Thermostable UCRT VI reverse transcriptase mutant with improved thermal stability, its construction method and application

Abstract

The application belongs to the technical field of biology, and specifically provides a UCRT VI heat-resistant reverse transcriptase mutant with improved thermal stability, which comprises four single-point mutants, six double mutants and four triple mutants, and the half-life of the mutants at 65 DEG C is longer than that of wild-type UCRT VI heat-resistant reverse transcriptase; the double mutants have better effects, and the half-life is about 3 times that of the wild-type DNA polymerase. The application also provides a genetically engineered bacterium comprising the mutants. The UCRT heat-resistant reverse transcriptase mutant obtained by the construction method has better thermal stability, and when reverse transcription and DNA synthesis are performed at a higher temperature, the mutant has higher thermal stability and great application potential.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a thermostable UCRT VI reverse transcriptase mutant with improved thermal stability, its construction method, and its application. Background Technology

[0002] UCRT thermostable reverse transcriptase is derived from a virus isolated from thermostable bacteria in Yellowstone National Park (the functional gene pol(A) was screened out through metagenomic sequencing). UCRT thermostable reverse transcriptase has the following functions: ① Reverse transcription activity, catalyzing dNTP polymerization using RNA as a template to generate DNA-RNA hybrid double strands; ② RNase H activity, hydrolyzing RNA in the hybrid double strand to obtain complementary DNA single strands (cDNA); ③ DNA-directed DNA polymerase activity, using cDNA single strands as templates and dNTPs as substrates to synthesize double-stranded DNA. UCRT thermostable reverse transcriptase possesses multiple enzyme activities and high-temperature resistance, making it suitable for reverse transcription of RNA to cDNA. Higher reaction temperatures facilitate the opening of RNA secondary structures, improving reverse transcription efficiency. It is widely favored in the scientific community; however, its thermal stability is generally limited, restricting its shelf life and operating temperature. Further targeted modification of its performance is needed for long-term stable use and storage.

[0003] The relationship between protein sequence and structure is often so ambiguous that it allows individual sequences to adopt alternative, stable folds. Therefore, computer-aided molecular models combined with site-directed mutagenesis can be used to optimize protein function, such as improving catalytic activity, thermal stability, and acid / alkali resistance. The Consensus Concept theory, based on the amino acid sequence information of homologous proteins, analyzes information from an evolutionary perspective that can improve enzyme thermal stability. This invention, guided by the Consensus Concept theory and aided by bioinformatics and crystallography, integrates and analyzes reverse transcriptase family sequences to obtain novel reverse transcriptase mutants with high stability. No related research has been reported to date. Summary of the Invention

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

[0005] Therefore, the present invention provides a thermostable UCRT VI reverse transcriptase mutant with improved thermal stability, wherein the thermostable UCRT reverse transcriptase mutant is as follows (a1) or (a2):

[0006] (a1) A derivative protein having the same function as the amino acid sequence shown in SEQ ID NO.2 by substituting, deleting or adding one or more amino acids;

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

[0008] Specifically, the amino acid sequence of the above-mentioned UCRT VI thermostable reverse transcriptase mutant is configured as the amino acid sequence after one or more combinations of mutation sites I417F, I404P, A120E, and G440A on SEQ ID NO.2 are mutated.

[0009] Specifically, the above mutation sites are I417F, I404P, A120E, G440A, I417F / I404P, I417F / A120E, I417F / G440A, I404P / A120E, I404P / G440A, A120E / G440A, I417F / I404P / A120E, I417F / I404P / G440A, I417F / A120E / G440A, I404P / A120E / G440A, or I417F / I404P / A120E / G440A.

[0010] Specifically, the amino acid sequence of the single-point mutant corresponding to I417F is SEQ ID NO.3;

[0011] The amino acid sequence of the single-point mutant corresponding to I404P is SEQ ID NO.4;

[0012] The amino acid sequence of the single-point mutant corresponding to A120E is SEQ ID NO.5;

[0013] The amino acid sequence of the single-point mutant corresponding to G440A is SEQ ID NO.6;

[0014] The amino acid sequence of the combined mutant I417F / I404P is SEQ ID NO.7;

[0015] The amino acid sequence of the combined mutant corresponding to I417F / A120E is SEQ ID NO.8;

[0016] The amino acid sequence of the combined mutant I417F / G440A is SEQ ID NO.9;

[0017] The amino acid sequence of the combined mutant corresponding to I404P / A120E is SEQ ID NO.10;

[0018] The amino acid sequence of the combined mutant corresponding to I404P / G440A is SEQ ID NO.11;

[0019] The amino acid sequence of the combined mutant A120E / G440A is SEQ ID NO.12;

[0020] The amino acid sequence of the combined mutant I417F / I404P / A120E is SEQ ID NO.13;

[0021] The amino acid sequence of the combined mutant I417F / I404P / G440A is SEQ ID NO.14;

[0022] The amino acid sequence of the combined mutant I417F / A120E / G440A is SEQ ID NO.15;

[0023] The amino acid sequence of the combined mutant I404P / A120E / G440A is SEQ ID NO.16;

[0024] The amino acid sequence of the combined mutant I417F / I404P / A120E / G440A is SEQ ID NO.17.

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

[0026] Search the database for amino acid sequences that have a greater than 30% similarity to the amino acid sequence shown in SEQ ID NO.2, then perform multiple sequence alignment, and generate a consensus sequence that can be edited later using software.

[0027] Three-dimensional protein structure prediction was performed on SEQ ID NO.2, and stability-related mutation sites were screened out: D280E, D169K, I580M, and G335F.

[0028] Specifically, the amplification primer sequences for the above-mentioned mutation site I417F are SEQ ID NO.20 and SEQ ID NO.21;

[0029] The amplification primer sequences for the mutation site I404P are SEQ ID NO.22 and SEQ ID NO.23;

[0030] The amplification primer sequences for the mutation site A120E are SEQ ID NO.24 and SEQ ID NO.25;

[0031] The amplification primer sequences for the mutation site G440A are SEQ ID NO.26 and SEQ ID NO.27.

[0032] The present invention also provides the gene for the above-mentioned thermostable UCRT VI thermoresistant reverse transcriptase mutant with improved thermal stability.

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

[0034] The present invention also provides soluble proteins, immobilized enzymes, or engineered bacteria comprising the above-mentioned thermostable UCRT VI thermostable reverse transcriptase mutant with improved thermal stability.

[0035] The thermally stable UCRT VI thermostable reverse transcriptase mutant provided by this invention can be used for reverse transcription catalyzing DNA synthesis.

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

[0037] 1. The thermostable UCRT VI reverse transcriptase mutants provided by this invention include single-point mutants and combined mutants. Compared with wild-type UCRT VI thermostable reverse transcriptase, both the single-point mutants and combined mutants have longer half-lives at 65°C; especially the combined mutants, which exhibit the synergistic effect of the single-point mutants' thermostability, with a half-life approximately three times that of the wild type. The UCRT VI thermostable reverse transcriptase mutants possess excellent catalytic activity and show promising application prospects.

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

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

[0040] Figure 1 This is a schematic diagram of the simulated crystal structure of the UCRT VI heat-resistant reverse transcriptase protein provided in Embodiment 2 of the present invention. Detailed Implementation

[0041] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0042] This invention provides a thermostable UCRT VI reverse transcriptase mutant with improved thermal stability, wherein the thermostable UCRT reverse transcriptase mutant is as follows (a1) or (a2):

[0043] (a1) A derivative protein having the same function as the amino acid sequence shown in SEQ ID NO.2 by substituting, deleting or adding one or more amino acids;

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

[0045] The amino acid sequence of the UCRT VI thermostable reverse transcriptase mutant was configured as the amino acid sequence after mutation of one or more combinations of the mutation sites I417F, I404P, A120E, and G440A on SEQ ID NO.2.

[0046] The mutation sites are I417F, I404P, A120E, G440A, I417F / I404P, I417F / A120E, I417F / G440A, I404P / A120E, I404P / G440A, A120E / G440A, I417F / I404P / A120E, I417F / I404P / G440A, I417F / A120E / G440A, I404P / A120E / G440A, or I417F / I404P / A120E / G440A.

