Thermostable reverse transcriptase ucrt vz mutant and construction method and application thereof

Abstract

The application belongs to the technical field of biology, and specifically provides a reverse transcriptase UCRT vZ mutant with improved thermal stability, the reverse transcriptase UCRT vZ mutant includes four single-point mutants, six double mutants and four triple mutants, compared with a wild-type reverse transcriptase UCRT vZ, the half-life of the mutants is longer at 65 DEG C, the effect of the double mutants is better, and the half-life is about 4 times that of the wild-type reverse transcriptase UCRT vZ. The application further provides a genetically engineered bacterium containing the mutant. The reverse transcriptase UCRT vZ mutant obtained by the construction method has better thermal stability, and when used for RNA reverse transcription into cDNA at a higher temperature, the reverse transcriptase UCRT vZ mutant has higher thermal stability and great application potential.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a thermostability-enhanced reverse transcriptase UCRT vZ mutant, its construction method, and its application. Background Technology

[0002] Reverse transcriptase UCRT vZ uses RNA as a template to catalyze dNTP polymerization, generating a DNA-RNA hybrid double strand. It possesses diverse enzymatic activities and thermostable properties, with an optimal activity temperature of 60-70℃. Under these conditions, the efficiency of opening RNA secondary structures is significantly improved, enhancing transcription efficiency and cDNA integrity. There are few competing reverse transcriptases under these conditions, giving it a significant advantage over commonly available reverse transcriptases. During cDNA synthesis, reverse transcription at higher temperatures reduces RNA secondary structures and increases transcription efficiency. Bioactive enzymes have wide applications in industrial production, and improving enzyme thermostability is one of the key research focuses for industrial enzymes.

[0003] Protein engineering is based on the structural regularities of protein molecules and their relationship with biological functions. It utilizes chemical, physical, and molecular biological methods to modify or synthesize existing proteins or create new ones to meet human needs for production and daily life. Rational design is the most commonly used method in protein engineering, employing computer-aided molecular models combined with site-directed mutagenesis to optimize protein function, such as improving catalytic activity, thermal stability, and acid / alkali resistance. To effectively optimize protein thermal stability, Markus Wyss et al. proposed the Consensus Concept theory in 2001. Unlike conventional rational protein design methods based on precise structure-function relationships, the Consensus Concept theory is based on the amino acid sequence information of homologous proteins, analyzing information from an evolutionary perspective that can improve enzyme thermal stability. This invention, guided by the Consensus Concept theory, integrates and analyzes the sequences of the UCRT vZ family of reverse transcriptases, and combines bioinformatics and crystallography methods to obtain novel UCRT vZ mutant reverse transcriptases with high stability. Summary of the Invention

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

[0005] Therefore, 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 having the same function as the amino acid sequence shown in SEQ ID NO.2 by substituting, deleting or adding one or more amino acids;

[0007] (a2) A derivative protein having at least 90% homology to the amino acid sequence shown in SEQ ID NO.2 by substituting, deleting or adding one or more amino acids.

[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 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 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 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 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 N606Y / K238W is SEQ ID NO.11;

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

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

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

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

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

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

[0025] This invention also provides a method for constructing the above-mentioned thermostability-enhanced reverse transcriptase UCRT vZ mutant, comprising the following steps:

[0026] Search the database for amino acid sequences that are more than 60% identical to the amino acid sequence shown in SEQ ID NO.2, then perform multiple sequence alignment, and use software to generate a consensus sequence that can be edited later.

[0027] Three-dimensional protein structure prediction was performed on SEQ ID NO.2, and stability-related mutation sites were screened out: Q319G, N606Y, V188F, and K238W.

[0028] Specifically, the amplification primer sequences for the above-mentioned 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 for the above-mentioned thermostability-enhanced reverse transcriptase UCRT vZ mutant.

[0033] The present invention also provides recombinant plasmids containing the above-mentioned genes.

[0034] The present invention also provides a soluble protein, immobilized enzyme, or engineered bacteria comprising the above-mentioned thermostable reverse transcriptase UCRT vZ mutant.

