Thermostable reverse transcriptase ucrt v l mutant and construction method and application thereof

Abstract

The application belongs to the technical field of biology, and specifically provides a reverse transcriptase UCRT vL mutant with improved thermal stability, which includes four single-point mutants, six double mutants and four triple mutants, and the half-life of the mutants is longer than that of the wild-type reverse transcriptase UCRT vL at 65 DEG C; the triple mutants have better effects, and the half-life is about 3 times that of the wild-type reverse transcriptase UCRT. The application also provides a genetically engineered bacterium containing the mutants. The reverse transcriptase UCRT vL mutant obtained by the construction method has better thermal stability, and when used for RNA reverse transcription into cDNA at a higher temperature, the reverse transcriptase UCRT vL mutant has higher thermal stability and great application potential.

Description

Technical Field

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

[0002] UCRT vL reverse transcriptase is derived from a virus isolated from thermostable bacteria in Yellowstone National Park (the functional gene pol(A) was identified through metagenomic sequencing of the thermostable bacteria and virus). It possesses multiple enzymatic activities and is thermostable, with an optimal activity temperature of 60-70℃. Used for reverse transcription of RNA to cDNA, it exhibits higher thermostable properties compared to traditional reverse transcriptases (MMLV), displaying higher reverse transcriptase activity in the 60-80℃ range. Therefore, UCRT vL reverse transcriptase can be specifically applied to complex templates (high GC content / numerous RNA secondary structures), improving the reverse transcription efficiency of such RNA templates.

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

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

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

[0006] (a1) A derivative protein 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 aforementioned reverse transcriptase UCRT vL mutant is configured as the amino acid sequence after one or more combinations of mutation sites G167N, D280E, F431Y, and S396T on SEQ ID NO.2 are mutated.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0025] This invention also provides a method for constructing the above-mentioned thermostability-enhanced reverse transcriptase UCRT vL mutant, 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 use software to generate a consensus sequence that can be edited later.

[0027] Three-dimensional protein structure prediction was performed on SEQ ID NO.2, and stability-related mutation sites were screened out: G167N, D280E, F431Y, and S396T.

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

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

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

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

[0032] The present invention also provides the gene for the above-mentioned thermostability-enhanced reverse transcriptase UCRT vL mutant.

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

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

[0035] The thermostability-enhanced reverse transcriptase UCRT vL mutant provided by this invention can be used to reverse transcribe RNA into cDNA.

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

[0037] 1. The thermostable reverse transcriptase UCRT vL mutants provided by this invention include single-point mutants and combined mutants. Compared with wild-type reverse transcriptase UCRT vL, 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 thermostable single-point mutants, with a half-life approximately three times that of the wild type. The UCRT vL mutants possess excellent catalytic activity and show promising application prospects.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0064] The three-dimensional structure of the protein shown in SEQ ID NO.2 was predicted using the Swissmodel online tool, and the crystal structure of the protein shown in SEQ ID NO.2 was observed using PyMOL. The mutation sites related to thermal stability were screened out as: G167N, D280E, F431Y, and S396T.

[0065] The amplification primer sequences for the mutation site G167N are SEQ ID NO.20 and SEQ ID NO.21.

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

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

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

[0069] The effects of the thermostability-enhanced reverse transcriptase UCRT vL mutant of the present invention are studied through specific embodiments below.

[0070] Example 1:

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

[0072] SEQ ID NO.1

[0073]

[0074] SEQ ID NO.2

[0075] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0076] The thermostability-enhanced reverse transcriptase UCRT vL mutant provided in this embodiment includes: a derivative protein having the same function as the amino acid sequence shown in SEQ ID NO.2 (i.e., reverse transcriptase UCRT vL protein) by substituting, deleting, or adding one or more amino acids; or a derivative protein having at least 90% homology with the amino acid sequence shown in SEQ ID NO.2 (i.e., reverse transcriptase UCRT vL protein) by substituting, deleting, or adding one or more amino acids.