[0047] The amino acid sequence of the single-point mutant corresponding to I417F is SEQ ID NO.3;

[0048] The amino acid sequence of the single-point mutant corresponding to I404P is SEQ ID NO.4;

[0049] The amino acid sequence of the single-point mutant corresponding to A120E is SEQ ID NO.5;

[0050] The amino acid sequence of the single-point mutant corresponding to G440A is SEQ ID NO.6;

[0051] The amino acid sequence of the combined mutant I417F / I404P is SEQ ID NO.7;

[0052] The amino acid sequence of the combined mutant corresponding to I417F / A120E is SEQ ID NO.8;

[0053] The amino acid sequence of the combined mutant I417F / G440A is SEQ ID NO.9;

[0054] The amino acid sequence of the combined mutant corresponding to I404P / A120E is SEQ ID NO.10;

[0055] The amino acid sequence of the combined mutant corresponding to I404P / G440A is SEQ ID NO.11;

[0056] The amino acid sequence of the combined mutant A120E / G440A is SEQ ID NO.12;

[0057] The amino acid sequence of the combined mutant I417F / I404P / A120E is SEQ ID NO.13;

[0058] The amino acid sequence of the combined mutant I417F / I404P / G440A is SEQ ID NO.14;

[0059] The amino acid sequence of the combined mutant I417F / A120E / G440A is SEQ ID NO.15;

[0060] The amino acid sequence of the combined mutant I404P / A120E / G440A is SEQ ID NO.16;

[0061] The amino acid sequence of the combined mutant I417F / I404P / A120E / G440A is SEQ ID NO.17.

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

[0063] By searching the Pfam and NCBI databases for the amino acid sequence shown in SEQ ID NO.2, removing duplicate sequences, and selecting amino acid sequences with a similarity greater than 30% to the amino acid sequence shown in SEQ ID NO.2, multiple sequence alignment was performed using Clustalx 1.83 software. The remaining amino acid sequences were then compiled into a FASTA file and uploaded to the Consensus Maker v2.0.0 server. After modifying the settings as needed, the online software will generate a consensus sequence that can be edited later.

[0064] The three-dimensional structure of the protein shown in SEQ ID NO.2 was predicted using the Swissmodel online tool, and the crystal structure of the protein shown in SEQ ID NO.2 was observed using PyMOL. The mutation sites related to thermal stability were screened out as: I417F, I404P, A120E, and G440A.

[0065] The amplification primer sequences for the mutation site I417F are SEQ ID NO.20 and SEQ ID NO.21.

[0066] The amplification primer sequences for the mutation site I404P are SEQ ID NO.22 and SEQ ID NO.23;

[0067] The amplification primer sequences for the mutation site A120E are SEQ ID NO.24 and SEQ ID NO.25;

[0068] The amplification primer sequences for the mutation site G440A are SEQ ID NO.26 and SEQ ID NO.27.

[0069] The effects of the UCRT VI thermostable reverse transcriptase mutant of the present invention will be studied through specific embodiments below.

[0070] Example 1:

[0071] This embodiment provides a thermostable UCRT VI thermostable reverse transcriptase mutant with improved thermal stability. The UCRT VI thermostable reverse transcriptase is a wild-type UCRT VI thermostable reverse transcriptase derived from thermostable bacteria in Yellowstone National Park, named protein UCRT VI thermostable reverse transcriptase. The nucleic acid sequence encoding the UCRT VI 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] ATGGTAAAAGTTAAGTTTAAGTATAAAGGTGAAGAATTACAGGTAGACACGAGCAAGATAAAGAAAGTTTGGCGTGTCGGAAAAGCTATCAGCTTCACATACGACCAAGGAAAGACGGGGAGAGGGGCCGTGTCAGAAAAGGATGCGCCGAAGGAACTCCTTGATATGCTAGCTCGGGCCGAGCGCGAAAAGAAAGGTAGCGCCGGCATGGGTGAAGACGGTTTATCTCTACCCAAAATGATGAATACGCCAAAACCCATCCTAAAACCACAACCAAAGGCGCTTGTAGAACCCGTCTTATGTGATAGTATCGATGAGATACCTGCTAAGTATAATGAACCAGTTTATTTTGATTTAGCGACAGATGAGGATAGACCAGTACTTGCGTCAATCTATCAGCCTCATTTTGAGCGCAAAGTGTATTGTCTTAACCTACTCAAGGAAAAGGTAGCCCGATTCAAGGATTGGCTTTTAAAGTTTAGTGAAATCCGTGGTTGGGGCCTAGACTTCGATCTTAGAGTA

[0074] TTAGGGTACACCTATGAACAACTCCGAAATAAGAAAATTGTTGACGTA

[0075] CAATTAGCGATAAAGGTGCAGCACTATGAAAGGTTCAAACAAGGCGG

[0076] AACCAAGGGGGAGGGCTTTCGGCTAGACGATGTTGCCCGAGACCTCC

[0077] TAGGGATAGAATATCCAATGAATAAGACAAAAATACGTGAGACATTCA

[0078] AAAACAACATGTTTCATTCCTTTTCCAATGAACAGCTGCTATATGCATC

[0079] TCTTGACGCATATATACCGCACTTACTTTATGAACAACTCACATCATCC

[0080] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAGTG

[0081] GTAATAGAGACGTCCCAGCATGGTATGCCGGTTAAATTAAAAAGCATTG

[0082] GAAGAAGAGATCCATCGGCTGACTCAGCTCCGCTCGGAGATGCAAAA

[0083] ACAGATCCCCTTTAATTATAACTCCCCGAAACAGACTGCGAAATTTTTC

[0084] GGTGTAAATTCGTCCAGCAAAGACGTTCTAATGGACTTAGCTTTGCAA

[0085] GGGAACGAAATGGCAAAAAAAGTTTAGAGGCCAGACAGATTGAAA

[0086] AATCACTTGCCTTTGCCAAGGACCTCTATGACATAGCAAAGCGCTCCG

[0087] GGGGGCGTATCTATGGCAACTTCTTCACTACCACCGCCCGTCAGGGC

[0088] GCATGAGCTGCTCTGACATAAATCTGCAACAAATTCCACGAAGGTTAC

[0089] GGTCGTTCATTGGGTTCGACACCGAAGACAAAAATTGATAACTGCA

[0090] GACTTCCCCCAGATTGAACTTCGTCTCGCCGGTGTAATCTGGAACGAA

[0091] CCTAAGTTCATCGAGGCATTTCGGCAAGGGATTGATTTACATAAGTTA

[0092] ACTGCTAGCATCTTGTTCGACAAGAATATCGAGGAGGTAAGTAAGAAAA

[0093] AGAGCGTCAGATTGGTAAATCCGCAAACTTCGGTTTAATCTACGGTAT

[0094] CGCCCCCAAAGGCTTCGCCGAATATTGTATTGCTAATGGAATCAACAT

[0095] GACCGAGGAGCAGGCCTATGAGATCGTTCGCAAATGGAAAAAGTATT

[0096] ATACTAAGATTGCAGAACAGCACCAGGTGGCATATGAGAGATTCAAAT

[0097] ATAATGAGTATGTGGACAATGAAACCTGGTTAAATCGGACCTACCGCG

[0098] CCTGGAAACCGCAAGACTTGCTAAACTATCAGATACAGGGATCCGGC

[0099] GCCGAATTGTTCAAAAAAGCCATCGTCCTACTAAAGGAGACTAAGCC

[0100] AGATTTAAAGATAGTAAATCTAGTACATGATGAGATTGTGGTAGAAGC

[0101] TGATTCGAAGGAAGCTCAGGATCTAGCCAAACTAATCAAGGAAAAAA

[0102] TGGAGGAAGCCTGGGACTGGTGTTTGGAAAAGGCAGAGGAGTTTGG

[0103] GAATAGGGTCGCTAAGATCAAGTTAGAAGTGGAGGAGCCGCACGTGG

[0104] GTAACACATGGGAGAAGCCC

[0105] SEQ ID NO.2

[0106] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0107] The thermostable UCRT VI reverse transcriptase mutant provided in this embodiment includes: a derivative protein having the same function as the amino acid sequence shown in SEQ ID NO.2 (i.e., the UCRT VI thermostable reverse transcriptase protein) by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2; or a derivative protein having at least 90% homology with the amino acid sequence shown in SEQ ID NO.2 (i.e., the UCRT VI thermostable reverse transcriptase protein) by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2.

