UCRT X heat-resistant reverse transcriptase mutant with improved heat stability, and construction method and application of UCRT X heat-resistant reverse transcriptase mutant

Abstract

The invention belongs to the technical field of biology, and particularly provides UCRT X heat-resistant reverse transcriptase mutants with improved thermal stability, the UCRT heat-resistant reverse transcriptase mutants provided by the invention comprise four single-point mutants, six double mutants and four three mutants, and compared with wild-type UCRT X heat-resistant reverse transcriptase, the mutant has longer half-life period at 65 DEG C; the double mutant has a better effect, and the half-life period of the double mutant is about four times of that of wild type DNA polymerase. The invention also provides a genetically engineered bacterium containing the mutant. The UCRT heat-resistant reverse transcriptase mutant obtained by the construction method provided by the invention has better thermal stability, shows higher thermal stability when being used for synthesizing DNA (Deoxyribonucleic Acid) by reverse transcription at 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 UCRT X thermostable reverse transcriptase mutant with improved thermal stability, a construction method and an application thereof. Background Art

[0002] UCRT thermostable reverse transcriptase has reverse transcription activity and RNase H enzyme activity. It can use RNA as a template to catalyze dNTP polymerization to generate DNA-RNA hybrid double strands, and hydrolyze the RNA in the hybrid double strands to obtain a single strand of DNA (cDNA) complementary to RNA. UCRT thermostable reverse transcriptase is conducive to opening the RNA secondary structure at higher temperatures, improving reverse transcription efficiency and cDNA integrity, which has a great advantage over common reverse transcriptases on the market; however, its thermal stability is average, and in order to facilitate long-term stable use and storage, further targeted modification of its performance is needed.

[0003] 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 its guiding ideology, and combines bioinformatics and crystallographic methods to assist, using computer-assisted molecular models combined with site-directed mutagenesis to achieve protein function optimization, and integrates and analyzes the reverse transcriptase family sequence to obtain a novel reverse transcriptase mutant with high stability. Summary of the invention

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

[0005] To this end, the present invention provides a UCRT X thermostable reverse transcriptase mutant with improved thermal stability, wherein the UCRT thermostable reverse transcriptase 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 UCRT VI thermostable reverse transcriptase mutant is configured as the amino acid sequence after one or more combinations of mutation sites E574D, G167N, K298D, and T330L on SEQ ID NO.2 are mutated.

[0009] Specifically, the above mutation sites are E574D, G167N, K298D, T330L, E574D / G167N, E574D / K298D, E574D / T330L, G167N / K298D, G167N / T330L, K298D / T330L, E574D / G167N / K298D, E574D / G167N / T330L, E574D / K298D / T330L, G167N / K298D / T330L or E574D / G167N / K298D / T330L.

[0010] Specifically, the amino acid sequence of the single point mutant corresponding to the above E574D is SEQ ID NO.3;

[0011] The amino acid sequence of the single point mutant corresponding to G167N is SEQ ID NO.4;

[0012] The amino acid sequence of the single point mutant corresponding to K298D is SEQ ID NO.5;

[0013] The amino acid sequence of the single point mutant corresponding to T330L is SEQ ID NO.6;

[0014] The amino acid sequence of the combined mutant corresponding to E574D / G167N is SEQ ID NO.7;

[0015] The amino acid sequence of the combined mutant corresponding to E574D / K298D is SEQ ID NO.8;

[0016] The amino acid sequence of the combined mutant corresponding to E574D / T330L is SEQ ID NO.9;

[0017] The amino acid sequence of the combined mutant corresponding to G167N / K298D is SEQ ID NO. 10;

[0018] The amino acid sequence of the combined mutant corresponding to G167N / T330L is SEQ ID NO.11;

[0019] The amino acid sequence of the combined mutant corresponding to K298D / T330L is SEQ ID NO. 12;