[0035] The thermostability-enhanced reverse transcriptase UCRT vZ mutant provided by this 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 thermostable reverse transcriptase UCRT vZ mutant provided by this invention includes single-point mutants and combined mutants. Compared with wild-type reverse transcriptase UCRT vZ, both the single-point mutants and combined mutants have longer half-lives at 66°C; especially the combined mutants, which exhibit the synergistic effect of the thermostable single-point mutants, with a half-life approximately four times that of the wild type. The UCRT vZ mutants possess excellent catalytic activity and show promising application prospects.

[0038] 2. The method for constructing a thermostability-enhanced reverse transcriptase UCRT vZ mutant provided by this invention differs from rational design based on the precise structure-function relationship of proteins. This invention is guided by the Consensus Concept theory, analyzes information that can improve enzyme thermostability from an evolutionary perspective, integrates and analyzes the reverse transcriptase UCRT vZ family sequences, and combines bioinformatics and crystallography methods to obtain a novel UCRT vZ mutant with high stability.

[0039] The present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0040] Figure 1 This 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 Implementation

[0041] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand 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 defined by the appended claims and their equivalents.

[0042] This 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 having the same function as the amino acid sequence shown in SEQ ID NO.2 by substituting, deleting or adding one or more amino acids;

[0044] (a2) A derivative protein having at least 90% homology to the amino acid sequence shown in SEQ ID NO.2 by substituting, deleting or adding one or more amino acids.

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

[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] 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 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 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 N606Y / K238W is SEQ ID NO.11;

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

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

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

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

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

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

[0062] This invention also provides a method for constructing the above-mentioned thermostability-enhanced reverse transcriptase UCRT vZ mutant, comprising the following steps:

[0063] By searching the Pfam and NCBI databases for the amino acid sequence shown in SEQ ID NO.2, removing duplicate sequences, and selecting amino acid sequences with a similarity greater than 30% to the amino acid sequence shown in SEQ ID NO.2, multiple sequence alignment was performed using Clustalx 1.83 software. The remaining amino acid sequences were then compiled into a FASTA file and uploaded to the Consensus Maker v2.0.0 server. After modifying the settings 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 using the Swissmodel online tool, and the crystal structure of the protein shown in SEQ ID NO.2 was observed using PyMOL. The mutation sites related to thermal stability were screened out as: Q319G, N606Y, V188F, and K238W.

[0065] The amplification primer sequences for 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 effects of the thermostability-enhanced reverse transcriptase UCRT vZ mutant of the present invention are studied through specific embodiments below.

[0070] Example 1:

[0071] This embodiment provides a thermostable reverse transcriptase UCRT vZ mutant, wherein the reverse transcriptase UCRTvZ is an uncultured virus isolated from thermostable bacteria in Yellowstone National Park, the wild-type reverse transcriptase is named UCRTvZ, and 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 thermostability-enhanced reverse transcriptase UCRT vZ mutant provided in this embodiment includes: a derivative protein having the same function as the amino acid sequence shown in SEQ ID NO.2 (i.e., reverse transcriptase UCRT vZ protein) by substituting, deleting, or adding one or more amino acids; or a derivative protein having at least 90% homology with the amino acid sequence shown in SEQ ID NO.2 (i.e., reverse transcriptase UCRT vZ protein) by substituting, deleting, or adding one or more amino acids.

[0077] Specifically, a single-point mutation was performed at a site selected in the amino acid sequence shown in SEQ ID NO.2, resulting in four single-point mutants of the reverse transcriptase UCRT vZ. The mutation sites were Q319G, N606Y, V188F, and K238W. The activity of these four reverse transcriptase UCRT vZ single-point mutants was measured, 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 can be selected and combined within the amino acid sequence shown in SEQ ID NO. 2. For example, selecting two mutation sites from the above four mutation sites and combining them yields the following six thermostability-enhanced reverse transcriptase UCRTvZ mutants. 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, and Q319G / N606Y / V188F / K238W, with amino acid sequences of SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9, respectively. 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 thermostability-enhanced reverse transcriptase UCRT vZ mutants are obtained. The combined mutation sites are: Q319G / N606Y / V188F, Q319G / N606Y / K238W, Q319G / V188F / K238W, and N606Y / V188F / K238W, with amino acid sequences of 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] If four mutation sites are selected from the above four mutation sites and combined, a reverse transcriptase UCRT vZ mutant with improved thermostability is obtained. The 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] Example 2:

