptase UCRT vZ mutants with improved thermal stability can be constructed. The combined mutation sites are: Q319G / N606Y, Q319G / V188F, Q319G / K238W, N606Y / V188F, N606Y / K238W, V188F / K238W, and the amino acid sequences of the six reverse transcriptase UCRT vZ combined mutants with improved thermal stability are SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively;

[0174] (2) Three mutation sites were selected for combination to construct four reverse transcriptase UCRT vZ combination mutants with improved thermal stability, wherein the combination mutation sites were Q319G / N606Y / V188F, Q319G / N606Y / K238W, N606Y / V188F / K238W, and Q319G / V188F / K238W. The amino acid sequences of the four reverse transcriptase UCRT vZ combination mutants with improved thermal stability were SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.16, and SEQ ID NO.16, respectively;

[0175] (3) Four mutation sites are selected for combination to construct a reverse transcriptase UCRT vZ combination mutant with improved thermal stability, wherein the combined mutation sites are: Q319G / N606Y / V188F / K238W, and the amino acid sequence of the reverse transcriptase UCRT vZ combination mutant with improved thermal stability is SEQ ID NO.17.

[0176] Embodiment 3:

[0177] This example provides a gene encoding the reverse transcriptase UCRT vZ mutant with improved thermal stability as described in Example 1:

[0178] (1) The nucleic acid sequence encoding the reverse transcriptase UCRT vZ mutant with a mutation site of Q319G is SEQ ID NO. 28;

[0179] SEQ ID NO.28

[0180]

[0181] (2) The nucleic acid sequence of the reverse transcriptase UCRT vZ mutant with the coding mutation site N606Y is SEQ ID NO.29;

[0182] SEQ ID NO.29

[0183] ATGGTAAAAGTTAAGTTTAAGTATAAAGGTGAAGAATTACAGGTAGACACGAGCAAGATAAAGAAAGTTTGGCGTGTCGGAAAAGCTATCAGCTTCACATACGACCAAGGAAAGACGGGGAGAGGGGCCGTGTCAGAAAAGGATGCGCCGAAGGAACTCCTTGATATGCTAGCTCGGGCCGAGCGCGAAAAGAAAGGTAGCGCCGGCATGGGTGAAGACGGTTTATCTCTACCCAAAATGATGAATACGCCAAAACCCATCCTAAAACCACAACCAAAGGCGCTTGTAGAACCCGTCTTATGTGATAGTATCGATGAGATACCTGCTAAGTATAATGAACCAGTTTATTTTGATTTAGCGACAGATGAGGATAGACCAGTACTTGCGTCAATCTATCAGCCTCATTTTGAGCGCAAAGTGTATTGT

[0184] CTTAACCTACTCAAGGAAAAGGTAGCCCGATTCAAGGATTGGCTTTTA

[0185] AAGTTTAGTGAAATCCGTGGTTGGGGCCTAGACTTCGATCTTAGAGTA

[0186] TTAGGGTACACCTATGAACAACTCCGAAATAAGAAAATTGTTGACGTA

[0187] CAATTAGCGATAAAGGTGCAGCACTATGAAAGGTTCAAACAAGGCGG

[0188] AACCAAGGGGGAGGGCTTTCGGCTAGACGATGTTGCCCGAGACCTCC

[0189] TAGGGATAGAATATCCAATGAATAAGACAAAATACGTGGAGACATTCA

[0190] AAAACAACATGTTTCATTCCTTTTCCAATGAACAGCTGCTATATGCATC

[0191] TCTTGACGCATATATACCGCACTTACTTTATGAACACTCACATCATCC

[0192] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAGTG

[0193] GTAATAGAGACGTCCCAGCATGGTATGCCGGTTAAATTAAAAAGCATTG

[0194] GAAGAAGAGATCCATCGGCTGACTCAGCTCCGCTCGGAGATGCAAAA

[0195] ACAGATCCCCTTTAATTATAACTCCCCGAAACAGACTGCGAAATTTTTC

[0196] GGTGTAAATTCGTCCAGCAAAGACGTTCTAATGGACTTAGCTTTGCAA

[0197] GGGAACGAAATGGCAAAAAAAGTTTAGAGGCCAGACAGATTGAAA

[0198] AATCACTTGCCTTTGCCAAGGACCTCTATGACATAGCAAAGCGCTCCG

[0199] GGGGGCGTATCTATGGCAACTTCTTCACTACCACCGCCCGTCAGGGC

[0200] GCATGAGCTGCTCTGACATAAATCTGCAACAAATTCCACGAAGGTTAC

[0201] GGTCGTTCATTGGGTTCGACACCGAAGACAAAAATTGATAACTGCA

[0202] GACTTCCCCCAGATTGAACTTCGTCTCGCCGGTGTAATCTGGAACGAA

[0203] CCTAAGTTCATCGAGGCATTTCGGCAAGGGATTGATTTACATAAGTTA

[0204] ACTGCTAGCATCTTGTTCGACAAGAATATCGAGGAGGTAAGTAAAGA

[0205] AGAGCGTCAGATTGGTAAATCCGCAAACTTCGGTTTAATCTACGGTAT

[0206] CGCCCCCAAAGGCTTCGCCGAATATTGTATTGCTTATGGAATCAACATG

[0207] ACCGAGGAGCAGGCCTATGAGATCGTTCGCAAATGGAAAAAGTATTAT

[0208] ACTAAGATTGCAGAACAGCACCAGGTGGCATATGAGAGATTCAAATAT

[0209] AATGAGTATGTGGACAATGAAACCTGGTTAAATCGGACCTACCGCGCC

[0210] TGGAAACCGCAAGACTTGCTAAACTATCAGATACAGGGATCCGGCGC

[0211] CGAATTGTTCAAAAAAGCCATCGTCCTACTAAAGGAGACTAAGCCAG

[0212] ATTTAAAGATAGTAAATCTAGTACATGATGAGATTGTGGTAGAAGCTGA

[0213] TTCGAAGGAAGCTCAGGATCTAGCCAAACTAATCAAGGAAAAAATGG

[0214] AGGAAGCCTGGGACTGGTGTTTGGAAAAGGCAGAGGAGTTTGGGAA

[0215] TAGGGTCGCTAAGATCAAGTTAGAAGTGGAGGAGCCGCACGTGGGTA

[0216] ACACATGGGAGAAGCCC

[0217] (3) The nucleic acid sequence encoding the reverse transcriptase UCRT vZ mutant with mutation site V188F is SEQ ID NO.30;

[0218] SEQ ID NO.30

[0219]

