Thermostable UCRTX thermostable reverse transcriptase mutants, their construction methods and applications

Abstract

The application belongs to the technical field of biology, and specifically provides a UCRT X heat-resistant reverse transcriptase mutant with improved thermal stability. The application provides four single-point mutants, six double mutants and four triple mutants of the UCRT heat-resistant reverse transcriptase mutant. Compared with the wild-type UCRT X heat-resistant reverse transcriptase, the mutants have a longer half-life at 65 DEG C. The double mutants have a better effect, and the half-life is about 4 times that of the wild-type DNA polymerase. The application further provides a genetically engineered bacterium containing the mutant. The UCRT heat-resistant reverse transcriptase mutant obtained by the construction method has better thermal stability, and shows high thermal stability when reverse transcribing and synthesizing DNA at a high temperature, thus having great application potential.

Description

Technical Field

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

[0002] UCRT thermostable reverse transcriptase possesses both reverse transcription and RNase H enzyme activities. Using RNA as a template, it catalyzes dNTP polymerization to generate a DNA-RNA hybrid double helix, and then hydrolyzes the RNA within the hybrid double helix to obtain a single-stranded DNA (cDNA) complementary to the RNA. The higher temperatures facilitate the opening of RNA secondary structures, improving reverse transcription efficiency and cDNA integrity, offering significant advantages over commercially available reverse transcriptases. However, its thermal stability is generally poor, and further targeted modification is needed to ensure stable long-term use and storage.

[0003] The Consensus Concept theory is based on the amino acid sequence information of homologous proteins, analyzing information that can improve the thermal stability of enzymes from an evolutionary perspective. This invention, guided by the Consensus Concept theory and aided by bioinformatics and crystallography, utilizes computer-aided molecular models combined with site-directed mutagenesis to optimize protein function. Through integrated analysis of reverse transcriptase family sequences, novel reverse transcriptase mutants with high stability are obtained. Summary of the Invention

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

[0005] Therefore, the present invention provides a thermostable UCRT X 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 E574D, G167N, K298D, and T330L on SEQ ID NO.2 are mutated.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0026] Search the database for amino acid sequences that are more than 50% identical to the amino acid sequence shown in SEQ ID NO.2, then perform multiple sequence alignment, and generate a consensus sequence that can be edited later using software.

[0027] Three-dimensional protein structure prediction was performed on SEQ ID NO.2, and stability-related mutation sites were screened out: E574D, G167N, K298D, and T330L.

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

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

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

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

[0032] This invention also provides the gene for the above-mentioned thermostable UCRTX 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 UCRTX thermostable reverse transcriptase mutant with improved thermal stability.

[0035] The thermally stable UCRT X 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 X reverse transcriptase mutants provided by this invention include single-point mutants and combined mutants. Compared with wild-type UCRT X 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 mutants possess excellent catalytic activity and show promising application prospects.

[0038] 2. The method for constructing a thermostable UCRTX 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 thermostable UCRTX 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 X 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 X 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 E574D, G167N, K298D, and T330L on SEQ ID NO.2.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0062] This invention also provides a method for constructing the above-mentioned thermostable UCRT X 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 following mutation sites related to thermal stability were screened out: E574D, G167N, K298D, and T330L.

[0065] The amplification primer sequences for the mutation site E574D are SEQ ID NO.20 and SEQ ID NO.21.

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

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

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

[0069] The following specific examples illustrate the effects of the UCRTX thermostable reverse transcriptase mutant with improved thermal stability according to the present invention.

[0070] Example 1:

[0071] This embodiment provides a thermostable UCRT X thermoresistant reverse transcriptase mutant with improved thermal stability. The UCRT X thermoresistant reverse transcriptase is a wild-type UCRT X thermoresistant reverse transcriptase derived from thermostable bacteria in Yellowstone National Park, named protein UCRT X thermoresistant reverse transcriptase. The nucleic acid sequence encoding the UCRT X thermoresistant reverse transcriptase polymerase protein is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO.2.

[0072] SEQ ID NO.1

[0073]

[0074] SEQ ID NO.2

[0075] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0076] The thermostable UCRT X 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 X thermostable reverse transcriptase protein) by substituting, deleting, or adding one or more amino acids of 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 X thermostable reverse transcriptase protein) by substituting, deleting, or adding one or more amino acids of the amino acid sequence shown in SEQ ID NO.2.