[0112] This embodiment provides a method for constructing a thermostable reverse transcriptase UCRT vZ mutant, including 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 the host cell to obtain the 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. Using SEQ ID NO.1 as the target gene, the target gene was amplified using upstream amplification primer SEQ ID NO.18 and downstream amplification primer SEQ ID NO.19.

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

[0116] 6'-ACTGCT CATATG ATGGTAAAAGTTAAGTTTAAGTATAAAG-3' (where the underlined part is the NdeI restriction enzyme recognition site);

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

[0118] 6'-TCAGCT CTCGAG GGGCTTCTCCCATGTGTTACC-3' (where the underlined part is the XhoI restriction enzyme recognition site).

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

[0120] After the reaction was complete, the PCR amplification product was detected by 1.6% agarose gel electrophoresis, yielding a 1.0 kb band, the length of which was in line with the expected result. Following the standard procedure of the kit, the target fragment was recovered and purified. The target fragment and the pET28a plasmid were double-digested using restriction endonucleases XhoI and NdeI, and then ligated using T4 DNA ligase. The ligation product was transformed into *E. coli* BL21(DE3) competent cells. The transformed cells were plated on LB plates containing 60 μg / ml kanamycin, and positive clone plasmids were extracted and sequenced. The results showed that the cloned reverse transcriptase UCRT vZ gene sequence was correct and had been correctly inserted into the pET28a plasmid, yielding the recombinant plasmid pET28a-Bst.

[0121] Among them, the wild-type reverse transcriptase UCRT vZ was derived from an uncultured virus isolated from thermostable bacteria in Yellowstone National Park.

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

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

[0124] 2. Expression and purification of reverse transcriptase UCRT vZ protein

[0125] The engineered bacteria from the glycerol tube were inoculated at a volume ratio of 1% into a 4 mL LB medium tube containing 100 μg / mL Kan, and cultured at 37℃ and 220 rpm for 12 h. 4 mL of the bacterial suspension was then transferred to a 1 L LB medium shake flask containing 60 μg / mL Kan, and cultured at 37℃ and 220 rpm for 2.6 h until the OD600 reached approximately 0.9. 0.1 mM IPTG inducer was then added, and the culture was induced at 26℃ and 200 rpm for 14 h. The harvested *E. coli* bacterial suspension was ultrasonically disrupted, followed by a one-step Ni-NTA affinity chromatography to obtain the reverse transcriptase UCRT vZ protein with a purity >96%, the amino acid sequence of which is SEQ ID NO. 2.

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

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

[0128] 3.2. Input the amino acid sequence shown in SEQ ID NO.2 into the NCBI protein database and Pfam database. Use the Blast tool to find all protein sequences with an amino acid sequence identity greater than 30% with the reverse transcriptase UCRT vZ protein (SEQ ID NO.2). Delete any duplicate sequences. Organize the remaining amino acid sequences into fasta. format and input them into Clustalx 1.83 software for multiple sequence alignment. The alignment results are output in alan., dnd., and fasta. formats. The dnd. file is the phylogenetic tree file, while the alan. and fasta. files are sequence files in different formats.

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

[0130] 3.3. The amino acid sequence (SEQ ID NO.2) of the reverse transcriptase UCRT vZ protein 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 of the reverse transcriptase UCRT vZ protein (amino acid sequence SEQ ID NO.2) was predicted 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. Based on the structural information, the above-mentioned candidate mutation sites and mutation modes were reviewed to screen for the mutant sites most likely to improve the thermal stability of the reverse transcriptase UCRT vZ protein. The screening criteria are as follows:

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

[0135] ①Most proteins in this family have a generally high amino acid abundance at this site;

[0136] ②The amino acid at this site is conserved;

[0137] ③ The amino acids that appear most frequently at this site have significant differences in physicochemical properties compared to the amino acids at this site in the reverse transcriptase UCRT vZ protein, such as differences in charge, polarity, and steric hindrance.