[0108] Specifically, a single-point mutation was performed at a site selected in the amino acid sequence shown in SEQ ID NO.2 to obtain four single-point mutants of UCRT VI thermostable reverse transcriptase. The mutation sites are I417F, I404P, A120E, and G440A. The activity of these four UCRT VI thermostable reverse transcriptase single-point mutants was measured, and their amino acid sequences are SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0109] SEQ ID NO.3

[0110] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFFGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0111] SEQ ID NO.4

[0112] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDPNLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0113] SEQ ID NO.5

[0114] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLETDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0115] SEQ ID NO.6

[0116] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAAVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0117] Alternatively, multiple mutation sites can be selected and combined within the amino acid sequence shown in SEQ ID NO.2. For example, two mutation sites can be selected from the above four mutation sites for combination to obtain the following six thermostable UCRT VI thermoresistant reverse transcriptase mutants. The combined mutation sites are: I417F / I404P, I417F / A120E, I417F / G440A, I404P / A120E, I404P / G440A, and A120E / G440A, with amino acid sequences of 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.

[0118] SEQ ID NO.7

[0119] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDPNLQQIPRRLRSFFGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0120] SEQ ID NO.8

[0121] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLETDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFFGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0122] SEQ ID NO.9

[0123] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFFGFDTEDKKLITADFPQIELRLAAVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0124] SEQ ID NO.10

[0125] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLETDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDPNLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0126] SEQ ID NO.11

[0127] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDPNLQQIPRRLRSFFGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0128] SEQ ID NO.12

[0129] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLETDEDDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAAVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0130] If three mutation sites are selected from the above four mutation sites for combination, four thermostable UCRT VI reverse transcriptase mutants with improved thermostability are obtained. The combined mutation sites are: I417F / I404P / A120E, I417F / I404P / G440A, I417F / A120E / G440A, and I404P / A120E / G440A, with amino acid sequences of SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively.

[0131] SEQ ID NO.13

[0132] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLETDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDPNLQQIPRRLRSFFGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0133] SEQ ID NO.14

[0134] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDPNLQQIPRRLRSFFGFDTEDKKLITADFPQIELRLAAVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0135] SEQ ID NO.15

[0136] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLETDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFFGFDTEDKKLITADFPQIELRLAAVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0137] SEQ ID NO.16

[0138] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLETDEDDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDPNLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAAVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0139] If four mutation sites are selected from the above four mutation sites and combined, a thermostable UCRTVI reverse transcriptase with improved thermal stability can be obtained. The combined mutation sites are: I417F / I404P / A120E / G440A, and its amino acid sequence is SEQ ID NO.17.

[0140] Example 2:

[0141] This embodiment provides a method for constructing a thermostable UCRT VI reverse transcriptase mutant with improved thermal stability, including the following steps:

[0142] 1. Cloning of the wild-type UCRT VI thermostable reverse transcriptase gene

[0143] The wild-type UCRT VI thermostable reverse transcriptase gene was codon optimized using Escherichia coli as the host cell to obtain the optimized UCRT VI thermostable reverse transcriptase gene, whose nucleic acid sequence is SEQ ID NO.1 and the expressed amino acid sequence is SEQ ID NO.2. Using SEQ ID NO.1 as the target gene, the target gene was amplified using upstream amplification primer SEQ ID NO.18 and downstream amplification primer SEQ ID NO.19.

[0144] The nucleic acid sequence of SEQ ID NO.18 is:

[0145] 5'-ACTGCT CATATG ATGGTAAAAGTTAAGTTTAAGTATAAA-3' (where the underlined part is the NdeI restriction enzyme recognition site);

[0146] The nucleic acid sequence of SEQ ID NO.19 is:

[0147] 5'-TCAGCT CTCGAG GGGCTTCTCCCATGTGTTACCCAC-3' (where the underlined part is the XhoI restriction enzyme recognition site).

[0148] The amplification conditions were as follows: amplification at 95℃ for 2 min, then at 56℃ for 20 sec, then at 72℃ for 90 sec, for a total of 30 cycles, and finally at 72℃ for 10 min.

[0149] After the reaction was complete, the PCR amplification product was detected by 1.5% agarose gel electrophoresis, yielding a 1.0 kb band, the length of which was in line with the expected result. Following the standard procedure of the kit, the target fragment was recovered and purified. The target fragment and the pET28a plasmid were double-digested using restriction endonucleases XhoI and NdeI, and then ligated using T4 DNA ligase. The ligation product was transformed into *E. coli* BL21(DE3) competent cells. The transformed cells were plated on LB plates containing 50 μg / ml kanamycin, and positive clone plasmids were extracted and sequenced. The results showed that the cloned UCRT VI thermostable reverse transcriptase gene sequence was correct and correctly inserted into the pET28a plasmid, yielding the recombinant plasmid pET28a-Bst.

[0150] Among them, the wild-type UCRT VI thermostable reverse transcriptase was derived from a virus isolated from thermostable bacteria in Yellowstone National Park;

[0151] The UCRT VI thermostable reverse transcriptase gene was provided by Suzhou Genewise Biotechnology Co., Ltd.

[0152] The PCR amplification enzyme was a KOD high-fidelity polymerase provided by Toyobo.

[0153] 2. Expression and purification of UCRT VI thermostable reverse transcriptase protein

[0154] The engineered bacteria from the glycerol tube were inoculated at a volume ratio of 1% into a 4 mL LB medium tube containing 100 μg / mL Kan, and cultured at 37℃ and 220 rpm for 12 h. 4 mL of the bacterial suspension was then transferred to a 1 L LB medium shake flask containing 50 μg / mL Kan, and cultured at 37℃ and 220 rpm for 2.5 h until the OD600 reached approximately 0.9. Then, 0.1 mM IPTG inducer was added, and the culture was induced at 25℃ and 200 rpm for 14 h. The harvested *E. coli* bacterial suspension was ultrasonically disrupted, followed by a one-step Ni-NTA affinity chromatography to obtain UCRT VI thermostable reverse transcriptase protein with a purity >95%, the amino acid sequence of which is SEQ ID NO.2.

[0155] 3. Multiple sequence alignment and Consensus analysis of UCRT VI thermostable reverse transcriptase homologs

[0156] 3.1. Go to the Pfam database homepage (http: / / pfam.xfam.org / ), enter the amino acid sequence of UCRT VI reverse transcriptase in the SEQUENCE SEARCH tool, and the server will directly return the alignment results of the amino acid sequences of the entire protein family, displaying the abundance of various amino acids at each mutation site in the form of a bar chart. The website can also automatically generate the consensus sequence of the protein family.

[0157] 3.2. Input the amino acid sequence shown in SEQ ID NO.2 into the NCBI protein database and Pfam database. Use the Blast tool to find all protein sequences with an amino acid sequence identity greater than 30% with the UCRT VI reverse transcriptase protein (SEQ ID NO.2). Delete any duplicate sequences. Organize the remaining amino acid sequences into fasta. format and input them into Clustalx 1.83 software for multiple sequence alignment. The alignment results are output in alan., dnd., and fasta. formats. The dnd. file is the phylogenetic tree file, while the alan. and fasta. files are sequence files in different formats.

[0158] Upload the aforementioned fasta. file to the Consensus Maker v2.0.0 server (http: / / www.hiv.lanl.gov / content / sequence / CONSENSUS / consensus.html). After modifying the settings as needed, the online software will generate a consensus sequence that can be edited later.

[0159] 3.3. The amino acid sequence (SEQ ID NO.2) of the UCRT VI thermostable reverse transcriptase protein was compared with the consensus sequence of the family and the amino acid abundance map of each site.

[0160] 4. Simulation of the three-dimensional structure of UCRT VI thermostable reverse transcriptase protein and selection of mutation hotspots

[0161] 4.1. The three-dimensional structure of the UCRT VI thermostable reverse transcriptase protein (amino acid sequence SEQ ID NO.2) was predicted using the Swissmodel online tool;

[0162] 4.2. The crystal structure of the thermostable UCRT VI reverse transcriptase (amino acid sequence SEQ ID NO.2) was observed using PyMOL. Based on the structural information, the above-mentioned candidate mutation sites and mutation modes were reviewed to screen for the mutant sites most likely to improve the thermostability of the UCRT VI thermostable reverse transcriptase. The screening criteria are as follows:

[0163] (1) The criteria for determining a site as a candidate site are:

[0164] ①Most proteins in this family have a generally high amino acid abundance at this site;

[0165] ②The amino acid at this site is conserved;

[0166] ③ The amino acids that appear most frequently at this site have significant differences in physicochemical properties compared to the amino acids at this site in the thermostable reverse transcriptase of UCRT VI, such as differences in charge, polarity, and steric hindrance.