[0220] (4) The nucleic acid sequence of the reverse transcriptase UCRT vZ mutant with the coding mutation site K238W is SEQ ID NO.31;

[0221] SEQ ID NO.31

[0222] ATGGTAAAAGTTAAGTTTAAGTATAAAGGTGAAGAATTACAGGTAGACACGAGCAAGATAAAGAAAGTTTGGCGTGTCGGAAAAGCTATCAGCTTCACATACGACCAAGGAAAGACGGGGAGAGGGGCCGTGTCAGAA

[0223] AAGGATGCGCCGAAGGAACTCCTTGATATGCTAGCTCGGGCCGAGCG

[0224] CGAAAAGAAAGGTAGCGCCGGCATGGGTGAAGACGGTTTATCTCTAC

[0225] CCAAAATGATGAATACGCCAAAACCCATCCTAAAACCACAACCAAAG

[0226] GCGCTTGTAGAACCCGTCTTATGTGATAGTATCGATGAGATACCTGCTA

[0227] AGTATAATGAACCAGTTTATTTTGATTTAGCGACAGATGAGGATAGACC

[0228] AGTACTTGCGTCAATCTATCAGCCTCATTTTGAGCGCAAAGTGTATTGT

[0229] CTTAACCTACTCAAGGAAAAGGTAGCCCGATTCAAGGATTGGCTTTTA

[0230] AAGTTTAGTGAAATCCGTGGTTGGGGCCTAGACTTCGATCTTAGAGTA

[0231] TTAGGGTACACCTATGAACAACTCCGAAATAAGAAAATTGTTGACGTA

[0232] CAATAGCGATAAAGGTGCAGCACTATGAAAGGTTCAAACAAGGCGG

[0233] AACCAAGGGGGAGGGCTTTCGGCTAGACGATGTTGCCCGAGACCTCCC

[0234] TAGGGATAGAATATCCAATGAATAAGACAAAATACGTGGAGACATTCT

[0235] GGAAACATGTTTCATTCCTTTTCCAATGAACAGCTGCTATATGCATC

[0236] TCTTGACGCATATATACCGCACTTACTTTATGAACACTCACATCATCC

[0237] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAGTG

[0238] GTAATAGAGACGTCCCAGCATGGTATGCCGGTTAAATTAAAAAGCATTG

[0239] GAAGAAGAGATCCATCGGCTGACTCAGCTCCGCTCGGAGATGCAAAA

[0240] ACAGATCCCCTTTAATTATAACTCCCCGAAACAGACTGCGAAATTTTTC

[0241] GGTGTAAATTCGTCCAGCAAAGACGTTCTAATGGACTTAGCTTTGCAA

[0242] GGGAACGAAATGGCAAAAAAAGTTTAGAGGCCAGACAGATTGAAA

[0243] AATCACTTGCCTTTGCCAAGGACCTCTATGACATAGCAAAGCGCTCCG

[0244] GGGGGCGTATCTATGGCAACTTCTTCACTACCACCGCCCGTCAGGGC

[0245] GCATGAGCTGCTCTGACATAAATCTGCAACAAATTCCACGAAGGTTAC

[0246] GGTCGTTCATTGGGTTCGACACCGAAGACAAAAAATTGATAACTGCA

[0247] GACTTCCCCCAGATTGAACTTCGTCTCGCCGGTGTAATCTGGAACGAA

[0248] CCTAAGTTCATCGAGGCATTTCGGCAAGGGATTGATTTACATAAGTTA

[0249] ACTGCTAGCATCTTGTTCGACAAGAATATCGAGGAGGTAAGTAAAGA

[0250] AGAGCGTCAGATTGGTAAATCCGCAAACTTCGGTTTAATCTACGGTAT

[0251] CGCCCCCAAAGGCTTCGCCGAATATTGTATTGCTAATGGAATCAACAT

[0252] GACCGAGGAGCAGGCCTATGAGATCGTTCGCAAATGGAAAAAGTATT

[0253] ATACTAAGATTGCAGAACAGCACCAGGTGGCATATGAGAGATTCAAAT

[0254] ATAATGAGTATGTGGACAATGAAACCTGGTTAAATCGGACCTACCGCG

[0255] CCTGGAAACCGCAAGACTTGCTAAACTATCAGATACAGGGATCCGGC

[0256] GCCGAATTGTTCAAAAAAGCCATCGTCCTACTAAAGGAGACTAAGCC

[0257] AGATTTAAAGATAGTAAATCTAGTACATGATGAGATTGTGGTAGAAGC

[0258] TGATTCGAAGGAAGCTCAGGATCTAGCCAAACTAATCAAGGAAAAAA

[0259] TGGAGGAAGCCTGGGACTGGTGTTTGGAAAAGGCAGAGGAGTTTGG

[0260] GAATAGGGTCGCTAAGATCAAGTTAGAAGTGGAGGAGCCGCACGTGG

[0261] GTAACACATGGGAGAAGCCC

[0262] (6) The nucleic acid sequence encoding the reverse transcriptase UCRT vZ mutant with mutation site Q319G / N606Y is SEQ ID NO.32;

[0263] SEQ ID NO.32

[0264] ATGGTAAAAGTTAAGTTTAAGTATAAAGGTGAAGAATTACAGGTAGACACGAGCAAGATAAAGAAAGTTTGGCGTGTCGGAAAAGCTATCAGCTTCACATACGACCAAGGAAAGACGGGGAGAGGGGCCGTGTCAGAAAAGGATGCGCCGAAGGAACTCCTTGATATGCTAGCTCGGGCCGAGCGCGAAAAGAAAGGTAGCGCCGGCATGGGTGAAGACGGTTTATCTCTACCCAAAATGATGAATACGCCAAAACCCATCCTAAAACCACAACCAAAGGCGCTTGTAGAACCCGTCTTATGTGATAGTATCGATGAGATACCTGCTAAGTATAATGAACCAGTTTATTTTGATTTAGCGACAGATGAGGATAGACCAGTACTTGCGTCAATCTATCAGCCTCATTTTGAGCGCAAAGTGTATTGTCTTAACCTACTCAAGGAAAAGGTAGCCCGATTCAAGGATTGGCTTTTAAAGTTTAGTGAAATCCGTGGTTGGGGCCTAGACTTCGATCTTAGAGTATTAGGGTACACCTATGAACAACTCCGAAATAAGAAAATTGTTGACGTACAATTAGCGATAAAGGTGCAGCACTATGAAAGGTTCAAACAAGGCGGAACCAAGGGGGAGGGCTTTCGGCTAGACGATGTTGCCCGAGACCTCC