[0077] Specifically, a single-point mutation was performed at a site selected in the amino acid sequence shown in SEQ ID NO.2, resulting in four single-point mutants of the UCRTX thermostable reverse transcriptase. The mutation sites were E574D, G167N, K298D, and T330L. The activity of these four UCRTX thermostable reverse transcriptase single-point mutants was determined, 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 GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGADLFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0080] SEQ ID NO.4

[0081] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0082] SEQ ID NO.5

[0083] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVDLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0084] SEQ ID NO.6

[0085] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QLAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0086] Alternatively, multiple mutation sites 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 UCRTX thermoresistant reverse transcriptase mutants. The combined mutation sites are: E574D / G167N, E574D / K298D, E574D / T330L, G167N / K298D, G167N / T330L, and K298D / T330L, 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.

[0087] SEQ ID NO.7

[0088] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGADLFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0089] SEQ ID NO.8

[0090] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVDLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGADLFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0091] SEQ ID NO.9

[0092] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQLAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGADLFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0093] SEQ ID NO.10

[0094] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVDLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0095] SEQ ID NO.11

[0096] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQLAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0097] SEQ ID NO.12

[0098] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVDLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QLAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0099] If three mutation sites are selected from the above four mutation sites for combination, four thermostable UCRT X reverse transcriptase mutants with improved thermostability are obtained. The combined mutation sites are: E574D / G167N / K298D, E574D / G167N / T330L, E574D / K298D / T330L, and G167N / K298D / T330L, with amino acid sequences of SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively.

[0100] SEQ ID NO.13

[0101] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVDLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGADLFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0102] SEQ ID NO.14

[0103] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQLAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGADLFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0104] SEQ ID NO.15

[0105] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVDLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQLAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGADLFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0106] SEQ ID NO.16

[0107] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLLVYQLDQQAQKVVIETSQHGMPVDLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QLAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0108] If four mutation sites are selected from the above four mutation sites and combined, a thermostable UCRTX reverse transcriptase with improved thermal stability can be obtained. The combined mutation sites are: E574D / G167N / K298D / T330L, and its amino acid sequence is SEQ ID NO.17.

[0109] SEQ ID NO.17

[0110] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLLVYQLDQQAQKVVIETSQHGMPVDLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QLAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGADLFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0111] Example 2:

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

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

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

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

[0116] 5'-ACTGCT CATATG ATGGTAAAAGTTAAGTTTAAGTATAAA-3' (where the underlined part is the NdeI restriction enzyme recognition site);

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

[0118] 5'-TCAGCT CTCGAG GGGCTTCTCCCATGTGTTACCCAC-3' (where the underlined part is the XhoI restriction enzyme recognition site).

[0119] 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.

[0120] 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 X thermostable reverse transcriptase gene sequence was correct and had been correctly inserted into the pET28a plasmid, yielding the recombinant plasmid pET28a-Bst.

[0121] Among them, the wild-type UCRT X thermostable reverse transcriptase was derived from a virus isolated from thermostable bacteria in Yellowstone National Park;

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

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

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

[0125] The engineered bacteria from the glycerol tube were inoculated at a volume ratio of 1% into a 4 mL LB medium tube containing 100 μg / mL Kan, and cultured at 37℃ and 220 rpm for 12 h. 4 mL of the bacterial suspension was then transferred to a 1 L LB medium shake flask containing 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 UCRTX thermostable reverse transcriptase protein with a purity >95%, the amino acid sequence of which is SEQ ID NO.2.

[0126] 3. Multiple sequence alignment and Consensus analysis of UCRT X thermostable reverse transcriptase homologs

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

[0128] 3.2. Input the amino acid sequence shown in SEQ ID NO.2 into the NCBI protein database and Pfam database. Use the Blast tool to find all protein sequences with an amino acid sequence identity greater than 30% with the UCRTX 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.

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

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

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

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

[0133] 4.2. The crystal structure of the thermostable UCRT X 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 thermostable UCRT X reverse transcriptase. The screening criteria are as follows:

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

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

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

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

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

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

[0140] (3) Based on the crystal structure of the UCRT X 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 X thermostable reverse transcriptase protein were screened out.

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

[0142] Four single-point mutants were designed, with mutation sites of E574D, G167N, K298D, and T330L.