[0167] (2) Remove the area near the active site, i.e., the distance from the catalytic residue ( The amino acid residues within the range, excluding amino acid residues that are embedded or partially embedded.

[0168] After the above two screening steps, a total of 10 differential sites remain, most of which are located on the surface of the UCRT VI thermostable reverse transcriptase protein molecule, such as... Figure 1 As shown, the arrow points to the mutation site.

[0169] (3) Based on the crystal structure of the UCRT VI thermostable reverse transcriptase protein, the above 10 mutation forms were analyzed in detail one by one, and mutants that may improve the thermal stability of the UCRT VI thermostable reverse transcriptase protein were screened out.

[0170] The main criteria for judgment are: ① Mutations should eliminate existing forces that are detrimental to thermal stability, such as electrostatic repulsion and charge accumulation; ② Mutations should not destroy existing forces that are beneficial to thermal stability and stable protein structures; ③ Mutations should introduce new forces that are beneficial to thermal stability, such as hydrogen bonds, salt bridges, and hydrophobic interactions.

[0171] Four single-point mutants were designed, with mutation sites of I417F, I404P, A120E, and G440A.

[0172] The activity of the four thermostable UCRT VI reverse transcriptase mutants was determined, and four thermostable UCRT VI reverse transcriptases with improved thermostability were screened out. The mutation sites were I417F, I404P, A120E, and G440A, 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.

[0173] 5. Construction, expression, and purification of mutants

[0174] 5.1. Construction of a single-point mutant of the thermostable reverse transcriptase UCRT VI

[0175] Using the recombinant plasmid pET28a-Bst from step 1 as a template, and a pair of complementary oligonucleotides with mutation sites as amplification primers, KOD high-fidelity enzyme was used to perform full plasmid PCR amplification to obtain a recombinant plasmid with specific mutation sites.

[0176] The amplification primer pairs used are:

[0177] (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 I417F are as follows:

[0178] SEQ ID NO.20:

[0179] 5'-TCTTCGGGTTCGACACCGAAGACA-3'

[0180] SEQ ID NO.21:

[0181] 5'-TGTCTTCGGTGTCGAACCCGAAGA-3'

[0182] (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 I404P are as follows:

[0183] SEQ ID NO.22:

[0184] 5'-TCTGACCCAAATCTGCAACAAATTCCA-3';

[0185] SEQ ID NO.23:

[0186] 5'-TGGAATTTGTTGCAGATTTGGGTCAGA-3';

[0187] (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 A120E are as follows:

[0188] SEQ ID NO.24:

[0189] 5'-GATTTAGAGACAGATGAGGATAGACCAGTA-3';

[0190] SEQ ID NO.25:

[0191] 5'-TACTGGTCTATCCTCATCTGTCTCTAAATC-3';

[0192] (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 G440A are as follows:

[0193] SEQ ID NO.26:

[0194] 5'-GCCGCCGTAATCTGGAACGAACCTAAG-3';

[0195] SEQ ID NO.27:

[0196] 5'-CTTAGGTTCGTTCCAGATTACGGCGGC-3';

[0197] The amplification conditions were as follows: amplification at 95℃ for 2 min, followed by amplification at 56℃ for 20 sec, amplification at 72℃ for 90 sec, for a total of 30 cycles, and finally amplification at 72℃ for 10 min. The PCR amplification products were recovered by gel extraction, and the gel-recovered products were digested with DpnI enzyme at 37℃ for 2 h to degrade the initial template. The digested products were transformed into Escherichia coli BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / mL kanamycin, and cultured overnight at 37℃. Positive clones were screened and sequenced to verify the results, yielding recombinant bacteria containing a single-point mutant of the UCRT VI thermostable reverse transcriptase.

[0198] The aforementioned high-fidelity KOD enzyme was provided by TakaRa.

[0199] The DpnI enzyme mentioned above was provided by Fermentas.

[0200] 5.2. Construction of the UCRT VI thermostable reverse transcriptase protein combinatorial mutant

[0201] Using a construction method similar to that for single-point mutants, the single-point mutants with improved stability were cumulatively combined. Multiple mutation sites were selected from the amino acid sequence shown in SEQ ID NO.2 for combination. For example, 2 to 4 mutation sites were selected from the above 4 mutation sites for combination to obtain different DUCRT VI thermostable reverse transcriptase combination mutants:

[0202] (1) By selecting two mutation sites for combination, six thermostable UCRT VI reverse transcriptase mutants can be constructed. The combined mutation sites are: I417F / I404P, I417F / A120E, I417F / G440A, I404P / A120E, I404P / G440A, and A120E / G440A. The amino acid sequences of these six thermostable UCRT VI reverse transcriptase combined mutants are SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively.

[0203] (2) By selecting three mutation sites for combination, four thermostable UCRT VI thermoresistant reverse transcriptase combination mutants with improved thermostability can be constructed. The combination mutation sites are: I417F / I404P / A120E, I417F / I404P / G440A, I417F / A120E / G440A, and I404P / A120E / G440A. The amino acid sequences of these four thermostable UCRT VI thermoresistant reverse transcriptase combination mutants are SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively.

[0204] (3) By selecting four mutation sites for combination, a thermostable UCRT VI thermostable reverse transcriptase combination mutant with improved thermal stability can be constructed. The combination mutation sites are: I417F / I404P / A120E / G440A. The amino acid sequence of this thermostable UCRT VI thermostable reverse transcriptase combination mutant is SEQ ID NO.17.

[0205] Example 3:

[0206] This embodiment provides a gene encoding a thermostable UCRT VI reverse transcriptase with improved thermal stability as described in Example 1:

[0207] (1) The nucleic acid sequence encoding the UCRT VI thermostable reverse transcriptase mutant with mutation site I417F is SEQ ID NO.28;

[0208] SEQ ID NO.28

[0209]

[0210] (2) The nucleic acid sequence of the UCRT VI thermostable reverse transcriptase mutant encoding the mutation site I404P is SEQ ID NO.29;

[0211] SEQ ID NO.29

[0212]

[0213] (3) The nucleic acid sequence of the heat-resistant reverse transcriptase mutant of UCRT VI with the coding mutation site A120E is SEQ ID NO.30;

[0214] SEQ ID NO.30

[0215] ATGGTAAAAGTTAAGTTTAAGTATAAAGGTGAAGAATTACAGGTAGACACGAGCAAGATAAAGAAAGTTTGGCGTGTCGGAAAAGCTATCAGCTTCACATACGACCAAGGAAAGACGGGGAGAGGGGCCGTGTCAGAAAAGGATGCGCCGAAGGAACTCCTTGATATGCTAGCTCGGGCCGAGCGCGAAAAGAAAGGTAGCGCCGGCATGGGTGAAGACGGTTTATCTCTACCCAAAATGATGAATACGCCAAAACCCATCCTAAAACCACAACCAAAGGCGCTTGTAGAACCCGTCTTATGTGATAGTATCGATGAGATACCTGCTAAGTATAATGAACCAGTTTATTTTGATTTAGAGACAGATGAGGATAGACCAGTACTTGCGTCAATCTATCAGCCTCATTTTGAGCGCAAAGTGTATTGTCTTAACCTACTCAAGGAAAAGGTAGCCCGATTCAAGGATTGGCTTTTAAAGTTTAGTGAAATCCGTGGTTGGGGCCTAGACTTCGATCTTAGAGTA