[0020] The amino acid sequence of the combined mutant corresponding to E574D / G167N / K298D is SEQ ID NO. 13;

[0021] The amino acid sequence of the combined mutant corresponding to E574D / G167N / T330L is SEQ ID NO. 14;

[0022] The amino acid sequence of the combined mutant corresponding to E574D / K298D / T330L is SEQ ID NO. 15;

[0023] The amino acid sequence of the combined mutant corresponding to G167N / K298D / T330L is SEQ ID NO. 16;

[0024] The amino acid sequence of the combined mutant corresponding to E574D / G167N / K298D / T330L is SEQ ID NO.17.

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

[0026] 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;

[0027] The protein three-dimensional structure of SEQ ID NO.2 was predicted, and the stability-related mutation sites were screened out: E574D, G167N, K298D, and T330L.

[0028] Specifically, the amplification primer sequences for the mutation site E574D are SEQ ID NO.20 and SEQ ID NO.21;

[0029] The amplification primer sequences for the mutation site G167N are SEQ ID NO.22 and SEQ ID NO.23;

[0030] The amplification primer sequences for the mutation site K298D are SEQ ID NO.24 and SEQ ID NO.25;

[0031] The amplification primer sequences for the mutation site T330L are SEQ ID NO.26 and SEQ ID NO.27.

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

[0035] The UCRT X thermostable reverse transcriptase mutant provided by the invention can be used for reverse transcription catalysis of DNA synthesis.

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

[0037] 1. The UCRTX thermostable reverse transcriptase mutants with improved thermal stability provided by the present invention include single point mutants and combined mutants. Compared with the wild-type UCRTX thermostable reverse transcriptase, the single point mutants and combined mutants have longer half-lives at 65°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 3 times that of the wild type. The mutants have excellent catalytic activity and good application prospects.

[0038] 2. The method for constructing a thermostable UCRTX reverse transcriptase 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 thermostable stability of the enzyme from an evolutionary perspective, conducts an integrated analysis of the thermostable reverse transcriptase family sequence, and combines bioinformatics and crystallography methods to obtain a novel thermostable UCRTX reverse transcriptase 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 UCRT X thermostable reverse transcriptase 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 UCRT X thermostable reverse transcriptase mutant with improved thermal stability, wherein the UCRT X thermostable reverse transcriptase 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 UCRT VI thermostable reverse transcriptase mutant is configured as the amino acid sequence after one or more combinations of mutation sites E574D, G167N, K298D, and T330L on SEQ ID NO.2 are mutated.

[0046] The mutation sites are E574D, G167N, K298D, T330L, E574D / G167N, E574D / K298D, E574D / T330L, G167N / K298D, G167N / T330L, K298D / T330L, E574D / G167N / K298D, E574D / G167N / T330L, E574D / K298D / T330L, G167N / K298D / T330L or E574D / G167N / K298D / T330L.

[0047] Among them, the amino acid sequence of the single point mutant corresponding to E574D is SEQ ID NO.3;

[0048] The amino acid sequence of the single point mutant corresponding to G167N is SEQ ID NO.4;

[0049] The amino acid sequence of the single point mutant corresponding to K298D is SEQ ID NO.5;

[0050] The amino acid sequence of the single point mutant corresponding to T330L is SEQ ID NO.6;

[0051] The amino acid sequence of the combined mutant corresponding to E574D / G167N is SEQ ID NO.7;

[0052] The amino acid sequence of the combined mutant corresponding to E574D / K298D is SEQ ID NO.8;

[0053] The amino acid sequence of the combined mutant corresponding to E574D / T330L is SEQ ID NO.9;

[0054] The amino acid sequence of the combined mutant corresponding to G167N / K298D is SEQ ID NO. 10;

[0055] The amino acid sequence of the combined mutant corresponding to G167N / T330L is SEQ ID NO.11;

[0056] The amino acid sequence of the combined mutant corresponding to K298D / T330L is SEQ ID NO. 12;

