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

Abstract

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

Description

Technical Field

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

[0002] Reverse transcriptase UCRT vL is derived from a virus isolated from heat-resistant bacteria in Yellowstone Park (functional gene pol(A) was screened out through metagenomic sequencing of heat-resistant bacterial viruses). It has multiple enzyme activities and high temperature resistance, with the best enzyme activity temperature at 60-70°C. It is used for reverse transcription of RNA into cDNA. Compared with traditional reverse transcriptase (MMLV), it has higher heat resistance and higher reverse transcriptase activity in the range of 60-80°C. Therefore, UCRT vL reverse transcriptase can be specifically applied to complex templates (high GC content / more RNA secondary structures) to improve the reverse transcription efficiency of such RNA templates.

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

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

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

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

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

[0008] Specifically, the amino acid sequence of the above-mentioned reverse transcriptase UCRT vL mutant is configured as the amino acid sequence after one or more combinations of mutation sites G167N, D280E, F431Y, and S396T on SEQ ID NO.2 are mutated.

[0009] Specifically, the above mutation sites are G167N, D280E, F431Y, S396T, G167N / D280E, G167N / F431Y, G167N / S396T, D280E / F431Y, D280E / S396T, F431Y / S396T, G167N / D280E / F431Y, G167N / D280E / S396T, D280E / F431Y / S396T, G167N / F431Y / S396T or G167N / D280E / F431Y / S396T.

[0010] Specifically, the amino acid sequence of the single point mutant corresponding to the above G167N is SEQ ID NO.3;

[0011] The amino acid sequence of the single point mutant corresponding to D280E is SEQ ID NO.4;

[0012] The amino acid sequence of the single point mutant corresponding to F431Y is SEQ ID NO.5;

[0013] The amino acid sequence of the single point mutant corresponding to S396T is SEQ ID NO.6;

[0014] The amino acid sequence of the combined mutant corresponding to G167N / D280E is SEQ ID NO.7;

[0015] The amino acid sequence of the combined mutant corresponding to G167N / F431Y is SEQ ID NO.8;

[0016] The amino acid sequence of the combined mutant corresponding to G167N / S396T is SEQ ID NO.9;

[0017] The amino acid sequence of the combined mutant corresponding to D280E / F431Y is SEQ ID NO. 10;

[0018] The amino acid sequence of the combined mutant corresponding to D280E / S396T is SEQ ID NO.11;

[0019] The amino acid sequence of the combined mutant corresponding to F431Y / S396T is SEQ ID NO. 12;

[0020] The amino acid sequence of the combined mutant corresponding to G167N / D280E / F431Y is SEQ ID NO. 13;

[0021] The amino acid sequence of the combined mutant corresponding to G167N / D280E / S396T is SEQ ID NO. 14;

[0022] The amino acid sequence of the combined mutant corresponding to D280E / F431Y / S396T is SEQ ID NO.15;

[0023] The amino acid sequence of the combined mutant corresponding to G167N / F431Y / S396T is SEQ ID NO. 16;

[0024] The amino acid sequence of the combined mutant corresponding to G167N / D280E / F431Y / S396T is SEQ ID NO.17.

[0025] The present invention also provides a method for constructing the above-mentioned reverse transcriptase UCRT vL 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: G167N, D280E, F431Y, and S396T.

[0028] Specifically, the amplification primer sequences for the mutation site G167N are SEQ ID NO.20 and SEQ ID NO.21;

[0029] The amplification primer sequences for mutation site D280E are SEQ ID NO.22 and SEQ ID NO.23;

[0030] The amplification primer sequences for mutation site F431Y are SEQ ID NO.24 and SEQ ID NO.25;

[0031] The amplification primer sequences for the mutation site S396T are SEQ ID NO.26 and SEQ ID NO.27.

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

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

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

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

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

[0037] 1. The reverse transcriptase UCRT vL mutant with improved thermal stability provided by the present invention includes single point mutants and combined mutants. Compared with the wild-type reverse transcriptase UCRT vL, 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 UCRT vL mutant has excellent catalytic activity and has good application prospects.

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

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

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

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

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

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

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

[0045] The amino acid sequence of the reverse transcriptase UCRT vL mutant is configured as the amino acid sequence after one or more combinations of mutation sites G167N, D280E, F431Y, and S396T on SEQ ID NO.2 are mutated.