[0216] TTAGGGTACACCTATGAACAACTCCGAAATAAGAAAATTGTTGACGTA

[0217] CAATTAGCGATAAAGGTGCAGCACTATGAAAGGTTCAAACAAGGCGG

[0218] AACCAAGGGGGAGGGCTTTCGGCTAGACGATGTTGCCCGAGACCTCC

[0219] TAGGGATAGAATATCCAATGAATAAGACAAAAATACGTGAGACATTCA

[0220] AAAACAACATGTTTCATTCCTTTTCCAATGAACAGCTGCTATATGCATC

[0221] TCTTGACGCATATATACCGCACTTACTTTATGAACACTCACATCATCC

[0222] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAGTG

[0223] GTAATAGAGACGTCCCAGCATGGTATGCCGGTTAAATTAAAAAGCATTG

[0224] GAAGAAGAGATCCATCGGCTGACTCAGCTCCGCTCGGAGATGCAAAA

[0225] ACAGATCCCCTTTAATTATAACTCCCCGAAACAGACTGCGAAATTTTTC

[0226] GGTGTAAATTCGTCCAGCAAAGACGTTCTAATGGACTTAGCTTTGCAA

[0227] GGGAACGAAATGGCAAAAAAAGTTTAGAGGCCAGACAGATTGAAA

[0228] AATCACTTGCCTTTGCCAAGGACCTCTATGACATAGCAAAGCGCTCCG

[0229] GGGGGCGTATCTATGGCAACTTCTTCACTACCACCGCCCGTCAGGGC

[0230] GCATGAGCTGCTCTGACATAAATCTGCAACAAATTCCACGAAGGTTAC

[0231] GGTCGTTCATTGGGTTCGACACCGAAGACAAAAATTGATAACTGCA

[0232] GACTTCCCCCAGATTGAACTTCGTCTCGCCGGTGTAATCTGGAACGAA

[0233] CCTAAGTTCATCGAGGCATTTCGGCAAGGGATTGATTTACATAAGTTA

[0234] ACTGCTAGCATCTTGTTCGACAAGAATATCGAGGAGGTAAGTAAAGA

[0235] AGAGCGTCAGATTGGTAAATCCGCAAACTTCGGTTTAATCTACGGTAT

[0236] CGCCCCCAAAGGCTTCGCCGAATATTGTATTGCTAATGGAATCAACAT

[0237] GACCGAGGAGCAGGCCTATGAGATCGTTCGCAAATGGAAAAAGTATT

[0238] ATACTAAGATTGCAGAACAGCACCAGGTGGCATATGAGAGATTCAAAT

[0239] ATAATGAGTATGTGGACAATGAAACCTGGTTAAATCGGACCTACCGCG

[0240] CCTGGAAACCGCAAGACTTGCTAAACTATCAGATACAGGGATCCGGC

[0241] GCCGAATTGTTCAAAAAAGCCATCGTCCTACTAAAGGAGACTAAGCC

[0242] AGATTTAAAGATAGTAAATCTAGTACATGATGAGATTGTGGTAGAAGC

[0243] TGATTCGAAGGAAGCTCAGGATCTAGCCAAACTAATCAAGGAAAAAA

[0244] TGGAGGAAGCCTGGGACTGGTGTTTGGAAAAGGCAGAGGAGTTTGG

[0245] GAATAGGGTCGCTAAGATCAAGTTAGAAGTGGAGGAGCCGCACGTGG

[0246] GTAACACATGGGAGAAGCCC

[0247] (4) The nucleic acid sequence of the UCRT VI thermostable reverse transcriptase mutant encoding the mutation site G440A is SEQ ID NO.31;

[0248] SEQ ID NO.31

[0249]

[0250] (5) The nucleic acid sequence of the UCRT VI heat-resistant reverse transcriptase mutant with coding mutation sites I417F / I404P is SEQ ID NO.32;

[0251] SEQ ID NO.32

[0252] ATGGTAAAAGTTAAGTTTAAGTATAAAGGTGAAGAATTACAGGTAGACACGAGCAAGATAAAGAAAGTTTGGCGTGTCGGAAAAGCTATCAGCTTCACATACGACCAAGGAAAGACGGGGAGAGGGGCCGTGTCAGAAAAGGATGCGCCGAAGGAACTCCTTGATATGCTAGCTCGGGCCGAGCGCGAAAAGAAAGGTAGCGCCGGCATGGGTGAAGACGGTTTATCTCTAC

[0253] CCAAAATGATGAATACGCCAAAACCCATCCTAAAACCACAACCAAAG

[0254] GCGCTTGTAGAACCCGTCTTATGTGATAGTATCGATGAGATACCTGCTA

[0255] AGTATAATGAACCAGTTTATTTTGATTTAGCGACAGATGAGGATAGACC

[0256] AGTACTTGCGTCAATCTATCAGCCTCATTTTGAGCGCAAAGTGTATTGT

[0257] CTTAACCTACTCAAGGAAAAGGTAGCCCGATTCAAGGATTGGCTTTTA

[0258] AAGTTTAGTGAAATCCGTGGTTGGGGCCTAGACTTCGATCTTAGAGTA

[0259] TTAGGGTACACCTATGAACAACTCCGAAATAAGAAAATTGTTGACGTA

[0260] CAATTAGCGATAAAGGTGCAGCACTATGAAAGGTTCAAACAAGGCGG

[0261] AACCAAGGGGGAGGGCTTTCGGCTAGACGATGTTGCCCGAGACCTCCC

[0262] TAGGGATAGAATATCCAATGAATAAGACAAAATACGTGGAGACATTCA

[0263] AAAACAACATGTTTCATTCCTTTTCCAATGAACAGCTGCTATATGCATC

[0264] TCTTGACGCATATATACCGCACTTACTTTATGAACACTCACATCATCC

[0265] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAGTG

[0266] GTAATAGAGACGTCCCAGCATGGTATGCCGGTTAAATTAAAAAGCATTG

[0267] GAAGAAGAGATCCATCGGCTGACTCAGCTCCGCTCGGAGATGCAAAA

[0268] ACAGATCCCCTTTAATTATAACTCCCCGAAACAGACTGCGAAATTTTTC

[0269] GGTGTAAATTCGTCCAGCAAAGACGTTCTAATGGACTTAGCTTTGCAA

[0270] GGGAACGAAATGGCAAAAAAAGTTTAGAGGCCAGACAGATTGAAA

[0271] AATCACTTGCCTTTGCCAAGGACCTCTATGACCCAAGCAAAGCGCTCCG

[0272] GGGGGCGTATCTATGGCAACTTCTTCACTACCACCGCCCGTCAGGGC

[0273] GCATGAGCTGCTCTGACATAAATCTGCAACAAATTCCACGAAGGTTAC

[0274] GGTCGTTCTTCGGGTTCGACACCGAAGACAAAAAATTGATAACTGCA

[0275] GACTTCCCCCAGATTGAACTTCGTCTCGCCGGTGTAATCTGGAACGAA

[0276] CCTAAGTTCATCGAGGCATTTCGGCAAGGGATTGATTTACATAAGTTA

[0277] ACTGCTAGCATCTTGTTCGACAAGAATATCGAGGAGGTAAGTAAAGA

[0278] AGAGCGTCAGATTGGTAAATCCGCAAACTTCGGTTTAATCTACGGTAT

[0279] CGCCCCCAAAGGCTTCGCCGAATATTGTATTGCTAATGGAATCAACAT

[0280] GACCGAGGAGCAGGCCTATGAGATCGTTCGCAAATGGAAAAAGTATT

[0281] ATACTAAGATTGCAGAACAGCACCAGGTGGCATATGAGAGATTCAAAT

[0282] ATAATGAGTATGTGGACAATGAAACCTGGTTAAATCGGACCTACCGCG

[0283] CCTGGAAACCGCAAGACTTGCTAAACTATCAGATACAGGGATCCGGC

[0284] GCCGAATTGTTCAAAAAAGCCATCGTCCTACTAAAGGAGACTAAGCC

[0285] AGATTTAAAGATAGTAAATCTAGTACATGATGAGATTGTGGTAGAAGC

[0286] TGATTCGAAGGAAGCTCAGGATCTAGCCAAACTAATCAAGGAAAAAA

[0287] TGGAGGAAGCCTGGGACTGGTGTTTGGAAAAGGCAGAGGAGTTTGG

[0288] GAATAGGGTCGCTAAGATCAAGTTAGAAGTGGAGGAGCCGCACGTGG

[0289] GTAACACATGGGAGAAGCCC

[0290] (6) The nucleic acid sequence encoding the UCRT VI thermostable reverse transcriptase mutant with the mutation site I417F / A120E is SEQ ID NO.33;

[0291] SEQ ID NO.33

[0292]