[0057] The amino acid sequence of the combined mutant corresponding to E574D / G167N / K298D is SEQ ID NO. 13;

[0058] The amino acid sequence of the combined mutant corresponding to E574D / G167N / T330L is SEQ ID NO. 14;

[0059] The amino acid sequence of the combined mutant corresponding to E574D / K298D / T330L is SEQ ID NO. 15;

[0060] The amino acid sequence of the combined mutant corresponding to G167N / K298D / T330L is SEQ ID NO. 16;

[0061] The amino acid sequence of the combined mutant corresponding to E574D / G167N / K298D / T330L is SEQ ID NO.17.

[0062] The present invention also provides a method for constructing the above-mentioned UCRT X thermostable reverse transcriptase 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: E574D, G167N, K298D, and T330L.

[0065] Among them, the amplification primer sequences of the mutation site E574D are SEQ ID NO.20 and SEQ ID NO.21;

[0066] The amplification primer sequences for the mutation site G167N are SEQ ID NO.22 and SEQ ID NO.23;

[0067] The amplification primer sequences for the mutation site K298D are SEQ ID NO.24 and SEQ ID NO.25;

[0068] The amplification primer sequences for the mutation site T330L are SEQ ID NO.26 and SEQ ID NO.27.

[0069] The effects of the UCRT X thermostable reverse transcriptase mutant with improved thermal stability of the present invention are studied below through specific examples.

[0070] Embodiment 1:

[0071] This embodiment provides a UCRT X thermostable reverse transcriptase mutant with improved thermal stability, wherein the UCRT X thermostable reverse transcriptase is a wild-type UCRT X thermostable reverse transcriptase derived from the Yellowstone Park thermotolerant bacteria, named as protein UCRT X thermostable reverse transcriptase, the nucleic acid sequence encoding the UCRT X thermostable reverse transcriptase polymerase 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 UCRTX thermostable reverse transcriptase mutant with improved thermal stability provided in this embodiment comprises: 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 having the same function as the amino acid sequence shown in SEQ ID NO.2 (i.e., UCRTX thermostable reverse transcriptase 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 having at least 90% homology with the amino acid sequence shown in SEQ ID NO.2 (i.e., UCRTX thermostable reverse transcriptase 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 single point mutants of UCRT X thermostable reverse transcriptase, wherein the mutation sites were E574D, G167N, K298D, and T330L. The activity of the four single point mutants of UCRT X thermostable reverse transcriptase was determined, and the amino acid sequences thereof were SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0078] SEQ ID NO.3

[0079] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGADLFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0080] SEQ ID NO.4

[0081] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0082] SEQ ID NO.5

[0083] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVDLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0084] SEQ ID NO.6

[0085] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QLAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[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 UCRT X thermostable reverse transcriptase mutants with improved thermal stability, wherein the combined mutation sites are: E574D / G167N, E574D / K298D, E574D / T330L, G167N / K298D, G167N / T330L, K298D / T330L, 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, and SEQ ID NO.12, respectively.

[0087] SEQ ID NO.7

[0088] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGADLFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0089] SEQ ID NO.8

[0090] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVDLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGADLFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0091] SEQ ID NO.9

[0092] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQLAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGADLFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0093] SEQ ID NO.10

[0094] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVDLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0095] SEQ ID NO.11

[0096] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQLAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0097] SEQ ID NO.12

[0098] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVDLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QLAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0099] For example, by selecting three mutation sites from the above four mutation sites for combination, four UCRT X thermostable reverse transcriptase mutants with improved thermal stability were obtained, and their combined mutation sites were: E574D / G167N / K298D, E574D / G167N / T330L, E574D / K298D / T330L, G167N / K298D / T330L, and their amino acid sequences were SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively.