[0046] The mutation sites are G167N, D280E, F431Y, S396T, G167N / D280E, G167N / F431Y, G167N / S396T, D280E / F431Y, D280E / S396T, F431Y / S396T, G167N / D280E / F431Y, G167N / D280E / S396T, D280E / F431Y / S396T, G167N / F431Y / S396T or G167N / D280E / F431Y / S396T.

[0047] Among them, the amino acid sequence of the single point mutant corresponding to G167N is SEQ ID NO.3;

[0048] The amino acid sequence of the single point mutant corresponding to D280E is SEQ ID NO.4;

[0049] The amino acid sequence of the single point mutant corresponding to F431Y is SEQ ID NO.5;

[0050] The amino acid sequence of the single point mutant corresponding to S396T is SEQ ID NO.6;

[0051] The amino acid sequence of the combined mutant corresponding to G167N / D280E is SEQ ID NO.7;

[0052] The amino acid sequence of the combined mutant corresponding to G167N / F431Y is SEQ ID NO.8;

[0053] The amino acid sequence of the combined mutant corresponding to G167N / S396T is SEQ ID NO.9;

[0054] The amino acid sequence of the combined mutant corresponding to D280E / F431Y is SEQ ID NO. 10;

[0055] The amino acid sequence of the combined mutant corresponding to D280E / S396T is SEQ ID NO.11;

[0056] The amino acid sequence of the combined mutant corresponding to F431Y / S396T is SEQ ID NO. 12;

[0057] The amino acid sequence of the combined mutant corresponding to G167N / D280E / F431Y is SEQ ID NO. 13;

[0058] The amino acid sequence of the combined mutant corresponding to G167N / D280E / S396T is SEQ ID NO. 14;

[0059] The amino acid sequence of the combined mutant corresponding to D280E / F431Y / S396T is SEQ ID NO. 15;

[0060] The amino acid sequence of the combined mutant corresponding to G167N / F431Y / S396T is SEQ ID NO. 16;

[0061] The amino acid sequence of the combined mutant corresponding to G167N / D280E / F431Y / S396T is SEQ ID NO.17.

[0062] The present invention also provides a method for constructing the above-mentioned reverse transcriptase UCRT vL 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. The mutation sites related to thermal stability were screened out as follows: G167N, D280E, F431Y, and S396T.

[0065] Among them, the amplification primer sequences of the mutation site G167N are SEQ ID NO.20 and SEQ ID NO.21;

[0066] The amplification primer sequences for mutation site D280E are SEQ ID NO.22 and SEQ ID NO.23;

[0067] The amplification primer sequences for mutation site F431Y are SEQ ID NO.24 and SEQ ID NO.25;

[0068] The amplification primer sequences for the mutation site S396T are SEQ ID NO.26 and SEQ ID NO.27.

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

[0070] Embodiment 1:

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

[0072] SEQ ID NO.1

[0073]

[0074] SEQ ID NO.2

[0075] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

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

[0077] Specifically, a single point mutation was performed on a certain site in the amino acid sequence shown in SEQ ID NO.2 to obtain four reverse transcriptase UCRT vL single point mutants, whose mutation sites were: G167N, D280E, F431Y, and S396T. The activity of the four reverse transcriptase UCRT vL single point mutants was tested, and their amino acid sequences 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 GWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0080] SEQ ID NO.4

[0081] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0082] SEQ ID NO.5

[0083] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADYPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0084] SEQ ID NO.6

[0085] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPTGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0086] Alternatively, multiple mutation sites are selected and combined in the amino acid sequence shown in SEQ ID NO.2, such as selecting 2 mutation sites from the above 4 mutation sites for combination, to obtain the following 6 reverse transcriptase UCRTvL mutants with improved thermal stability, wherein the combined mutation sites are: G167N / D280E, G167N / F431Y, G167N / S396T, D280E / F431Y, D280E / S396T, F431Y / S396T, G167N / D280E / F431Y, G167N / D280E / S396T, D280E / F431Y / S396T, G167N / F431Y / S396T, G167N / D280E / F431Y / S396T, and the amino acid sequences are SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12.