[0293] GTAACACATGGGAGAAGCCC

[0294] CTCGAG

[0295] (7) The nucleic acid sequence encoding the UCRT VI thermostable reverse transcriptase mutant with the mutation site I417F / G440A is SEQ ID NO.34;

[0296] SEQ ID NO.34

[0297]

[0298] (8) The nucleic acid sequence of the thermotolerant reverse transcriptase mutant of UCRT VI with the coding mutation sites I404P / A120E is SEQ ID NO.35;

[0299] ATGGTAAAAGTTAAGTTTAAGTATAAAGGTGAAGAATTACAGGTAGACACGAGCAAGATAAAGAAAGTTTGGCGTGTCGGAAAAGCTATCAGCTTCACATACGACCAAGGAAAGACGGGGAGAGGGGCCGTGTCAGAAAAGGATGCGCCGAAGGAACTCCTTGATATGCTAGCTCGGGCCGAGCGCGAAAAGAAAGGTAGCGCCGGCATGGGTGAAGACGGTTTATCTCTACCCAAAATGATGAATACGCCAAAACCCATCCTAAAACCACAACCAAAGGCGCTTGTAGAACCCGTCTTATGTGATAGTATCGATGAGATACCTGCTAAGTATAATGAACCAGTTTATTTTGATTTAGAGACAGATGAGGATAGACCAGTACTTGCGTCAATCTATCAGCCTCATTTTGAGCGCAAAGTGTATTGTCTTAACCTACTCAAGGAAAAGGTAGCCCGATTCAAGGATTGGCTTTTA

[0300] AAGTTTAGTGAAATCCGTGGTTGGGGCCTAGACTTCGATCTTAGAGTA

[0301] TTAGGGTACACCTATGAACAACTCCGAAATAAGAAAATTGTTGACGTA

[0302] CAATTAGCGATAAAGGTGCAGCACTATGAAAGGTTCAAACAAGGCGG

[0303] AACCAAGGGGGAGGGCTTTCGGCTAGACGATGTTGCCCGAGACCTCC

[0304] TAGGGATAGAATATCCAATGAATAAGACAAAAATACGTGAGACATTCA

[0305] AAAACAACATGTTTCATTCCTTTTCCAATGAACAGCTGCTATATGCATC

[0306] TCTTGACGCATATATACCGCACTTACTTTATGAACACTCACATCATCC

[0307] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAGTG

[0308] GTAATAGAGACGTCCCAGCATGGTATGCCGGTTAAATTAAAAAGCATTG

[0309] GAAGAAGAGATCCATCGGCTGACTCAGCTCCGCTCGGAGATGCAAAA

[0310] ACAGATCCCCTTTAATTATAACTCCCCGAAACAGACTGCGAAATTTTTC

[0311] GGTGTAAATTCGTCCAGCAAAGACGTTCTAATGGACTTAGCTTTGCAA

[0312] GGGAACGAAATGGCAAAAAAAGTTTAGAGGCCAGACAGATTGAAA

[0313] AATCACTTGCCTTTGCCAAGGACCTCTATGACCCAAGCAAAGCGCTCCG

[0314] GGGGGCGTATCTATGGCAACTTCTTCACTACCACCGCCCGTCAGGGC

[0315] GCATGAGCTGCTCTGACATAAATCTGCAACAAATTCCACGAAGGTTAC

[0316] GGTCGTTCATTGGGTTCGACACCGAAGACAAAAATTGATAACTGCA

[0317] GACTTCCCCCAGATTGAACTTCGTCTCGCCGGTGTAATCTGGAACGAA

[0318] CCTAAGTTCATCGAGGCATTTCGGCAAGGGATTGATTTACATAAGTTA

[0319] ACTGCTAGCATCTTGTTCGACAAGAATATCGAGGAGGTAAGTAAAGA

[0320] AGAGCGTCAGATTGGTAAATCCGCAAACTTCGGTTTAATCTACGGTAT

[0321] CGCCCCCAAAGGCTTCGCCGAATATTGTATTGCTAATGGAATCAACAT

[0322] GACCGAGGAGCAGGCCTATGAGATCGTTCGCAAATGGAAAAAGTATT

[0323] ATACTAAGATTGCAGAACAGCACCAGGTGGCATATGAGAGATTCAAAT

[0324] ATAATGAGTATGTGGACAATGAAACCTGGTTAAATCGGACCTACCGCG

[0325] CCTGGAAACCGCAAGACTTGCTAAACTATCAGATACAGGGATCCGGC

[0326] GCCGAATTGTTCAAAAAAGCCATCGTCCTACTAAAGGAGACTAAGCC

[0327] AGATTTAAAGATAGTAAATCTAGTACATGATGAGATTGTGGTAGAAGC

[0328] TGATTCGAAGGAAGCTCAGGATCTAGCCAAACTAATCAAGGAAAAAA

[0329] TGGAGGAAGCCTGGGACTGGTGTTTGGAAAAGGCAGAGGAGTTTGG

[0330] GAATAGGGTCGCTAAGATCAAGTTAGAAGTGGAGGAGCCGCACGTGG

[0331] GTAACACATGGGAGAAGCCC

[0332] (9) The nucleic acid sequence encoding the UCRT VI thermostable reverse transcriptase mutant with the mutation site I404P / G440A is SEQ ID NO.36;

[0333]

[0334] (10) The nucleic acid sequence of the UCRT VI heat-resistant reverse transcriptase mutant with coding mutation sites A120E / G440A is SEQ ID NO.37;

[0335] ATGGTAAAAGTTAAGTTTAAGTATAAAGGTGAAGAATTACAGGTAGACACGAGCAAGATAAAGAAAGTTTGGCGTGTCGGAAAAGCTATCAGCTTCACATACGACCAAGGAAAGACGGGGAGAGGGGCCGTGTCAGAAAAGGATGCGCCGAAGGAACTCCTTGATATGCTAGCTCGGGCCGAGCGCGAAAAGAAAGGTAGCGCCGGCATGGGTGAAGACGGTTTATCTCTACCCAAAATGATGAATACGCCAAAACCCATCCTAAAACCACAACCAAAG