[0100] SEQ ID NO.13

[0101] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVDLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGADLFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0102] SEQ ID NO.14

[0103] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQLAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGADLFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0104] SEQ ID NO.15

[0105] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVDLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQLAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGADLFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0106] SEQ ID NO.16

[0107] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLLVYQLDQQAQKVVIETSQHGMPVDLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QLAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0108] For example, by selecting 4 mutation sites from the above 4 mutation sites and combining them, a UCRTX thermostable reverse transcriptase with improved thermal stability is obtained, and its combined mutation sites are: E574D / G167N / K298D / T330L, and its amino acid sequence is SEQ ID NO.17.

[0109] SEQ ID NO.17

[0110] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLLVYQLDQQAQKVVIETSQHGMPVDLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QLAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGADLFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0111] Embodiment 2:

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

[0113] 1. Cloning of wild-type UCRT X thermostable reverse transcriptase gene

[0114] The wild-type UCRT X thermostable reverse transcriptase gene was codon optimized using Escherichia coli as a host cell to obtain an optimized UCRT X thermostable reverse transcriptase 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] 5'-ACTGCT CATATG ATGGTAAAAGTTAAGTTTAAGTATAAA-3' (where the underline indicates the restriction endonuclease NdeI recognition site);

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

[0118] 5'-TCAGCT CTCGAG GGGCTTCTCCCATGTGTTACCCAC-3' (the underline indicates the restriction endonuclease XhoI recognition site).

[0119] The amplification conditions were as follows: amplification at 95°C for 2 min, followed by amplification at 56°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, 1.5% 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 50 μg / ml kanamycin, and the positive clone plasmid was extracted and sequenced. The results showed that the cloned UCRT X thermostable reverse transcriptase 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 UCRT X thermostable reverse transcriptase is derived from a virus isolated from thermostable bacteria in Yellowstone Park;

[0122] UCRT X thermostable reverse transcriptase 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 UCRT X thermostable reverse transcriptase 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 50μg / mL Kan, and cultured at 37°C and 220rpm for 2.5h until the OD600 reached about 0.9, and 0.1mM IPTG inducer was added, and induced culture was carried out at 25°C and 200rpm for 14h. The Escherichia coli cell suspension harvested after fermentation was ultrasonically disrupted, and then subjected to a step of Ni-NTA affinity chromatography to obtain a UCRT X thermostable reverse transcriptase protein with a purity of >95%, and the amino acid sequence was SEQID NO.2.

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

[0127] 3.1. Enter the Pfam database homepage (http: / / pfam.xfam.org / ), enter the amino acid sequence of UCRT X reverse transcriptase in the SEQUENCE SEARCH tool for search, and 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 (SEQ ID NO.2) of the UCRT X reverse transcriptase protein, delete the repeated identical sequences, and organize the remaining amino acid sequences into fasta. format, input them into 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 UCRT X thermostable reverse transcriptase 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 UCRT X thermostable reverse transcriptase protein and selection of mutation hotspots

[0132] 4.1. The three-dimensional structure prediction of UCRT X thermostable reverse transcriptase protein (amino acid sequence SEQ ID NO. 2) was obtained by using Swissmodel online tool;

[0133] 4.2. The crystal structure of UCRT X thermostable reverse transcriptase (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 UCRT X thermostable reverse transcriptase. 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 UCRT X thermostable reverse transcriptase, 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 a buried or semi-buried state.