[0087] SEQ ID NO.7

[0088] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0089] SEQ ID NO.8

[0090] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADYPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0091] SEQ ID NO.9

[0092] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPTGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0093] SEQ ID NO.10

[0094] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADYPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0095] SEQ ID NO.11

[0096] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPTGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0097] SEQ ID NO.12

[0098] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPTGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADYPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0099] If three mutation sites are selected from the above four mutation sites and combined, four reverse transcriptase UCRT vL mutants with improved thermal stability are obtained, and their combined mutation sites are: G167N / D280E / F431Y, G167N / D280E / S396T, G167N / F431Y / S396T, D280E / F431Y / S396T, and their amino acid sequences are SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively.

[0100] SEQ ID NO.13

[0101] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADYPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0102] SEQ ID NO.14

[0103] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPTGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0104] SEQ ID NO.15

[0105] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPTGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADYPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0106] SEQ ID NO.16

[0107] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPTGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADYPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0108] For example, by selecting 4 mutation sites from the above 4 mutation sites and combining them, a reverse transcriptase UCRT vL mutant with improved thermal stability is obtained, and its combined mutation sites are: G167N / D280E / F431Y / S396T, and its amino acid sequence is SEQ ID NO.17.

[0109] SEQ ID NO.17

[0110] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPTGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADYPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0111] Embodiment 2:

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

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

[0114] The wild-type reverse transcriptase UCRT vL gene was codon optimized using Escherichia coli as a host cell to obtain an optimized reverse transcriptase UCRT vL 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 ATGGTAAAAGTTAAGTTTAAGTATAAAG-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 GGGCTTCTCCCATGTGTTACC-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 reverse transcriptase UCRT vL gene sequence was correct and had been correctly connected to the pET28a plasmid to obtain the recombinant plasmid pET28a-Bst;

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

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

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

[0124] 2. Expression and purification of reverse transcriptase UCRT vL 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 one-step Ni-NTA affinity chromatography treatment to obtain a reverse transcriptase UCRT vL protein with a purity of >95%, and the amino acid sequence was SEQ ID NO.2.

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

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

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

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

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

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

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

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

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

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

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

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

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

[0139] After the above two steps of screening, there are 10 differential sites remaining, most of which are located on the surface of the reverse transcriptase UCRT vL 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 vL protein, the above 10 mutation forms were analyzed in detail one by one to screen out mutants that may improve the thermal stability of the reverse transcriptase UCRT vL 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: G167N, D280E, F431Y, and S396T;

[0143] The activity of the four reverse transcriptase UCRT vL single point mutants was determined, and four reverse transcriptase UCRT vL mutants with improved thermal stability were screened out, whose mutation sites were: G167N, D280E, F431Y, and S396T, and the corresponding amino acid sequences of the 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 reverse transcriptase UCRT vL protein

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

[0147] The amplification primer pairs used were:

[0148] (1) The nucleic acid sequences of the upstream amplification primer SEQ ID NO.20 and the downstream amplification primer SEQ ID NO.21 of the mutation site G167N are as follows:

[0149] SEQ ID NO.20:

[0150] 5'-CGTGGTTGGAATCTAGACTTCGAT-3';

[0151] SEQ ID NO.21:

[0152] 5'-TAAGATCGAAGTCTAGATTCCAACCACG-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 D280E are as follows:

[0154] SEQ ID NO.22:

[0155] 5'-TATCAGCTTGAACAACAGGCGCAAAAAG-3';

[0156] SEQ ID NO.23:

[0157] 5'-GCGCCTGTTGTTCAAGCTGATACACGAGA-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 F431Y are as follows:

[0159] SEQ ID NO.24:

[0160] 5'-TGCAGACTACCCCCAGATTGAACTTC-3';

[0161] SEQ ID NO.25:

[0162] 5'-CTGGGGGGTAGTCTGCAGTTATCA-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 S396T are as follows:

[0164] SEQ ID NO.26:

[0165] 5′-CCGACTGGGCGCATGAGCT-3′;

[0166] SEQ ID NO.27:

[0167] 5'-CCAGTCGGGGCGGTGGTAGTGA-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; gel recovery of PCR amplification products, digestion of gel recovery products with DpnI enzyme at 37°C for 2 h, degradation of the initial template; transformation of the digestion products into competent Escherichia coli BL21 (DE3) cells, plating on LB agar plates containing 50 μg / mL kanamycin, culturing at 37°C overnight, screening positive clones, and sequencing verification to obtain recombinant bacteria containing a single-point mutant of reverse transcriptase UCRT vL;

[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 reverse transcriptase UCRT vL protein combination mutants