[0336] GCGCTTGTAGAACCCGTCTTATGTGATAGTATCGATGAGATACCTGCTA

[0337] AGTATAATGAACCAGTTTATTTTGATTTAGAGACAGATGAGGATAGACC

[0338] AGTACTTGCGTCAATCTATCAGCCTCATTTTGAGCGCAAAGTGTATTGT

[0339] CTTAACCTACTCAAGGAAAAGGTAGCCCGATTCAAGGATTGGCTTTTA

[0340] AAGTTTAGTGAAATCCGTGGTTGGGGCCTAGACTTCGATCTTAGAGTA

[0341] TTAGGGTACACCTATGAACAACTCCGAAATAAGAAAATTGTTGACGTA

[0342] CAATTAGCGATAAAGGTGCAGCACTATGAAAGGTTCAAACAAGGCGG

[0343] AACCAAGGGGGAGGGCTTTCGGCTAGACGATGTTGCCCGAGACCTCC

[0344] TAGGGATAGAATATCCAATGAATAAGACAAAATACGTGGAGACATTCA

[0345] AAAACAACATGTTTCATTCCTTTTCCAATGAACAGCTGCTATATGCATC

[0346] TCTTGACGCATATATACCGCACTTACTTTATGAACACTCACATCATCC

[0347] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAGTG

[0348] GTAATAGAGACGTCCCAGCATGGTATGCCGGTTAAATTAAAAAGCATTG

[0349] GAAGAAGAGATCCATCGGCTGACTCAGCTCCGCTCGGAGATGCAAAA

[0350] ACAGATCCCCTTTAATTATAACTCCCCGAAACAGACTGCGAAATTTTTC

[0351] GGTGTAAATTCGTCCAGCAAAGACGTTCTAATGGACTTAGCTTTGCAA

[0352] GGGAACGAAATGGCAAAAAAAGTTTAGAGGCCAGACAGATTGAAA

[0353] AATCACTTGCCTTTGCCAAGGACCTCTATGACATAGCAAAGCGCTCCG

[0354] GGGGGCGTATCTATGGCAACTTCTTCACTACCACCGCCCGTCAGGGC

[0355] GCATGAGCTGCTCTGACATAAATCTGCAACAAATTCCACGAAGGTTAC

[0356] GGTCGTTCATTGGGTTCGACACCGAAGACAAAAATTGATAACTGCA

[0357] GACTTCCCCCAGATTGAACTTCGTCTCGCCGCCGTAATCTGGAACGAA

[0358] CCTAAGTTCATCGAGGCATTTCGGCAAGGGATTGATTTACATAAGTTA

[0359] ACTGCTAGCATCTTGTTCGACAAGAATATCGAGGAGGTAAGTAAAGA

[0360] AGAGCGTCAGATTGGTAAATCCGCAAACTTCGGTTTAATCTACGGTAT

[0361] CGCCCCCAAAGGCTTCGCCGAATATTGTATTGCTAATGGAATCAACAT

[0362] GACCGAGGAGCAGGCCTATGAGATCGTTCGCAAATGGAAAAAGTATT

[0363] ATACTAAGATTGCAGAACAGCACCAGGTGGCATATGAGAGATTCAAAT

[0364] ATAATGAGTATGTGGACAATGAAACCTGGTTAAATCGGACCTACCGCG

[0365] CCTGGAAACCGCAAGACTTGCTAAACTATCAGATACAGGGATCCGGC

[0366] GCCGAATTGTTCAAAAAAGCCATCGTCCTACTAAAGGAGACTAAGCC

[0367] AGATTTAAAGATAGTAAATCTAGTACATGATGAGATTGTGGTAGAAGC

[0368] TGATTCGAAGGAAGCTCAGGATCTAGCCAAACTAATCAAGGAAAAAA

[0369] TGGAGGAAGCCTGGGACTGGTGTTTGGAAAAGGCAGAGGAGTTTGG

[0370] GAATAGGGTCGCTAAGATCAAGTTAGAAGTGGAGGAGCCGCACGTGG

[0371] GTAACACATGGGAGAAGCCC

[0372] (11) The nucleic acid sequence of the UCRT VI thermostable reverse transcriptase mutant encoding the mutation site I417F / I404P / A120E is SEQ ID NO.38;

[0373] SEQ ID NO.38

[0374]

[0375] (12) The nucleic acid sequence of the UCRT VI thermostable reverse transcriptase mutant encoding the mutation site I417F / I404P / G440A is SEQ ID NO.39;

[0376] SEQ ID NO.39

[0377]

[0378] (13) The nucleic acid sequence of the UCRT VI heat-resistant reverse transcriptase mutant with coding mutation sites I404P / A120E / G440A is SEQ ID NO.40;

[0379] SEQ ID NO.40

[0380] ATGGTAAAAGTTAAGTTTAAGTATAAAGGTGAAGAATTACAGGTAGACACGAGCAAGATAAAGAAAGTTTGGCGTGTCGGAAAAGCTATCAGCTTCACATACGACCAAGGAAAGACGGGGAGAGGGGCCGTGTCAGAAAAGGATGCGCCGAAGGAACTCCTTGATATGCTAGCTCGGGCCGAGCGCGAAAAGAAAGGTAGCGCCGGCATGGGTGAAGACGGTTTATCTCTACCCAAAATGATGAATACGCCAAAACCCATCCTAAAACCACAACCAAAGGCGCTTGTAGAACCCGTCTTATGTGATAGTATCGATGAGATACCTGCTAAGTATAATGAACCAGTTTATTTTGATTTAGAGACAGATGAGGATAGACCAGTACTTGCGTCAATCTATCAGCCTCATTTTGAGCGCAAAGTGTATTGTCTTAACCTACTCAAGGAAAAGGTAGCCCGATTCAAGGATTGGCTTTTAAAGTTTAGTGAAATCCGTGGTTGGGGCCTAGACTTCGATCTTAGAGTA

[0381] TTAGGGTACACCTATGAACAACTCCGAAATAAGAAAATTGTTGACGTA

[0382] CAATTAGCGATAAAGGTGCAGCACTATGAAAGGTTCAAACAAGGCGG

[0383] AACCAAGGGGGAGGGCTTTCGGCTAGACGATGTTGCCCGAGACCTCC

[0384] TAGGGATAGAATATCCAATGAATAAGACAAAAATACGTGAGACATTCA

[0385] AAAACAACATGTTTCATTCCTTTTCCAATGAACAGCTGCTATATGCATC

[0386] TCTTGACGCATATATACCGCACTTACTTTATGAACACTCACATCATCC

[0387] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAGTG

[0388] GTAATAGAGACGTCCCAGCATGGTATGCCGGTTAAATTAAAAAGCATTG

[0389] GAAGAAGAGATCCATCGGCTGACTCAGCTCCGCTCGGAGATGCAAAA

[0390] ACAGATCCCCTTTAATTATAACTCCCCGAAACAGACTGCGAAATTTTTC

[0391] GGTGTAAATTCGTCCAGCAAAGACGTTCTAATGGACTTAGCTTTGCAA

[0392] GGGAACGAAATGGCAAAAAAAGTTTAGAGGCCAGACAGATTGAAA

[0393] AATCACTTGCCTTTGCCAAGGACCTCTATGACATAGCAAAGCGCTCCG

[0394] GGGGGCGTATCTATGGCAACTTCTTCACTACCACCGCCCGTCAGGGC

[0395] GCATGAGCTGCTCTGACATAAATCTGCAACAAATTCCACGAAGGTTAC

[0396] GGTCGTTCTTCGGGTTCGACACCGAAGACAAAAATTGATAACTGCA

[0397] GACTTCCCCCAGATTGAACTTCGTCTCGCCGCCGTAATCTGGAACGAA

[0398] CCTAAGTTCATCGAGGCATTTCGGCAAGGGATTGATTTACATAAGTTA

[0399] ACTGCTAGCATCTTGTTCGACAAGAATATCGAGGAGGTAAGTAAAGA

[0400] AGAGCGTCAGATTGGTAAATCCGCAAACTTCGGTTTAATCTACGGTAT

[0401] CGCCCCCAAAGGCTTCGCCGAATATTGTATTGCTAATGGAATCAACAT

[0402] GACCGAGGAGCAGGCCTATGAGATCGTTCGCAAATGGAAAAAGTATT

[0403] ATACTAAGATTGCAGAACAGCACCAGGTGGCATATGAGAGATTCAAAT

[0404] ATAATGAGTATGTGGACAATGAAACCTGGTTAAATCGGACCTACCGCG

[0405] CCTGGAAACCGCAAGACTTGCTAAACTATCAGATACAGGGATCCGGC

[0406] GCCGAATTGTTCAAAAAAGCCATCGTCCTACTAAAGGAGACTAAGCC

[0407] AGATTTAAAGATAGTAAATCTAGTACATGATGAGATTGTGGTAGAAGC

[0408] TGATTCGAAGGAAGCTCAGGATCTAGCCAAACTAATCAAGGAAAAAA

[0409] TGGAGGAAGCCTGGGACTGGTGTTTGGAAAAGGCAGAGGAGTTTGG

[0410] GAATAGGGTCGCTAAGATCAAGTTAGAAGTGGAGGAGCCGCACGTGG

[0411] GTAACACATGGGAGAAGCCC

[0412] (14) The nucleic acid sequence of the UCRT VI thermostable reverse transcriptase mutant encoding the mutation site I404P / A120E / G440A is SEQ ID NO.41;

[0413] SEQ ID NO.41

[0414]

[0415] (15) The nucleic acid sequence of the UCRT VI heat-resistant reverse transcriptase mutant with coding mutation sites I417F / I404P / A120E / G440A is SEQ ID NO.42.