[0138] (2) Remove the area near the active site, i.e., the distance from the catalytic residue ( The amino acid residues within the range, excluding amino acid residues that are embedded or partially embedded.

[0139] After the above two screening steps, a total of 10 differentially expressed sites remain, 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, and mutants that may improve the thermal stability of the reverse transcriptase UCRT vZ protein were screened out.

[0141] The main criteria for judgment are: ① Mutations should eliminate existing forces that are detrimental to thermal stability, such as electrostatic repulsion and charge accumulation; ② Mutations should not destroy existing forces that are beneficial to thermal stability and stable protein structures; ③ Mutations should introduce new forces that are beneficial to thermal stability, such as hydrogen bonds, salt bridges, and hydrophobic interactions.

[0142] Four single-point mutants were designed, with mutation sites of 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 thermostability were screened out. The mutation sites are Q319G, N606Y, V188F, and K238W. The amino acid sequences of the corresponding single-point mutants are 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 a single-point mutant of the reverse transcriptase UCRT vZ protein

[0146] Using the recombinant plasmid pET28a-Bst from step 1 as a template, and a pair of complementary oligonucleotides with mutation sites as amplification primers, KOD high-fidelity enzyme was used to perform full plasmid PCR amplification to obtain a recombinant plasmid with specific mutation sites.

[0147] The amplification primer pairs used are:

[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℃ for 2 min, followed by amplification at 66℃ for 20 sec, amplification at 72℃ for 90 sec, for a total of 30 cycles, and finally amplification at 72℃ for 10 min. The PCR amplification products were recovered by gel extraction, and the gel products were digested with DpnI enzyme at 37℃ for 2 h to degrade the initial template. The digested products were transformed into Escherichia coli BL21(DE3) competent cells, plated on LB agar plates containing 60 μg / mL kanamycin, and cultured overnight at 37℃. Positive clones were screened and sequenced to verify the results, yielding recombinant bacteria containing the reverse transcriptase UCRT vZ single-point mutant.

[0169] The aforementioned high-fidelity KOD enzyme was provided by TakaRa.

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

[0171] 6.2. Construction of a combinatorial mutant of the reverse transcriptase UCRT vZ protein

[0172] Using a construction method similar to that for single-point mutants, single-point mutants with improved stability were cumulatively combined. Multiple mutation sites were selected from the amino acid sequence shown in SEQ ID NO.2 for combination. For example, 2 to 4 mutation sites were selected from the above 4 mutation sites for combination to obtain different reverse transcriptase UCRT vZ combined mutants:

[0173] (1) By selecting two mutation sites for combination, six thermostability-enhanced reverse transcriptase UCRT vZ mutants can be constructed. The combined mutation sites are: Q319G / N606Y, Q319G / V188F, Q319G / K238W, N606Y / V188F, N606Y / K238W, and V188F / K238W. The amino acid sequences of these six thermostability-enhanced reverse transcriptase UCRT vZ combined mutants 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) By selecting three mutation sites for combination, four thermostability-enhanced reverse transcriptase UCRT vZ combination mutants can be constructed. The combination mutation sites are: Q319G / N606Y / V188F, Q319G / N606Y / K238W, N606Y / V188F / K238W, and Q319G / V188F / K238W. The amino acid sequences of these four thermostability-enhanced reverse transcriptase UCRT vZ combination mutants are SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.16, and SEQ ID NO.16, respectively.

[0175] (3) By selecting four mutation sites for combination, a thermo-stable reverse transcriptase UCRT vZ combined mutant can be constructed. The combined mutation sites are Q319G / N606Y / V188F / K238W. The amino acid sequence of this thermo-stable reverse transcriptase UCRT vZ combined mutant is SEQ ID NO.17.