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

[0173] (1) Selecting two mutation sites for combination, six reverse transcriptase UCRT vL mutants with improved thermal stability can be constructed. The combined mutation sites are G167N / D280E, G167N / F431Y, G167N / S396T, D280E / F431Y, D280E / S396T, and F431Y / S396T. The amino acid sequences of the six reverse transcriptase UCRT vL 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 combined mutants of reverse transcriptase UCRT vL with improved thermal stability, wherein the combined mutation sites were G167N / D280E / F431Y, G167N / D280E / S396T, D280E / F431Y / S396T, and G167N / F431Y / S396T. The amino acid sequences of the four combined mutants of reverse transcriptase UCRT vL 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 reverse transcriptase UCRT vL combination mutant with improved thermal stability, wherein the combination mutation sites are: G167N / D280E / F431Y / S396T. The amino acid sequence of the reverse transcriptase UCRT vL combination mutant with improved thermal stability is SEQ ID NO.17.

[0176] Embodiment 3:

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

[0178] (1) The nucleic acid sequence encoding the reverse transcriptase UCRT vL mutant with mutation site G167N is SEQ ID NO. 28;

[0179] SEQ ID NO.28

[0180]

[0181] (2) The nucleic acid sequence encoding the reverse transcriptase UCRT vL mutant with mutation site D280E is SEQ ID NO. 29;

[0182] SEQ ID NO.29

[0183]

[0184] (3) The nucleic acid sequence encoding the reverse transcriptase UCRT vL mutant with mutation site F431Y is SEQ ID NO.30;

[0185] SEQ ID NO.30

[0186]

[0187] (4) The nucleic acid sequence encoding the reverse transcriptase UCRT vL mutant with a mutation site of S396T is SEQ ID NO.31;

[0188] SEQ ID NO.31

[0189]

[0190] (5) The nucleic acid sequence encoding the reverse transcriptase UCRT vL mutant with mutation site G167N / D280E is SEQ ID NO.32;

[0191] SEQ ID NO.32

[0192]

[0193] (6) The nucleic acid sequence encoding the reverse transcriptase UCRT vL mutant with mutation site G167N / F431Y is SEQ ID NO.33;

[0194] SEQ ID NO.33

[0195]

[0196] (7) The nucleic acid sequence encoding the reverse transcriptase UCRT vL mutant with mutation site G167N / S396T is SEQ ID NO.34;

[0197] SEQ ID NO.34

[0198]

[0199] (8) The nucleic acid sequence encoding the reverse transcriptase UCRT vL mutant with mutation site D280E / F431Y is SEQ ID NO.35;

[0200] SEQ ID NO.35

[0201]

[0202] (9) The nucleic acid sequence encoding the reverse transcriptase UCRT vL mutant with mutation site D280E / S396T is SEQ ID NO.36;

[0203] SEQ ID NO.36

[0204]

[0205] (10) The nucleic acid sequence encoding the reverse transcriptase UCRT vL mutant with mutation site F431Y / S396T is SEQ ID NO.37;

[0206] SEQ ID NO.37

[0207]

[0208] (11) The nucleic acid sequence encoding the reverse transcriptase UCRT vL mutant with mutation sites G167N / D280E / F431Y is SEQ ID NO.38;

[0209] SEQ ID NO.38

[0210]

[0211] (12) The nucleic acid sequence encoding the reverse transcriptase UCRT vL mutant with mutation sites G167N / D280E / S396T is SEQ ID NO. 39;

[0212] SEQ ID NO.39

[0213]

[0214] (13) The nucleic acid sequence encoding the reverse transcriptase UCRT vL mutant with mutation sites G167N / F431Y / S396T is SEQ ID NO.40;

[0215] SEQ ID NO.40

[0216]

[0217] (14) The nucleic acid sequence encoding the reverse transcriptase UCRT vL mutant with mutation sites D280E / F431Y / S396T is SEQ ID NO.41;

[0218] SEQ ID NO.41

[0219]

[0220] (15) The nucleic acid sequence encoding the reverse transcriptase UCRT vL mutant with mutation sites G167N / D280E / F431Y / S396T is SEQ ID NO.42.