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

[0140] (3) Based on the crystal structure of UCRTX thermostable reverse transcriptase protein, the above 10 mutation forms were analyzed in detail one by one to screen out mutants that may improve the thermal stability of UCRTX thermostable reverse transcriptase 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: E574D, G167N, K298D, and T330L;

[0143] The activity of the four UCRT X thermostable reverse transcriptase mutants was determined, and four UCRT X thermostable reverse transcriptases with improved thermal stability were screened out, whose mutation sites were: E574D, G167N, K298D, and T330L, and the amino acid sequences of the corresponding single point mutants were SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0144] 5. Construction, expression and purification of mutants

[0145] 5.1. Construction of single-point mutants of UCRTX thermostable reverse transcriptase

[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 E574D are as follows:

[0149] SEQ ID NO.20:

[0150] 5'-GGCGCCGATTTGTTCAAAAAAGCCATCGTC-3'

[0151] SEQ ID NO.21:

[0152] 5'-GACGATGGCTTTTTTGAACAAATCGGCGCC-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 G167N are as follows:

[0154] SEQ ID NO.22:

[0155] 5'-GGTTGGAATCTAGACTTCGATCTT-3';

[0156] SEQ ID NO.23:

[0157] 5'-AAGATCGAAGTCTAGATTCCAACC-3';

[0158] (3) The nucleic acid sequences of the upstream amplification primer SEQ ID NO.24 and the downstream amplification primer SEQ ID NO.25 of the mutation site K298D are as follows:

[0159] SEQ ID NO.24:

[0160] 5'-CCGGTTGATTTAAAAGCATTGGAAGAAGAGATC-3';

[0161] SEQ ID NO.25:

[0162] 5'-GATCTCTTCTTCCAATGCTTTTTAAATCAACCGG-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 T330L are as follows:

[0164] SEQ ID NO.26:

[0165] 5'-AAACAGTTGGCGAAATTTTTCGGTGTAAAT-3';

[0166] SEQ ID NO.27:

[0167] 5'-ATTTACACCGAAAAATTTCGCCAACTGTTT-3';

[0168] The amplification conditions were as follows: amplification at 95°C for 2 min, then amplification at 56°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; the PCR amplification product was recovered by gel, and the gel recovery 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 50 μg / mL kanamycin, cultured at 37°C overnight, positive clones were screened, and sequencing was performed to obtain recombinant bacteria containing a single-point mutant of the UCRT X thermostable reverse transcriptase;

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

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

[0171] 5.2. Construction of UCRT X thermostable reverse transcriptase 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 DUCRT X thermostable reverse transcriptase combination mutants:

[0173] (1) Two mutation sites are selected for combination to construct six UCRT X thermostable reverse transcriptase mutants with improved thermal stability, wherein the combined mutation sites are E574D / G167N, E574D / K298D, E574D / T330L, G167N / K298D, G167N / T330L, and K298D / T330L, and the amino acid sequences of the six UCRT X thermostable reverse transcriptase 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 UCRT X thermostable reverse transcriptase combination mutants with improved thermal stability, wherein the combination mutation sites were E574D / G167N / K298D, E574D / G167N / T330L, E574D / K298D / T330L, and G167N / K298D / T330L. The amino acid sequences of the four UCRT X thermostable reverse transcriptase combination mutants with improved thermal stability were SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively.

[0175] (3) Four mutation sites are selected for combination to construct a UCRT X thermostable reverse transcriptase combination mutant with improved thermal stability, wherein the combined mutation sites are: E574D / G167N / K298D / T330L. The amino acid sequence of the UCRT X thermostable reverse transcriptase combination mutant with improved thermal stability is SEQ ID NO.17.

[0176] Embodiment 3:

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

[0178] (1) The nucleic acid sequence encoding the UCRT X thermostable reverse transcriptase mutant with mutation site E574D is SEQ ID NO. 28;

[0179] SEQ ID NO.28

[0180]

[0181] (2) The nucleic acid sequence encoding the UCRT X thermostable reverse transcriptase mutant with mutation site G167N is SEQ ID NO. 29;

[0182] SEQ ID NO.29

[0183]

[0184] (3) The nucleic acid sequence encoding the UCRT X thermostable reverse transcriptase mutant with mutation site K298D is SEQ ID NO.30;

[0185] SEQ ID NO.30

[0186]

[0187] (4) The nucleic acid sequence encoding the UCRT X thermostable reverse transcriptase mutant with a mutation site of T330L is SEQ ID NO.31;