[0265] TAGGGATAGAATATCCAATGAATAAGACAAAAATACGTGAGACATTCA

[0266] AAAACAACATGTTTCATTCCTTTTCCAATGAACAGCTGCTATATGCATC

[0267] TCTTGACGCATATATACCGCACTTACTTTATGAACAACTCACATCATCC

[0268] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAGTG

[0269] GTAATAGAGACGTCCCAGCATGGTATGCCGGTTAAATTAAAAAGCATTG

[0270] GAAGAAGAGATCCATCGGCTGACTCAGCTCCGCTCGGAGATGCAAAA

[0271] AGAGATCCCCTTTAATTATAACTCCCCGAAACAGACTGCGAAATTTTT

[0272] CGGTGTAAATTCGTCCAGCAAAGACGTTCTAATGGACTTAGCTTTGCA

[0273] AGGGAACGAAATGGCAAAAAAAGTTTAGAGGCCAGACAGATTGAA

[0274] AAATCACTTGCCTTTGCCAAGGACCTCTATGACATAGCAAAGCGCTCC

[0275] GGGGGGCGTATCTATGGCAACTTCTTCACTACCACCGCCCCGTCAGGG

[0276] CGCATGAGCTGCTCTGACATAATCTGCAACAAATTCCACGAAGGTTA

[0277] CGGTCGTTCATTGGGTTCGACACCGAAGACAAAAATTGATAACTGC

[0278] AGACTTCCCCCAGATTGAACTTCGTCTCGCCGGTGTAATCTGGAACGA

[0279] ACCTAAGTTCATCGAGGCATTTCGGCAAGGGATTGATTTACATAAGTT

[0280] AACTGCTAGCATCTTTGTTCGACAAGAATATCGAGGGAGGTAAGTAAG

[0281] AAGAGCGTCAGATTGGTAAATCCGCAAACTTCGGTTTAATCTACGGTA

[0282] TCGCCCCCAAAGGCTTCGCCGAATATTGTATTGCTTATGGAATCAACAT

[0283] GACCGAGGAGCAGGCCTATGAGATCGTTCGCAAATGGAAAAAGTATT

[0284] ATACTAAGATTGCAGAACAGCACCAGGTGGCATATGAGAGATTCAAAT

[0285] ATAATGAGTATGTGGACAATGAAACCTGGTTAAATCGGACCTACCGCG

[0286] CCTGGAAACCGCAAGACTTGCTAAACTATCAGATACAGGGATCCGGC

[0287] GCCGAATTGTTCAAAAAAGCCATCGTCCTACTAAAGGAGACTAAGCC

[0288] AGATTTAAAGATAGTAAATCTAGTACATGATGAGATTGTGGTAGAAGC

[0289] TGATTCGAAGGAAGCTCAGGATCTAGCCAAACTAATCAAGGAAAAAA

[0290] TGGAGGAAGCCTGGGACTGGTGTTTGGAAAAGGCAGAGGAGTTTGG

[0291] GAATAGGGTCGCTAAGATCAAGTTAGAAGTGGAGGAGCCGCACGTGG

[0292] GTAACACATGGGAGAAGCCC

[0293] (6) The nucleic acid sequence encoding the reverse transcriptase UCRT vZ mutant with the coding mutation sites Q319G / V188F is SEQ ID NO.33;

[0294] SEQ ID NO.33

[0295]

[0296] (7) The nucleic acid sequence of the reverse transcriptase UCRT vZ mutant with the coding mutation sites Q319G / K238W is SEQ ID NO.34;

[0297] SEQ ID NO.34

[0298] ATGGTAAAAGTTAAGTTTAAGTATAAAGGTGAAGAATTACAGGTAGACACGAGCAAGATAAAGAAAGTTTGGCGTGTCGGAAAAGCTATCAGCTTCACATACGACCAAGGAAAGACGGGGAGAGGGGCCGTGTCAGAAAAGGATGCGCCGAAGGAACTCCTTGATATGCTAGCTCGGGCCGAGCGCGAAAAGAAAGGTAGCGCCGGCATGGGTGAAGACGGTTTATCTCTACCCAAAATGATGAATACGCCAAAACCCATCCTAAAACCACAACCAAAGGCGCTTGTAGAACCCGTCTTATGTGATAGTATCGATGAGATACCTGCTAAGTATAATGAACCAGTTTATTTTGATTTAGCGACAGATGAGGATAGACC