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

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

[0145] 5.1. Construction of a single-point mutant of the UCRT X thermostable reverse transcriptase

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

[0147] The amplification primer pairs used are:

[0148] (1) The nucleic acid sequences of the upstream amplification primer SEQ ID NO.20 and the downstream amplification primer SEQ ID NO.21 of the mutation site E574D are as follows:

[0149] SEQ ID NO.20:

[0150] 5'-GGCGCCGATTTGTTCAAAAAAGCCATCGTC-3'

[0151] SEQ ID NO.21:

[0152] 5'-GACGATGGCTTTTTTGAACAAATCGGCGCC-3'

[0153] (2) The nucleic acid sequences of the upstream amplification primer SEQ ID NO.22 and the downstream amplification primer SEQ ID NO.23 of the mutation site G167N are as follows:

[0154] SEQ ID NO.22:

[0155] 5'-GGTTGGAATCTAGACTTCGATCTT-3';

[0156] SEQ ID NO.23:

[0157] 5'-AAGATCGAAGTCTAGATTCCAACC-3';

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

[0159] SEQ ID NO.24:

[0160] 5'-CCGGTTGATTTAAAAGCATTGGAAGAAGAGATC-3';

[0161] SEQ ID NO.25:

[0162] 5'-GATCTCTTCTTCCAATGCTTTTTAAATCAACCGG-3';

[0163] (4) The nucleic acid sequences of the upstream amplification primer SEQ ID NO.26 and the downstream amplification primer SEQ ID NO.27 of the mutation site T330L are as follows:

[0164] SEQ ID NO.26:

[0165] 5'-AAACAGTTGGCGAAATTTTTCGGTGTAAAT-3';

[0166] SEQ ID NO.27:

[0167] 5'-ATTTACACCGAAAAATTTCGCCAACTGTTT-3';

[0168] The amplification conditions were as follows: amplification at 95℃ 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 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 UCRTX thermostable reverse transcriptase.

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

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

[0171] 5.2. Construction of the UCRT X thermostable reverse transcriptase protein combinatorial mutant

[0172] 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 X thermostable reverse transcriptase combination mutants:

[0173] (1) By selecting two mutation sites for combination, six thermostable UCRT X reverse transcriptase mutants can be constructed. The combined mutation sites are: E574D / G167N, E574D / K298D, E574D / T330L, G167N / K298D, G167N / T330L, and K298D / T330L. The amino acid sequences of these six thermostable UCRT X 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.

[0174] (2) By selecting three mutation sites for combination, four thermostable UCRT X thermoresistant reverse transcriptase combination mutants with improved thermostability can be constructed. The combination mutation sites are: E574D / G167N / K298D, E574D / G167N / T330L, E574D / K298D / T330L, and G167N / K298D / T330L. The amino acid sequences of these four thermostable UCRT X thermoresistant reverse transcriptase combination mutants are SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively.

[0175] (3) By selecting four mutation sites for combination, a thermostable UCRT X thermostable reverse transcriptase combination mutant with improved thermal stability can be constructed. The combination mutation sites are: E574D / G167N / K298D / T330L. The amino acid sequence of this thermostable UCRT X thermostable reverse transcriptase combination mutant is SEQ ID NO.17.

[0176] Example 3:

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

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

[0179] SEQ ID NO.28

[0180]

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

[0182] SEQ ID NO.29

[0183]

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

[0185] SEQ ID NO.30

[0186]

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

[0188] SEQ ID NO.31

[0189]

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

[0191] SEQ ID NO.32

[0192]

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

[0194] SEQ ID NO.33

[0195]

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

[0197] SEQ ID NO.34

[0198]

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

[0200] SEQ ID NO.35

[0201]

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

[0203] SEQ ID NO.36

[0204]

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

[0206] SEQ ID NO.37

[0207]

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

[0209] SEQ ID NO.38

[0210]

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

[0212] SEQ ID NO.39

[0213]

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

[0215] SEQ ID NO.40

[0216]

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

[0218] SEQ ID NO.41

[0219]

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

[0221] SEQ ID NO.42

[0222]

[0223] Example 4:

[0224] This embodiment investigated the enzymatic characterization of the UCRT X thermostable reverse transcriptase mutant.