[0416] SEQ ID NO.42

[0417] ATGGTAAAAGTTAAGTTTAAGTATAAAGGTGAAGAATTACAGGTAGACACGAGCAAGATAAAGAAAGTTTGGCGTGTCGGAAAAGCTATCAGCTTCACATACGACCAAGGAAAGACGGGGAGAGGGGCCGTGTCAGAAAAGGATGCGCCGAAGGAACTCCTTGATATGCTAGCTCGGGCCGAGCGCGAAAAGAAAGGTAGCGCCGGCATGGGTGAAGACGGTTTATCTCTAC

[0418] CCAAAATGATGAATACGCCAAAACCCATCCTAAAACCACAACCAAAG

[0419] GCGCTTGTAGAACCCGTCTTATGTGATAGTATCGATGAGATACCTGCTA

[0420] AGTATAATGAACCAGTTTATTTTGATTTAGAGACAGATGAGGATAGACC

[0421] AGTACTTGCGTCAATCTATCAGCCTCATTTTGAGCGCAAAGTGTATTGT

[0422] CTTAACCTACTCAAGGAAAAGGTAGCCCGATTCAAGGATTGGCTTTTA

[0423] AAGTTTAGTGAAATCCGTGGTTGGGGCCTAGACTTCGATCTTAGAGTA

[0424] TTAGGGTACACCTATGAACAACTCCGAAATAAGAAAATTGTTGACGTA

[0425] CAATTAGCGATAAAGGTGCAGCACTATGAAAGGTTCAAACAAGGCGG

[0426] AACCAAGGGGGAGGGCTTTCGGCTAGACGATGTTGCCCGAGACCTCCC

[0427] TAGGGATAGAATATCCAATGAATAAGACAAAATACGTGGAGACATTCA

[0428] AAAACAACATGTTTCATTCCTTTTCCAATGAACAGCTGCTATATGCATC

[0429] TCTTGACGCATATATACCGCACTTACTTTATGAACACTCACATCATCC

[0430] ACACTAAATTCTCTCGTGTATCAGCTTGATCAACAGGCGCAAAAAGTG

[0431] GTAATAGAGACGTCCCAGCATGGTATGCCGGTTAAATTAAAAAGCATTG

[0432] GAAGAAGAGATCCATCGGCTGACTCAGCTCCGCTCGGAGATGCAAAA

[0433] ACAGATCCCCTTTAATTATAACTCCCCGAAACAGACTGCGAAATTTTTC

[0434] GGTGTAAATTCGTCCAGCAAAGACGTTCTAATGGACTTAGCTTTGCAA

[0435] GGGAACGAAATGGCAAAAAAAGTTTAGAGGCCAGACAGATTGAAA

[0436] AATCACTTGCCTTTGCCAAGGACCTCTATGACCCAAGCAAAGCGCTCCG

[0437] GGGGGCGTATCTATGGCAACTTCTTCACTACCACCGCCCGTCAGGGC

[0438] GCATGAGCTGCTCTGACATAAATCTGCAACAAATTCCACGAAGGTTAC

[0439] GGTCGTTCTTCGGGTTCGACACCGAAGACAAAAAATTGATAACTGCA

[0440] GACTTCCCCCAGATTGAACTTCGTCTCGCCGCCGTAATCTGGAACGAA

[0441] CCTAAGTTCATCGAGGCATTTCGGCAAGGGATTGATTTACATAAGTTA

[0442] ACTGCTAGCATCTTGTTCGACAAGAATATCGAGGAGGTAAGTAAAGA

[0443] AGAGCGTCAGATTGGTAAATCCGCAAACTTCGGTTTAATCTACGGTAT

[0444] CGCCCCCAAAGGCTTCGCCGAATATTGTATTGCTAATGGAATCAACAT

[0445] GACCGAGGAGCAGGCCTATGAGATCGTTCGCAAATGGAAAAAGTATT

[0446] ATACTAAGATTGCAGAACAGCACCAGGTGGCATATGAGAGATTCAAAT

[0447] ATAATGAGTATGTGGACAATGAAACCTGGTTAAATCGGACCTACCGCG

[0448] CCTGGAAACCGCAAGACTTGCTAAACTATCAGATACAGGGATCCGGC

[0449] GCCGAATTGTTCAAAAAAGCCATCGTCCTACTAAAGGAGACTAAGCC

[0450] AGATTTAAAGATAGTAAATCTAGTACATGATGAGATTGTGGTAGAAGC

[0451] TGATTCGAAGGAAGCTCAGGATCTAGCCAAACTAATCAAGGAAAAAA

[0452] TGGAGGAAGCCTGGGACTGGTGTTTGGAAAAGGCAGAGGAGTTTGG

[0453] GAATAGGGTCGCTAAGATCAAGTTAGAAGTGGAGGAGCCGCACGTGG

[0454] GTAACACATGGGAGAAGCCC

[0455] Example 4:

[0456] This embodiment investigated the enzymatic characterization of the UCRT VI thermostable reverse transcriptase mutant.

[0457] The thermostability of wild-type UCRT VI thermostable reverse transcriptase and various UCRT VI thermostable reverse transcriptase mutants provided in Example 2 was tested according to the conventional method for determining the activity of UCRT VI thermostable reverse transcriptase.

[0458] The enzyme solution was incubated at a certain temperature, and samples were taken at different treatment times to determine the percentage of residual activity of UCRT VI thermostable reverse transcriptase or UCRT VI thermostable reverse transcriptase mutant. The ln value of the residual activity percentage was plotted against time t (min), and the slope of the straight line was the inactivation constant kinact. The half-life of the wild-type UCRT VI thermostable reverse transcriptase or UCRT VI thermostable reverse transcriptase mutant at this temperature was obtained by t1 / 2=ln2 / kinact.

[0459] Experimental results show that among the various UCRT VI thermostable reverse transcriptase mutants, the thermostability of 4 single-point mutants and 11 combined mutants was significantly improved, as shown in Table 1:

[0460] Table 1. Enzymatic properties of wild-type UCRT VI thermostable reverse transcriptase, single-point mutants, and combined mutants

[0461]

[0462]

[0463] As shown in Table 1, the UCRT VI thermostable reverse transcriptase mutants provided by this invention include single-point mutants and combined mutants. Compared with wild-type UCRT VI thermostable reverse transcriptase, both single-point mutants and combined mutants have longer half-lives at 65°C. In particular, the combined mutants exhibit the superimposed effect of the thermostability of the single-point mutants, and their half-life is about 3 times that of the wild type.

[0464] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.

Claims

1. A thermostable UCRT VI reverse transcriptase mutant with improved thermal stability, characterized in that, The UCRT thermostable reverse transcriptase mutant was configured as the amino acid sequence after mutation at the mutation site on SEQ ID NO.2; the mutation site is I417F, A120E, G440A, I417F / A120E, I417F / G440A, A120E / G440A, I417F / A120E / G440A.

2. The thermostable UCRT VI reverse transcriptase mutant with improved thermal stability as described in claim 1, characterized in that: The amino acid sequence of the single-point mutant corresponding to I417F is SEQ ID NO.3; The amino acid sequence of the single-point mutant corresponding to A120E is SEQ ID NO.5; The amino acid sequence of the single-point mutant corresponding to G440A is SEQ ID NO.6; The amino acid sequence of the combined mutant corresponding to I417F / A120E is SEQ ID NO.8; The amino acid sequence of the combined mutant I417F / G440A is SEQ ID NO.9; The amino acid sequence of the combined mutant A120E / G440A is SEQ ID NO.12; The amino acid sequence of the combined mutant I417F / A120E / G440A is SEQ ID NO.

15.

3. The method for constructing a thermostable UCRT VI reverse transcriptase mutant with improved thermal stability as described in any one of claims 1-2, characterized in that, Includes the following steps: Search the database for amino acid sequences that have a greater than 30% similarity to the amino acid sequence shown in SEQ ID NO.2, then perform multiple sequence alignment, and use software to generate a consensus sequence that can be edited later. Three-dimensional protein structure prediction was performed on SEQ ID NO.2, and stability-related mutation sites were screened out: I417F, A120E, and G440A.

4. The method for constructing the thermostable UCRT VI reverse transcriptase mutant with improved thermal stability as described in claim 3, characterized in that: The amplification primer sequences for the mutation site I417F are SEQ ID NO.20 and SEQ ID NO.21; The amplification primer sequences for the mutation site A120E are SEQ ID NO.24 and SEQ ID NO.25; The amplification primer sequences for the mutation site G440A are SEQ ID NO.26 and SEQ ID NO.

27.

5. A gene encoding a thermostable reverse transcriptase mutant of UCRT VI with enhanced thermal stability as described in any one of claims 1-2.

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

7. A soluble protein, immobilized enzyme, or engineered bacteria comprising a thermostable UCRT VI thermostable reverse transcriptase mutant with improved thermal stability as described in any one of claims 1-2.

8. The application of the thermostable UCRT VI reverse transcriptase mutant with improved thermal stability as described in any one of claims 1-2 in reverse transcription catalyzing DNA synthesis.

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