[0299] AGTACTTGCGTCAATCTATCAGCCTCATTTTGAGCGCAAAGTGTATTGT

[0300] CTTAACCTACTCAAGGAAAAGGTAGCCCGATTCAAGGATTGGCTTTTA

[0301] AAGTTTAGTGAAATCCGTGGTTGGGGCCTAGACTTCGATCTTAGAGTA

[0302] TTAGGGTACACCTATGAACAACTCCGAAATAAGAAAATTGTTGACGTA

[0303] CAATTAGCGATAAAGGTGCAGCACTATGAAAGGTTCAAACAAGGCGG

[0304] AACCAAGGGGGAGGGCTTTCGGCTAGACGATGTTGCCCGAGACCTCC

[0305] TAGGGATAGAATATCCAATGAATAAGACAAAATACGTGGAGACATTCT

[0306] GGAAACATGTTTCATTCCTTTTCCAATGAACAGCTGCTATATGCATC

[0307] TCTTGACGCATATATACCGCACTTACTTTATGAACACTCACATCATCC

[0308] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAGTG

[0309] GTAATAGAGACGTCCCAGCATGGTATGCCGGTTAAATTAAAAAGCATTG

[0310] GAAGAAGAGATCCATCGGCTGACTCAGCTCCGCTCGGAGATGCAAAA

[0311] AGAGATCCCCTTTAATTATAACTCCCCGAAACAGACTGCGAAATTTTT

[0312] CGGTGTAAATTCGTCCAGCAAAGACGTTCTAATGGACTTAGCTTTGCA

[0313] AGGGAACGAAATGGCAAAAAAAGTTTAGAGGCCAGACAGATTGAA

[0314] AAATCACTTGCCTTTGCCAAGGACCTCTATGACATAGCAAAGCGCTCC

[0315] GGGGGGCGTATCTATGGCAACTTCTTCACTACCACCGCCCCGTCAGGG

[0316] CGCATGAGCTGCTCTGACATAATCTGCAACAAATTCCACGAAGGTTA

[0317] CGGTCGTTCATTGGGTTCGACACCGAAGACAAAAATTGATAACTGC

[0318] AGACTTCCCCCAGATTGAACTTCGTCTCGCCGGTGTAATCTGGAACGA

[0319] ACCTAAGTTCATCGAGGCATTTCGGCAAGGGATTGATTTACATAAGTT

[0320] AACTGCTAGCATCTTGTTCGACAAGAATATCGAGGAGGTAAGTAAAG

[0321] AAGAGCGTCAGATTGGTAAATCCGCAAACTTCGGTTTAATCTACGGTA

[0322] TCGCCCCCAAAGGCTTCGCCGAATATTGTATTGCTAATGGAATCAACAT

[0323] GACCGAGGAGCAGGCCTATGAGATCGTTCGCAAATGGAAAAAGTATT

[0324] ATACTAAGATTGCAGAACAGCACCAGGTGGCATATGAGAGATTCAAAT

[0325] ATAATGAGTATGTGGACAATGAAACCTGGTTAAATCGGACCTACCGCG

[0326] CCTGGAAACCGCAAGACTTGCTAAACTATCAGATACAGGGATCCGGC

[0327] GCCGAATTGTTCAAAAAAGCCATCGTCCTACTAAAGGAGACTAAGCC

[0328] AGATTTAAAGATAGTAAATCTAGTACATGATGAGATTGTGGTAGAAGC

[0329] TGATTCGAAGGAAGCTCAGGATCTAGCCAAACTAATCAAGGAAAAAA

[0330] TGGAGGAAGCCTGGGACTGGTGTTTGGAAAAGGCAGAGGAGTTTGG

[0331] GAATAGGGTCGCTAAGATCAAGTTAGAAGTGGAGGAGCCGCACGTGG

[0332] GTAACACATGGGAGAAGCCC

[0333] (8) The nucleic acid sequence encoding the reverse transcriptase UCRT vZ mutant with mutation site N606Y / V188F is SEQ ID NO.36;

[0334] SEQ ID NO.36

[0335]

[0336] (9) The nucleic acid sequence of the reverse transcriptase UCRT vZ mutant with coding mutation sites N606Y / K238W is SEQ ID NO.36;

[0337] SEQ ID NO.36

[0338] ATGGTAAAAGTTAAGTTTAAGTATAAAGGTGAAGAATTACAGGTAGACACGAGCAAGATAAAGAAAGTTTGGCGTGTCGGAAAAGCTATCAG

[0339] CTTCACATACGACCAAGGAAAGACGGGGAGAGGGGCCGTGTCAGAA

[0340] AAGGATGCGCCGAAGGAACTCCTTGATATGCTAGCTCGGGCCGAGCG

[0341] CGAAAAGAAAGGTAGCGCCGGCATGGGTGAAGACGGTTTATCTCTAC

[0342] CCAAAATGATGAATACGCCAAAACCCATCCTAAAACCACAACCAAAG

[0343] GCGCTTGTAGAACCCGTCTTATGTGATAGTATCGATGAGATACCTGCTA

[0344] AGTATAATGAACCAGTTTATTTTGATTTAGCGACAGATGAGGATAGACC

[0345] AGTACTTGCGTCAATCTATCAGCCTCATTTTGAGCGCAAAGTGTATTGT

[0346] CTTAACCTACTCAAGGAAAAGGTAGCCCGATTCAAGGATTGGCTTTTA

[0347] AAGTTTAGTGAAATCCGTGGTTGGGGCCTAGACTTCGATCTTAGAGTA

[0348] TTAGGGTACACCTATGAACAACTCCGAAATAAGAAAATTGTTGACGTA

[0349] CAATAGCGATAAAGGTGCAGCACTATGAAAGGTTCAAACAAGGCGG

[0350] AACCAAGGGGGAGGGCTTTCGGCTAGACGATGTTGCCCGAGACCTCCC

[0351] TAGGGATAGAATATCCAATGAATAAGACAAAATACGTGGAGACATTCT

[0352] GGAAACATGTTTCATTCCTTTTCCAATGAACAGCTGCTATATGCATC

[0353] TCTTGACGCATATATACCGCACTTACTTTATGAACACTCACATCATCC

[0354] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAGTG

[0355] GTAATAGAGACGTCCCAGCATGGTATGCCGGTTAAATTAAAAAGCATTG

[0356] GAAGAAGAGATCCATCGGCTGACTCAGCTCCGCTCGGAGATGCAAAA

[0357] ACAGATCCCCTTTAATTATAACTCCCCGAAACAGACTGCGAAATTTTTC

[0358] GGTGTAAATTCGTCCAGCAAAGACGTTCTAATGGACTTAGCTTTGCAA

[0359] GGGAACGAAATGGCAAAAAAAGTTTAGAGGCCAGACAGATTGAAA

[0360] AATCACTTGCCTTTGCCAAGGACCTCTATGACATAGCAAAGCGCTCCG

[0361] GGGGGCGTATCTATGGCAACTTCTTCACTACCACCGCCCGTCAGGGC

[0362] GCATGAGCTGCTCTGACATAAATCTGCAACAAATTCCACGAAGGTTAC

[0363] GGTCGTTCATTGGGTTCGACACCGAAGACAAAAAATTGATAACTGCA

[0364] GACTTCCCCCAGATTGAACTTCGTCTCGCCGGTGTAATCTGGAACGAA

[0365] CCTAAGTTCATCGAGGCATTTCGGCAAGGGATTGATTTACATAAGTTA

[0366] ACTGCTAGCATCTTGTTCGACAAGAATATCGAGGAGGTAAGTAAAGA

[0367] AGAGCGTCAGATTGGTAAATCCGCAAACTTCGGTTTAATCTACGGTAT

[0368] CGCCCCCAAAGGCTTCGCCGAATATTGTATTGCTTATGGAATCAACATG

[0369] ACCGAGGAGCAGGCCTATGAGATCGTTCGCAAATGGAAAAAGTATTAT

[0370] ACTAAGATTGCAGAACAGCACCAGGTGGCATATGAGAGATTCAAATAT

[0371] AATGAGTATGTGGACAATGAAACCTGGTTAAATCGGACCTACCGCGCC

[0372] TGGAAACCGCAAGACTTGCTAAACTATCAGATACAGGGATCCGGCGC

[0373] CGAATTGTTCAAAAAAGCCATCGTCCTACTAAAGGAGACTAAGCCAG

[0374] ATTTAAAGATAGTAAATCTAGTACATGATGAGATTGTGGTAGAAGCTGA

[0375] TTCGAAGGAAGCTCAGGATCTAGCCAAACTAATCAAGGAAAAAATGG

[0376] AGGAAGCCTGGGACTGGTGTTTGGAAAAGGCAGAGGAGTTTGGGAA

[0377] TAGGGTCGCTAAGATCAAGTTAGAAGTGGAGGAGCCGCACGTGGGTA

[0378] ACACATGGGAGAAGCCC

[0379] (10) The nucleic acid sequence of the reverse transcriptase UCRT vZ mutant with the coding mutation sites V188F / K238W is SEQ ID NO.37;