[0176] Example 3:

[0177] This embodiment provides a gene encoding the thermostable reverse transcriptase UCRT vZ mutant as described in Example 1:

[0178] (1) The nucleic acid sequence of the reverse transcriptase UCRT vZ mutant encoding the Q319G mutation site 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 the 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] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAAGTG

[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 of the reverse transcriptase UCRT vZ mutant encoding the Q319G / N606Y mutation site 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 the mutation site N606Y / V188F is SEQ ID NO.36;

[0334] SEQ ID NO.36

[0335]

[0336] 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 of the reverse transcriptase UCRT vZ mutant encoding the mutation site Q319G / N606Y / V188F is SEQ ID NO.38;

[0412] SEQ ID NO.38

[0413]

[0414] (12) 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 of the reverse transcriptase UCRT vZ mutant encoding the Q319G / V188F / K238W mutation site 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 of the reverse transcriptase UCRT vZ mutant encoding the mutation site 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] Example 4:

[0531] This embodiment investigates the enzymatic characterization of the reverse transcriptase UCRT vZ mutant.

[0532] The thermostability of wild-type reverse transcriptase UCRT vZ and various reverse transcriptase UCRT vZ mutants provided in Example 2 was tested according to the conventional method for determining the activity of reverse transcriptase UCRT vZ.

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

[0534] Experimental results show that among the various reverse transcriptase UCRT vZ mutants, the thermostability of 4 single-point mutants and 11 combined mutants was significantly improved, as shown in Table 1:

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

[0536]

[0537]

[0538] As shown in Table 1, the reverse transcriptase UCRT vZ mutants provided by this invention include single-point mutants and combined mutants. Compared with wild-type reverse transcriptase UCRT vZ, both single-point mutants and combined mutants have longer half-lives at 66°C. In particular, the combined mutants exhibit the superimposed 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 scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.

Claims

1. A thermostable reverse transcriptase UCRT vZ mutant, characterized in that, The amino acid sequence of the reverse transcriptase UCRT vZ mutant is configured as SEQ ID. The amino acid sequence resulting from mutation of one or more combinations of the mutation sites Q319G, N505Y, V188F, and K238W on NO.2; wherein 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; 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 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 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 N505Y / K238W is SEQ ID NO.11; The amino acid sequence of the combined mutant V188F / K238W is SEQ ID NO.12; The amino acid sequence of the combined mutant Q319G / N505Y / V188F is SEQ ID NO.13; The amino acid sequence of the combined mutant Q319G / N505Y / K238W is SEQ ID NO.14; The amino acid sequence of the combined mutant N505Y / V188F / K238W is SEQ ID NO.15; The amino acid sequence of the combined mutant Q319G / V188F / K238W is SEQ ID NO.16; The amino acid sequence of the combined mutant Q319G / N505Y / V188F / K238W is SEQ ID NO.

17.

2. The method for constructing the thermostable reverse transcriptase UCRT vZ mutant as described in claim 1, characterized in that, Includes the following steps: Search the database for amino acid sequences that are more than 50% identical to the amino acid sequence shown in SEQ ID NO.2, then perform multiple sequence alignment, and generate a consensus sequence that can be edited later using software. Three-dimensional protein structure prediction was performed on SEQ ID NO.2, and stability-related mutation sites were screened out: Q319G, N505Y, V188F, and K238W.

3. The method for constructing the thermostable reverse transcriptase UCRT vZ mutant as described in claim 2, 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.

4. A gene encoding a reverse transcriptase UCRT vZ mutant with enhanced thermal stability as described in claim 1.

5. A recombinant plasmid comprising the gene as described in claim 4.

6. A soluble protein, immobilized enzyme, or engineered bacterium comprising the thermostable reverse transcriptase UCRT vZ mutant as described in claim 1.

7. The application of the thermostability-enhanced reverse transcriptase UCRT vZ mutant as described in claim 1 in the reverse transcription of RNA into cDNA.

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