[0225] The thermostability of wild-type UCRT X thermostable reverse transcriptase and various UCRT X thermostable reverse transcriptase mutants provided in Example 2 was tested according to the conventional UCRT X thermostable reverse transcriptase activity assay method, specifically as follows:

[0226] 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 X thermostable reverse transcriptase or UCRT X 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 X thermostable reverse transcriptase or UCRT X thermostable reverse transcriptase mutant at this temperature was obtained by t1 / 2=ln2 / kinact.

[0227] Experimental results show that among the various UCRT X thermostable reverse transcriptase mutants, the thermostability of 4 single-point mutants and 11 combined mutants was significantly improved, as shown in Table 1:

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

[0229]

[0230] As shown in Table 1, the UCRT X thermostable reverse transcriptase mutants provided by this invention include single-point mutants and combined mutants. Compared with wild-type UCRT X 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 4 times that of the wild type.

[0231] The above examples are merely illustrative of the present invention and do not constitute a limitation on the 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 UCRTX reverse transcriptase mutant with improved thermal stability, characterized in that: The amino acid sequence of the UCRTX thermostable reverse transcriptase mutant is configured as the amino acid sequence after mutation at the mutation site on SEQ ID NO.2; the mutation site is E574D, K298D, T330L, E574D / G167N, E574D / K298D, E574D / T330L, G167N / K298D, G167N / T330L, K298D / T330L, E574D / G167N / K298D, E574D / G167N / T330L, E574D / K298D / T330L, G167N / K298D / T330L, or E574D / G167N / K298D / T330L.

2. The UCRTX thermostable 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 E574D is SEQ ID NO. 3; The amino acid sequence of the single-point mutant corresponding to K298D is SEQ ID NO. 5; The amino acid sequence of the single-point mutant corresponding to T330L is SEQ ID NO. 6; The amino acid sequence of the combined mutant E574D / G167N is SEQ ID NO. 7; The amino acid sequence of the combined mutant E574D / K298D is SEQ ID NO. 8; The amino acid sequence of the combined mutant corresponding to E574D / T330L is SEQ ID NO. 9; The amino acid sequence of the combined mutant G167N / K298D is SEQ ID NO. 10; The amino acid sequence of the combined mutant corresponding to G167N / T330L is SEQ ID NO. 11; The amino acid sequence of the K298D / T330L combined mutant is SEQ ID NO. 12; The amino acid sequence of the combined mutant E574D / G167N / K298D is SEQ ID NO. 13; The amino acid sequence of the combined mutant E574D / G167N / T330L is SEQ ID NO. 14; The amino acid sequence of the combined mutant E574D / K298D / T330L is SEQ ID NO. 15; The amino acid sequence of the combined mutant G167N / K298D / T330L is SEQ ID NO. 16; The amino acid sequence of the combined mutant E574D / G167N / K298D / T330L is SEQ ID NO.

17.

3. As claimed in claim 1 2. The method for constructing the thermostable UCRTX reverse transcriptase mutant with improved thermal stability as described above, characterized in that, Includes the following steps: The database was searched for and amino acid sequences with greater than 30% similarity to the amino acid sequence shown in SEQ ID NO.2 were selected. Then, multiple sequence alignment was performed, and a consensus sequence that can be edited later was generated by software. The three-dimensional structure of the protein in SEQ ID NO.2 was predicted, and stability-related mutation sites were screened out: E574D, G167N, K298D, and T330L.

4. The method for constructing the thermostable UCRTX reverse transcriptase mutant with improved thermal stability as described in claim 3, characterized in that: The amplification primer sequences for the mutation site E574D are SEQ ID NO. 20 and SEQ ID NO. 21; The amplification primer sequences for the mutation site G167N are SEQ ID NO. 22 and SEQ ID NO. 23; The amplification primer sequences for the mutation site K298D are SEQ ID NO. 24 and SEQ ID NO. 25; The amplification primer sequences for the mutation site T330L are SEQ ID NO.26 and SEQ ID NO.

27.

5. An encoding as claimed in claim 1 2. Gene of any of the UCRTX thermostable reverse transcriptase mutants with improved thermal stability.

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

7. A device comprising as claimed in claim 1 2. Soluble protein, immobilized enzyme, or engineered bacteria of any of the thermally stable UCRTX thermostable reverse transcriptase mutants described above.

8. As claimed in claim 1 2. Application of the UCRTX thermostable reverse transcriptase mutant with improved thermal stability as described in any one of the above in reverse transcription catalyzing DNA synthesis.

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