[0380] SEQ ID NO.37

[0381] ATGGTAAAAGTTAAGTTTAAGTATAAAGGTGAAGAATTACAGGTAGACACGAGCAAGATAAAGAAAGTTTGGCGTGTCGGAAAAGCTATCAGCTTCACATACGACCAAGGAAAGACGGGGAGAGGGGCCGTGTCAGAAAAGGATGCGCCGAAGGAACTCCTTGATATGCTAGCTCGGGCCGAGCGCGAAAAGAAAGGTAGCGCCGGCATGGGTGAAGACGGTTTATCTCTACCCAAAATGATGAATACGCCAAAACCCATCCTAAAACCACAACCAAAGGCGCTTGTAGAACCCGTCTTATGTGATAGTATCGATGAGATACCTGCTAAGTATAATGAACCAGTTTATTTTGATTTAGCGACAGATGAGGATAGACCAGTACTTGCGTCAATCTATCAGCCTCATTTTGAGCGCAAAGTGTATTGTCTTAACCTACTCAAGGAAAAGGTAGCCCGATTCAAGGATTGGCTTTTAAAGTTTAGTGAAATCCGTGGTTGGGGCCTAGACTTCGATCTTAGAGTATTAGGGTACACCTATGAACAACTCCGAAATAAGAAAATTTTTGACGTACAATTAGCGATAAAGGTGCAGCACTATGAAAGGTTCAAACAAGGCGG

[0382] AACCAAGGGGGAGGGCTTTCGGCTAGACGATGTTGCCCGAGACCTCCC

[0383] TAGGGATAGAATATCCAATGAATAAGACAAAATACGTGGAGACATTCT

[0384] GGAAACATGTTTCATTCCTTTTCCAATGAACAGCTGCTATATGCATC

[0385] TCTTGACGCATATATACCGCACTTACTTTATGAACACTCACATCATCC

[0386] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAGTG

[0387] GTAATAGAGACGTCCCAGCATGGTATGCCGGTTAAATTAAAAAGCATTG

[0388] GAAGAAGAGATCCATCGGCTGACTCAGCTCCGCTCGGAGATGCAAAA

[0389] ACAGATCCCCTTTAATTATAACTCCCCGAAACAGACTGCGAAATTTTTC

[0390] GGTGTAAATTCGTCCAGCAAAGACGTTCTAATGGACTTAGCTTTGCAA

[0391] GGGAACGAAATGGCAAAAAAAGTTTAGAGGCCAGACAGATTGAAA

[0392] AATCACTTGCCTTTGCCAAGGACCTCTATGACATAGCAAAGCGCTCCG

[0393] GGGGGCGTATCTATGGCAACTTCTTCACTACCACCGCCCGTCAGGGC

[0394] GCATGAGCTGCTCTGACATAAATCTGCAACAAATTCCACGAAGGTTAC

[0395] GGTCGTTCATTGGGTTCGACACCGAAGACAAAAAATTGATAACTGCA

[0396] GACTTCCCCCAGATTGAACTTCGTCTCGCCGGTGTAATCTGGAACGAA

[0397] CCTAAGTTCATCGAGGCATTTCGGCAAGGGATTGATTTACATAAGTTA

[0398] ACTGCTAGCATCTTGTTCGACAAGAATATCGAGGAGGTAAGTAAAGA

[0399] AGAGCGTCAGATTGGTAAATCCGCAAACTTCGGTTTAATCTACGGTAT

[0400] CGCCCCCAAAGGCTTCGCCGAATATTGTATTGCTAATGGAATCAACAT

[0401] GACCGAGGAGCAGGCCTATGAGATCGTTCGCAAATGGAAAAAGTATT

[0402] ATACTAAGATTGCAGAACAGCACCAGGTGGCATATGAGAGATTCAAAT

[0403] ATAATGAGTATGTGGACAATGAAACCTGGTTAAATCGGACCTACCGCG

[0404] CCTGGAAACCGCAAGACTTGCTAAACTATCAGATACAGGGATCCGGC

[0405] GCCGAATTGTTCAAAAAAGCCATCGTCCTACTAAAGGAGACTAAGCC

[0406] AGATTTAAAGATAGTAAATCTAGTACATGATGAGATTGTGGTAGAAGC

[0407] TGATTCGAAGGAAGCTCAGGATCTAGCCAAACTAATCAAGGAAAAAA

[0408] TGGAGGAAGCCTGGGACTGGTGTTTGGAAAAGGCAGAGGAGTTTGG

[0409] GAATAGGGTCGCTAAGATCAAGTTAGAAGTGGAGGAGCCGCACGTGG

[0410] GTAACACATGGGAGAAGCCC

[0411] (11) The nucleic acid sequence encoding the reverse transcriptase UCRT vZ mutant with mutation sites Q319G / N606Y / V188F is SEQ ID NO.38;

[0412] SEQ ID NO.38

[0413]

[0414] The nucleic acid sequence of the reverse transcriptase UCRT vZ mutant with coding mutation sites Q319G / N606Y / K238W is SEQ ID NO.39;

[0415] SEQ ID NO.39

[0416] ATGGTAAAAGTTAAGTTTAAGTATAAAGGTGAAGAATTACAGGTAGACACGAGCAAGATAAAGAAAGTTTGGCGTGTCGGAAAAGCTATCAGCTTCACATACGACCAAGGAAAGACGGGGAGAGGGGCCGTGTCAGAAAAGGATGCGCCGAAGGAACTCCTTGATATGCTAGCTCGGGCCGAGCGCGAAAAGAAAGGTAGCGCCGGCATGGGTGAAGACGGTTTATCTCTACCCAAAATGATGAATACGCCAAAACCCATCCTAAAACCACAACCAAAGGCGCTTGTAGAACCCGTCTTATGTGATAGTATCGATGAGATACCTGCTA