[0221] SEQ ID NO.42

[0222]

[0223] Embodiment 4:

[0224] This example studies the enzymatic properties of the reverse transcriptase UCRT vL mutant

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

[0226] The enzyme solution was incubated at a certain temperature, samples were taken at different treatment times, and the residual activity percentage of reverse transcriptase UCRT vL or reverse transcriptase UCRT vL mutant was determined. The ln value of the residual activity percentage was plotted against the 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 vL or reverse transcriptase UCRT vL mutant at the temperature was obtained from t1 / 2=ln2 / kinact.

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

[0228] Table 1. Characterization of enzymatic properties of wild-type reverse transcriptase UCRT vL, single point mutants and combined mutants

[0229]

[0230]

[0231] As shown in Table 1, the reverse transcriptase UCRT vL mutants provided by the present invention include single point mutants and combined mutants. Compared with the wild-type reverse transcriptase UCRT vL, 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.

[0232] The above examples are merely illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. All designs that are the same or similar to the present invention fall within the protection scope of the present invention.

Claims

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

2.

2. The reverse transcriptase UCRT vL mutant with improved thermal stability according to claim 1, characterized in that: The amino acid sequence of the reverse transcriptase UCRT vL mutant is configured as an amino acid sequence after one or more combinations of mutations in the mutation sites G167N, D280E, F431Y, and S396T on SEQ ID NO.2 are mutated.

3. The reverse transcriptase UCRT vL mutant with improved thermal stability according to claim 2, characterized in that: The mutation sites are G167N, D280E, F431Y, S396T, G167N / D280E, G167N / F431Y, G167N / S396T, D280E / F431Y, D280E / S396T, F431Y / S396T, G167N / D280E / F431Y, G167N / D280E / S396T, D280E / F431Y / S396T, G167N / F431Y / S396T or G167N / D280E / F431Y / S396T.

4. The reverse transcriptase UCRT vL mutant with improved thermal stability according to claim 2, characterized in that: The amino acid sequence of the single point mutant corresponding to G167N is SEQ ID NO. 3; The amino acid sequence of the single point mutant corresponding to D280E is SEQ ID NO.4; The amino acid sequence of the single point mutant corresponding to F431Y is SEQ ID NO.5; The amino acid sequence of the single point mutant corresponding to S396T is SEQ ID NO.6; The amino acid sequence of the combined mutant corresponding to G167N / D280E is SEQ ID NO.7; The amino acid sequence of the combined mutant corresponding to G167N / F431Y is SEQ ID NO.8; The amino acid sequence of the combined mutant corresponding to G167N / S396T is SEQ ID NO.9; The amino acid sequence of the combined mutant corresponding to D280E / F431Y is SEQ ID NO. 10; The amino acid sequence of the combined mutant corresponding to D280E / S396T is SEQ ID NO.11; The amino acid sequence of the combined mutant corresponding to F431Y / S396T is SEQ ID NO. 12; The amino acid sequence of the combined mutant corresponding to G167N / D280E / F431Y is SEQ ID NO. 13; The amino acid sequence of the combined mutant corresponding to G167N / D280E / S396T is SEQ ID NO. 14; The amino acid sequence of the combined mutant corresponding to D280E / F431Y / S396T is SEQ ID NO. 15; The amino acid sequence of the combined mutant corresponding to G167N / F431Y / S396T is SEQ ID NO. 16; The amino acid sequence of the combined mutant corresponding to G167N / D280E / F431Y / S396T is SEQ ID NO.

17.

5. The method for constructing a reverse transcriptase UCRT vL mutant with improved thermal stability according to any one of claims 1 to 4, characterized in that: The following steps are involved: Search and select amino acid sequences with a consistency greater than 50% with the amino acid sequence shown in SEQ ID NO. 2 in the database, then perform multiple sequence alignment, and generate a consensus sequence that can be edited later using software; The protein three-dimensional structure of SEQ ID NO.2 was predicted, and the stability-related mutation sites were screened out: G167N, D280E, F431Y, and S396T.

6. The method for constructing a reverse transcriptase UCRT vL mutant with improved thermal stability according to claim 5, characterized in that: The amplification primer sequences for the mutation site G167N are SEQ ID NO.20 and SEQ ID NO.21; The amplification primer sequences for mutation site D280E are SEQ ID NO.22 and SEQ ID NO.23; The amplification primer sequences for mutation site F431Y are SEQ ID NO.24 and SEQ ID NO.25; The amplification primer sequences for the mutation site S396T are SEQ ID NO.26 and SEQ ID NO.

27.

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

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

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

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

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