[0188] SEQ ID NO.31

[0189]

[0190] (5) The nucleic acid sequence encoding the UCRT X thermostable reverse transcriptase mutant with mutation sites E574D / G167N is SEQ ID NO.32;

[0191] SEQ ID NO.32

[0192]

[0193] (6) The nucleic acid sequence encoding the UCRT X thermostable reverse transcriptase mutant with mutation sites E574D / K298D is SEQ ID NO. 33;

[0194] SEQ ID NO.33

[0195]

[0196] (7) The nucleic acid sequence encoding the UCRT X thermostable reverse transcriptase mutant with mutation site E574D / T330L is SEQ ID NO. 34;

[0197] SEQ ID NO.34

[0198]

[0199] (8) The nucleic acid sequence encoding the UCRT X thermostable reverse transcriptase mutant with mutation site G167N / K298D is SEQ ID NO.35;

[0200] SEQ ID NO.35

[0201]

[0202] (9) The nucleic acid sequence encoding the UCRT X thermostable reverse transcriptase mutant with mutation site G167N / T330L is SEQ ID NO. 36;

[0203] SEQ ID NO.36

[0204]

[0205] (10) The nucleic acid sequence encoding the UCRT X thermostable reverse transcriptase mutant with mutation site K298D / T330L is SEQ ID NO.37;

[0206] SEQ ID NO.37

[0207]

[0208] (11) The nucleic acid sequence encoding the UCRT X thermostable reverse transcriptase mutant with mutation sites E574D / G167N / K298D is SEQ ID NO. 38;

[0209] SEQ ID NO.38

[0210]

[0211] (12) The nucleic acid sequence encoding the UCRT X thermostable reverse transcriptase mutant with mutation sites E574D / G167N / T330L is SEQ ID NO. 39;

[0212] SEQ ID NO.39

[0213]

[0214] (13) The nucleic acid sequence encoding the UCRT X thermostable reverse transcriptase mutant with mutation sites G167N / K298D / T330L is SEQ ID NO.40;

[0215] SEQ ID NO.40

[0216]

[0217] (14) The nucleic acid sequence encoding the UCRT X thermostable reverse transcriptase mutant with mutation sites G167N / K298D / T330L is SEQ ID NO.41;

[0218] SEQ ID NO.41

[0219]

[0220] (15) The nucleic acid sequence encoding the UCRT X thermostable reverse transcriptase mutant with mutation sites E574D / G167N / K298D / T330L is SEQ ID NO.42.

[0221] SEQ ID NO.42

[0222]

[0223] Embodiment 4:

[0224] This example studies the enzymatic properties of the UCRT X thermostable reverse transcriptase mutant.

[0225] The wild-type UCRT X thermostable reverse transcriptase and the various UCRT X thermostable reverse transcriptase mutants provided in Example 2 were subjected to a thermal stability test according to a conventional UCRT X thermostable reverse transcriptase activity assay method, specifically:

[0226] The enzyme solution is incubated at a certain temperature, samples are taken at different treatment times, and the residual activity percentage of UCRT X thermostable reverse transcriptase or UCRT X thermostable reverse transcriptase mutant is determined. The ln value of the residual activity percentage is plotted against the time t (min), and the slope of the straight line is the inactivation constant kinact. The half-life of the wild-type UCRT X thermostable reverse transcriptase or UCRT X thermostable reverse transcriptase mutant at the temperature is obtained by t1 / 2=ln2 / kinact.

[0227] The experimental results showed that among the above UCRT X thermostable reverse transcriptase mutants, the thermal stability of 4 single-point mutants and 11 combined mutants was significantly improved, as shown in Table 1:

[0228] Table 1. Enzymatic properties of wild-type UCRT X thermostable reverse transcriptase, single point mutants and combined mutants

[0229]

[0230] As shown in Table 1, the UCRTX thermostable reverse transcriptase mutants provided by the present invention include single point mutants and combined mutants. Compared with the wild-type UCRTX thermostable reverse transcriptase, the single point mutants and combined mutants have longer half-lives at 65°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.