[0417] AGTATAATGAACCAGTTTATTTTGATTTAGCGACAGATGAGGATAGACC

[0418] AGTACTTGCGTCAATCTATCAGCCTCATTTTGAGCGCAAAGTGTATTGT

[0419] CTTAACCTACTCAAGGAAAAGGTAGCCCGATTCAAGGATTGGCTTTTA

[0420] AAGTTTAGTGAAATCCGTGGTTGGGGCCTAGACTTCGATCTTAGAGTA

[0421] TTAGGGTACACCTATGAACAACTCCGAAATAAGAAAATTGTTGACGTA

[0422] CAATTAGCGATAAAGGTGCAGCACTATGAAAGGTTCAAACAAGGCGG

[0423] AACCAAGGGGGAGGGCTTTCGGCTAGACGATGTTGCCCGAGACCTCC

[0424] TAGGGATAGAATATCCAATGAATAAGACAAAATACGTGGAGACATTCT

[0425] GGAAACATGTTTCATTCCTTTTCCAATGAACAGCTGCTATATGCATC

[0426] TCTTGACGCATATATACCGCACTTACTTTATGAACACTCACATCATCC

[0427] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAGTG

[0428] GTAATAGAGACGTCCCAGCATGGTATGCCGGTTAAATTAAAAAGCATTG

[0429] GAAGAAGAGATCCATCGGCTGACTCAGCTCCGCTCGGAGATGCAAAA

[0430] AGAGATCCCCTTTAATTATAACTCCCCGAAACAGACTGCGAAATTTTT

[0431] CGGTGTAAATTCGTCCAGCAAAGACGTTCTAATGGACTTAGCTTTGCA

[0432] AGGGAACGAAATGGCAAAAAAAGTTTAGAGGCCAGACAGATTGAA

[0433] AAATCACTTGCCTTTGCCAAGGACCTCTATGACATAGCAAAGCGCTCC

[0434] GGGGGGCGTATCTATGGCAACTTCTTCACTACCACCGCCCCGTCAGGG

[0435] CGCATGAGCTGCTCTGACATAATCTGCAACAAATTCCACGAAGGTTA

[0436] CGGTCGTTCATTGGGTTCGACACCGAAGACAAAAATTGATAACTGC

[0437] AGACTTCCCCCAGATTGAACTTCGTCTCGCCGGTGTAATCTGGAACGA

[0438] ACCTAAGTTCATCGAGGCATTTCGGCAAGGGATTGATTTACATAAGTT

[0439] AACTGCTAGCATCTTGTTCGACAAGAATATCGAGGAGGTAAGTAAAG

[0440] AAGAGCGTCAGATTGGTAAATCCGCAAACTTCGGTTTAATCTACGGTA

[0441] TCGCCCCCAAAGGCTTCGCCGAATATTGTATTGCTTATGGAATCAACAT

[0442] GACCGAGGAGCAGGCCTATGAGATCGTTCGCAAATGGAAAAAGTATT

[0443] ATACTAAGATTGCAGAACAGCACCAGGTGGCATATGAGAGATTCAAAT

[0444] ATAATGAGTATGTGGACAATGAAACCTGGTTAAATCGGACCTACCGCG

[0445] CCTGGAAACCGCAAGACTTGCTAAACTATCAGATACAGGGATCCGGC

[0446] GCCGAATTGTTCAAAAAAGCCATCGTCCTACTAAAGGAGACTAAGCC

[0447] AGATTTAAAGATAGTAAATCTAGTACATGATGAGATTGTGGTAGAAGC

[0448] TGATTCGAAGGAAGCTCAGGATCTAGCCAAACTAATCAAGGAAAAAA

[0449] TGGAGGAAGCCTGGGACTGGTGTTTGGAAAAGGCAGAGGAGTTTGG

[0450] GAATAGGGTCGCTAAGATCAAGTTAGAAGTGGAGGAGCCGCACGTGG

[0451] GTAACACATGGGAGAAGCCC

[0452] (13) The nucleic acid sequence encoding the reverse transcriptase UCRT vZ mutant with mutation sites Q319G / V188F / K238W is SEQ ID NO.40;

[0453] SEQ ID NO.40

[0454]

[0455] (14) The nucleic acid sequence of the reverse transcriptase UCRT vZ mutant with coding mutation sites N606Y / V188F / K238W is SEQ ID NO.41;

[0456] SEQ ID NO.41

[0457] ATGGTAAAAGTTAAGTTTAAGTATAAAGGTGAAGAATTACAGGTA

[0458] GACACGAGCAAGATAAAGAAAGTTTGGCGTGTCGGAAAAGCTATCAG

[0459] CTTCACATACGACCAAGGAAAGACGGGGAGAGGGGCCGTGTCAGAA

[0460] AAGGATGCGCCGAAGGAACTCCTTGATATGCTAGCTCGGGCCGAGCG

[0461] CGAAAAGAAAGGTAGCGCCGGCATGGGTGAAGACGGTTTATCTCTAC

[0462] CCAAAATGATGAATACGCCAAAACCCATCCTAAAACCACAACCAAAG

[0463] GCGCTTGTAGAACCCGTCTTATGTGATAGTATCGATGAGATACCTGCTA

[0464] AGTATAATGAACCAGTTTATTTTGATTTAGCGACAGATGAGGATAGACC

[0465] AGTACTTGCGTCAATCTATCAGCCTCATTTTGAGCGCAAAGTGTATTGT

[0466] CTTAACCTACTCAAGGAAAAGGTAGCCCGATTCAAGGATTGGCTTTTA

[0467] AAGTTTAGTGAAATCCGTGGTTGGGGCCTAGACTTCGATCTTAGAGTA

[0468] TTAGGGTACACCTATGAACAACTCCGAAATAAGAAAATTTTTGACGTA

[0469] CAATAGCGATAAAGGTGCAGCACTATGAAAGGTTCAAACAAGGCGG

[0470] AACCAAGGGGGAGGGCTTTCGGCTAGACGATGTTGCCCGAGACCTCCC

[0471] TAGGGATAGAATATCCAATGAATAAGACAAAATACGTGGAGACATTCT

[0472] GGAAACATGTTTCATTCCTTTTCCAATGAACAGCTGCTATATGCATC

[0473] TCTTGACGCATATATACCGCACTTACTTTATGAACACTCACATCATCC

[0474] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAGTG

[0475] GTAATAGAGACGTCCCAGCATGGTATGCCGGTTAAATTAAAAAGCATTG

[0476] GAAGAAGAGATCCATCGGCTGACTCAGCTCCGCTCGGAGATGCAAAA

[0477] ACAGATCCCCTTTAATTATAACTCCCCGAAACAGACTGCGAAATTTTTC

[0478] GGTGTAAATTCGTCCAGCAAAGACGTTCTAATGGACTTAGCTTTGCAA

[0479] GGGAACGAAATGGCAAAAAAAGTTTAGAGGCCAGACAGATTGAAA

[0480] AATCACTTGCCTTTGCCAAGGACCTCTATGACATAGCAAAGCGCTCCG

[0481] GGGGGCGTATCTATGGCAACTTCTTCACTACCACCGCCCCGTCAGGGC

[0482] GCATGAGCTGCTCTGACATAAATCTGCAACAAATTCCACGAAGGTTAC

[0483] GGTCGTTCATTGGGTTCGACACCGAAGACAAAAAATTGATAACTGCA

[0484] GACTTCCCCCAGATTGAACTTCGTCTCGCCGGTGTAATCTGGAACGAA

[0485] CCTAAGTTCATCGAGGCATTTCGGCAAGGGATTGATTTACATAAGTTA

[0486] ACTGCTAGCATCTTGTTCGACAAGAATATCGAGGAGGTAAGTAAAGA

[0487] AGAGCGTCAGATTGGTAAATCCGCAAACTTCGGTTTAATCTACGGTAT

[0488] CGCCCCCAAAGGCTTCGCCGAATATTGTATTGCTTATGGAATCAACATG

[0489] ACCGAGGAGCAGGCCTATGAGATCGTTCGCAAATGGAAAAAGTATTAT

[0490] ACTAAGATTGCAGAACAGCACCAGGTGGCATATGAGAGATTCAAATAT

[0491] AATGAGTATGTGGACAATGAAACCTGGTTAAATCGGACCTACCGCGCC

[0492] TGGAAACCGCAAGACTTGCTAAACTATCAGATACAGGGATCCGGCGC

[0493] CGAATTGTTCAAAAAAGCCATCGTCCTACTAAAGGAGACTAAGCCAG

[0494] ATTTAAAGATAGTAAATCTAGTACATGATGAGATTGTGGTAGAAGCTGA

[0495] TTCGAAGGAAGCTCAGGATTCTAGCCAAACTAATCAAGGAAAAAATGG

[0496] AGGAAGCCTGGGACTGGTGTTTGGAAAAGGCAGAGGAGTTTGGGAA

[0497] TAGGGTCGCTAAGATCAAGTTAGAAGTGGAGGAGCCGCACGTGGGTA

[0498] ACACATGGGAGAAGCCC

[0499] (16) The nucleic acid sequence encoding the reverse transcriptase UCRT vZ mutant with mutation sites Q319G / N606Y / V188F / K238W is SEQ ID NO.42.

[0500] SEQ ID NO.42

[0501] ATGGTAAAAGTTAAGTTTAAGTATAAAGGTGAAGAATTACAGGTAGACACGAGCAAGATAAAGAAAGTTTGGCGTGTCGGAAAAGCTATCAGCTTCACATACGACCAAGGAAAGACGGGGAGAGGGGCCGTGTCAGAAAAGGATGCGCCGAAGGAACTCCTTGATATGCTAGCTCGGGCCGAGCGCGAAAAGAAAGGTAGCGCCGGCATGGGTGAAGACGGTTTATCTCTACCCAAAATGATGAATACGCCAAAACCCATCCTAAAACCACAACCAAAGGCGCTTGTAGAACCCGTCTTATGTGATAGTATCGATGAGATACCTGCTAAGTATAATGAACCAGTTTATTTTGATTTAGCGACAGATGAGGATAGACCAGTACTTGCGTCAATCTATCAGCCTCATTTTGAGCGCAAAGTGTATTGTCTTAACCTACTCAAGGAAAAGGTAGCCCGATTCAAGGATTGGCTTTTAAAGTTTAGTGAAATCCGTGGTTGGGGCCTAGACTTCGATCTTAGAGTATTAGGGTACACCTATGAACAACTCCGAAATAAGAAAATTTTTGACGTA

[0502] CAATTAGCGATAAAGGTGCAGCACTATGAAAGGTTCAAACAAGGCGG

[0503] AACCAAGGGGGAGGGCTTTCGGCTAGACGATGTTGCCCGAGACCTCC

[0504] TAGGGATAGAATATCCAATGAATAAGACAAAAATACGTGAGACATTCT

[0505] GGAACAACATGTTTCATTCCTTTTCCAATGAACAGCTGCTATATGCATC

[0506] TCTTGACGCATATATACCGCACTTACTTTATGAACAACTCACATCATCC

[0507] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAGTG

[0508] GTAATAGAGACGTCCCAGCATGGTATGCCGGTTAAATTAAAAAGCATTG

[0509] GAAGAAGAGATCCATCGGCTGACTCAGCTCCGCTCGGAGATGCAAAA

[0510] AGAGATCCCCTTTAATTATAACTCCCCGAAACAGACTGCGAAATTTTT

[0511] CGGTGTAAATTCGTCCAGCAAAGACGTTCTAATGGACTTAGCTTTGCA

[0512] AGGGAACGAAATGGCAAAAAAAGTTTAGAGGCCAGACAGATTGAA

[0513] AAATCACTTGCCTTTGCCAAGGACCTCTATGACATAGCAAAGCGCTCC

[0514] GGGGGGCGTATCTATGGCAACTTCTTCACTACCACCGCCCCGTCAGGG

[0515] CGCATGAGCTGCTCTGACATAATCTGCAACAAATTCCACGAAGGTTA

[0516] CGGTCGTTCATTGGGTTCGACACCGAAGACAAAAATTGATAACTGC

[0517] AGACTTCCCCCAGATTGAACTTCGTCTCGCCGGTGTAATCTGGAACGA

[0518] ACCTAAGTTCATCGAGGCATTTCGGCAAGGGATTGATTTACATAAGTT

[0519] AACTGCTAGCATCTTTGTTCGACAAGAATATCGAGGGAGGTAAGTAAG

[0520] AAGAGCGTCAGATTGGTAAATCCGCAAACTTCGGTTTAATCTACGGTA

[0521] TCGCCCCCAAAGGCTTCGCCGAATATTGTATTGCTTATGGAATCAACAT

[0522] GACCGAGGAGCAGGCCTATGAGATCGTTCGCAAATGGAAAAAGTATT

[0523] ATACTAAGATTGCAGAACAGCACCAGGTGGCATATGAGAGATTCAAAT

[0524] ATAATGAGTATGTGGACAATGAAAACCTGGTTAAATCGGACCTACCGCG

[0525] CCTGGAAACCGCAAGACTTGCTAAACTATCAGATACAGGGATCCGGC

[0526] GCCGAATTGTTCAAAAAAGCCATCGTCCTACTAAAGGAGACTAAGCC

[0527] AGATTTAAAGATAGTAAATCTAGTACATGATGAGATTGTGGTAGAAGC

[0528] TGATTCGAAGGAAGCTCAGGATCTAGCCAAACTAATCAAGGAAAAAA

[0529] TGGAGGAAGCCTGGGACTGGTGTTTGGAAAAGGCAGAGGAGTTTGGGAATAGGGTCGCTAAGATCAAGTTAGAAGTGGAGGAGCCGCACGTGGGTAACACATGGGAGAAGCCC

[0530] Embodiment 4:

[0531] This example studies the enzymatic properties of the reverse transcriptase UCRT vZ mutant.