[0231] 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 UCRT X thermostable reverse transcriptase mutant with improved thermal stability, characterized in that: The UCRT thermostable reverse transcriptase 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 UCRT X thermostable reverse transcriptase mutant with improved thermal stability according to claim 1, characterized in that: The amino acid sequence of the UCRT VI thermostable reverse transcriptase mutant is configured as an amino acid sequence after one or more combinations of mutation sites E574D, G167N, K298D, and T330L on SEQ ID NO.2 are mutated.

3. The UCRT X thermostable reverse transcriptase mutant with improved thermal stability according to claim 2, characterized in that: The mutation sites are E574D, G167N, K298D, T330L, E574D / G167N, E574D / K298D, E574D / T330L, G167N / K298D, G167N / T330L, K298D / T330L, E574D / G167N / K298D, E574D / G167N / T330L, E574D / K298D / T330L, G167N / K298D / T330L or E574D / G167N / K298D / T330L.

4. The UCRT X thermostable reverse transcriptase mutant with improved thermal stability according to claim 2, characterized in that: The amino acid sequence of the single point mutant corresponding to E574D is SEQ ID NO. 3; The amino acid sequence of the single point mutant corresponding to G167N is SEQ ID NO.4; The amino acid sequence of the single point mutant corresponding to K298D is SEQ ID NO.5; The amino acid sequence of the single point mutant corresponding to T330L is SEQ ID NO.6; The amino acid sequence of the combined mutant corresponding to E574D / G167N is SEQ ID NO.7; The amino acid sequence of the combined mutant corresponding to E574D / K298D is SEQ ID NO.8; The amino acid sequence of the combined mutant corresponding to E574D / T330L is SEQ ID NO.9; The amino acid sequence of the combined mutant corresponding to G167N / K298D is SEQ ID NO.10; The amino acid sequence of the combined mutant corresponding to G167N / T330L is SEQ ID NO.11; The amino acid sequence of the combined mutant corresponding to K298D / T330L is SEQ ID NO. 12; The amino acid sequence of the combined mutant corresponding to E574D / G167N / K298D is SEQ ID NO. 13; The amino acid sequence of the combined mutant corresponding to E574D / G167N / T330L is SEQ ID NO. 14; The amino acid sequence of the combined mutant corresponding to E574D / K298D / T330L is SEQ ID NO. 15; The amino acid sequence of the combined mutant corresponding to G167N / K298D / T330L is SEQ ID NO. 16; The amino acid sequence of the combined mutant corresponding to E574D / G167N / K298D / T330L is SEQ ID NO.

17.

5. The method for constructing a UCRT X thermostable reverse transcriptase 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 of more than 30% 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: E574D, G167N, K298D, and T330L.

6. The method for constructing a UCRT X thermostable reverse transcriptase mutant with improved thermal stability according to claim 5, characterized in that: The amplification primer sequences for mutation site E574D are SEQ ID NO.20 and SEQ ID NO.21; The amplification primer sequences for the mutation site G167N are SEQ ID NO.22 and SEQ ID NO.23; The amplification primer sequences for the mutation site K298D are SEQ ID NO.24 and SEQ ID NO.25; The amplification primer sequences for the mutation site T330L are SEQ ID NO.26 and SEQ ID NO.

27.

7. A gene encoding the UCRTX thermostable reverse transcriptase mutant with improved thermal stability according to 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 UCRT X thermostable reverse transcriptase mutant with improved thermal stability according to any one of claims 1 to 4.

10. Use of the UCRTX thermostable reverse transcriptase mutant with improved thermal stability according to any one of claims 1 to 4 in reverse transcription catalysis of DNA synthesis.

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