[0532] The wild-type reverse transcriptase UCRT vZ and the various reverse transcriptase UCRT vZ mutants provided in Example 2 were subjected to a thermal stability test according to a conventional reverse transcriptase UCRT vZ activity assay method, specifically:

[0533] The enzyme solution is incubated at a certain temperature, and samples are taken at different treatment times to determine the residual activity percentage of the reverse transcriptase UCRT vZ or the reverse transcriptase UCRT vZ mutant. The ln value of the residual activity percentage is plotted against the time t (min). The slope of the straight line is the inactivation constant kinact. The half-life of the wild-type reverse transcriptase UCRT vZ or the reverse transcriptase UCRT vZ mutant at this temperature is obtained from t1 / 2=ln2 / kinact.

[0534] The experimental results show that among the above reverse transcriptase UCRT vZ mutants, the thermal stability of 4 single-point mutants and 11 combined mutants is significantly improved, as shown in Table 1:

[0535] Table 1. Characterization of enzymatic properties of wild-type reverse transcriptase UCRT vZ, single point mutants and combined mutants

[0536]

[0537]

[0538] As shown in Table 1, the reverse transcriptase UCRT vZ mutants provided by the present invention include single point mutants and combined mutants. Compared with the wild-type reverse transcriptase UCRT vZ, the single point mutants and the combined mutants have longer half-lives at 66°C; in particular, the combined mutants show the superposition effect of the thermal stability of the single point mutants, and their half-life is about 4 times that of the wild type.

[0539] The above examples are merely illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. All designs that are the same or similar to the present invention fall within the protection scope of the present invention.

Claims

1. A reverse transcriptase UCRT vZ mutant with improved thermal stability, characterized in that: The reverse transcriptase UCRT vZ mutant is as follows (a1) or (a2): (a1) a derivative protein in which one or more amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO.2 and which has the same function as the amino acid sequence shown in SEQ ID NO.2; (a2) A derivative protein in which one or more amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO. 2 and which has at least 90% homology with the amino acid sequence shown in SEQ ID NO.

2.

2. The reverse transcriptase UCRT vZ mutant with improved thermal stability according to claim 1, characterized in that: The amino acid sequence of the reverse transcriptase UCRT vZ mutant is configured as an amino acid sequence after one or more combinations of mutations at the mutation sites Q319G, N505Y, V188F, and K238W on SEQ ID NO.2 are mutated.

3. The reverse transcriptase UCRT vZ mutant with improved thermal stability according to claim 2, characterized in that: The mutation sites are Q319G, N505Y, V188F, K238W, Q319G / N505Y, Q319G / V188F, Q319G / K238W, N505Y / V188F, N505Y / K238W, V188F / K238W, Q319G / N505Y / V188F, Q319G / N505Y / K238W, N505Y / V188F / K238W, Q319G / V188F / K238W or Q319G / N505Y / V188F / K238W.

4. The reverse transcriptase UCRT vZ mutant with improved thermal stability according to claim 2, characterized in that: The amino acid sequence of the single point mutant corresponding to Q319G is SEQ ID NO. 3; The amino acid sequence of the single point mutant corresponding to N505Y is SEQ ID NO.4; The amino acid sequence of the single point mutant corresponding to V188F is SEQ ID NO.5; The amino acid sequence of the single point mutant corresponding to K238W is SEQ ID NO.6; The amino acid sequence of the combined mutant corresponding to Q319G / N505Y is SEQ ID NO.7; The amino acid sequence of the combined mutant corresponding to Q319G / V188F is SEQ ID NO.8; The amino acid sequence of the combined mutant corresponding to Q319G / K238W is SEQ ID NO.9; The amino acid sequence of the combined mutant corresponding to N505Y / V188F is SEQ ID NO.10; The amino acid sequence of the combined mutant corresponding to N505Y / K238W is SEQ ID NO.11; The amino acid sequence of the combined mutant corresponding to V188F / K238W is SEQ ID NO. 12; The amino acid sequence of the combined mutant corresponding to Q319G / N505Y / V188F is SEQ ID NO. 13; The amino acid sequence of the combined mutant corresponding to Q319G / N505Y / K238W is SEQ ID NO. 14; The amino acid sequence of the combined mutant corresponding to N505Y / V188F / K238W is SEQ ID NO. 15; The amino acid sequence of the combined mutant corresponding to Q319G / V188F / K238W is SEQ ID NO. 16; The amino acid sequence of the combined mutant corresponding to Q319G / N505Y / V188F / K238W is SEQ ID NO.

17.

5. The method for constructing a reverse transcriptase UCRT vZ mutant with improved thermal stability according to any one of claims 1 to 4, characterized in that: The following steps are involved: Search and select amino acid sequences with a consistency greater than 50% with the amino acid sequence shown in SEQ ID NO. 2 in the database, then perform multiple sequence alignment, and generate a consensus sequence that can be edited later using software; The protein three-dimensional structure of SEQ ID NO.2 was predicted, and the stability-related mutation sites were screened out: Q319G, N505Y, V188F, and K238W.

6. The method for constructing a reverse transcriptase UCRT vZ mutant with improved thermal stability according to claim 5, characterized in that: The amplification primer sequences for the mutation site Q319G are SEQ ID NO.20 and SEQ ID NO.21; The amplification primer sequences for the mutation site N505Y are SEQ ID NO.22 and SEQ ID NO.23; The amplification primer sequences for the mutation site V188F are SEQ ID NO.24 and SEQ ID NO.25; The amplification primer sequences for the mutation site K238W are SEQ ID NO.26 and SEQ ID NO.

27.

7. A gene encoding the reverse transcriptase UCRT vZ mutant with improved thermal stability as claimed in any one of claims 1 to 4.

8. A recombinant plasmid comprising the gene according to claim 7.

9. A soluble protein, immobilized enzyme or engineered bacteria comprising the reverse transcriptase UCRT vZ mutant with improved thermal stability as claimed in any one of claims 1 to 4.

10. Use of the reverse transcriptase UCRT vZ mutant with improved thermal stability according to any one of claims 1 to 4 in reverse transcribing RNA into cDNA.

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