[0077] Specifically, a single-point mutation was performed at a site selected in the amino acid sequence shown in SEQ ID NO.2, resulting in four single-point mutants of the reverse transcriptase UCRT vL. The mutation sites were G167N, D280E, F431Y, and S396T. The activity of these four single-point mutants of the reverse transcriptase UCRT vL 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 GWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0080] SEQ ID NO.4

[0081] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0082] SEQ ID NO.5

[0083] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADYPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0084] SEQ ID NO.6

[0085] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPTGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0086] Alternatively, multiple mutation sites can be selected and combined within the amino acid sequence shown in SEQ ID NO. 2. For example, selecting two mutation sites from the above four mutation sites and combining them yields the following six thermostability-enhanced reverse transcriptase UCRTVL mutants. The combined mutation sites are: G167N / D280E, G167N / F431Y, G167N / S396T, D280E / F431Y, D280E / S396T, F431Y / S396T, G167N / D280E / F431Y, G167N / D280E / S396T, D280E / F431Y / S396T, G167N / F431Y / S396T, and G167N / D280E / F431Y / S396T, with amino acid sequences of SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9, respectively. NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12.

[0087] SEQ ID NO.7

[0088] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0089] SEQ ID NO.8

[0090] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADYPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0091] SEQ ID NO.9

[0092] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPTGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0093] SEQ ID NO.10

[0094] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADYPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0095] SEQ ID NO.11

[0096] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPTGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0097] SEQ ID NO.12

[0098] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPTGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADYPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0099] If three mutation sites are selected from the above four mutation sites and combined, four thermostability-enhanced reverse transcriptase UCRT vL mutants are obtained. The combined mutation sites are: G167N / D280E / F431Y, G167N / D280E / S396T, G167N / F431Y / S396T, and D280E / F431Y / S396T, 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] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADYPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0102] SEQ ID NO.14

[0103] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPTGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0104] SEQ ID NO.15

[0105] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPTGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADYPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0106] SEQ ID NO.16

[0107] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPTGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADYPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0108] If four mutation sites are selected from the above four mutation sites and combined, a reverse transcriptase UCRT vL mutant with improved thermostability is obtained. The combined mutation sites are: G167N / D280E / F431Y / S396T, and its amino acid sequence is SEQ ID NO.17.

[0109] SEQ ID NO.17

[0110] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIR GWNLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPK QTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPTGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADYPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYG IAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0111] Example 2:

[0112] This embodiment provides a method for constructing a thermostable reverse transcriptase UCRT vL mutant, including the following steps:

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

[0114] The wild-type reverse transcriptase UCRT vL gene was codon optimized using Escherichia coli as the host cell to obtain the optimized reverse transcriptase UCRT vL gene, whose nucleic acid sequence is SEQ ID NO.1 and the expressed amino acid sequence is SEQ ID NO.2. 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 ATGGTAAAAGTTAAGTTTAAGTATAAAG-3' (where the underlined part is the NdeI restriction enzyme recognition site);

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

[0118] 5'-TCAGCT CTCGAG GGGCTTCTCCCATGTGTTACC-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 reverse transcriptase UCRT vL gene sequence was correct and had been correctly inserted into the pET28a plasmid, yielding the recombinant plasmid pET28a-Bst.

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

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

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

[0124] 2. Expression and purification of reverse transcriptase UCRT vL 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. 0.1 mM IPTG inducer was then 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 the reverse transcriptase UCRT vL 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 vL homologous proteins of reverse transcriptase

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

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

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

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

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

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

[0133] 4.2. The crystal structure of the reverse transcriptase UCRT vL protein (amino acid sequence SEQ ID NO.2) was observed using PyMOL. Based on the structural information, the above-mentioned candidate mutation sites and mutation modes were reviewed to screen for the mutant sites most likely to improve the thermal stability of the reverse transcriptase UCRT vL protein. The screening criteria are as follows:

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

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

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

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

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

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

[0140] (3) Based on the crystal structure of the reverse transcriptase UCRT vL protein, the above 10 mutation forms were analyzed in detail one by one, and mutants that may improve the thermal stability of the reverse transcriptase UCRT vL 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 G167N, D280E, F431Y, and S396T.

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

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

[0147] The amplification primer pairs used are:

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

[0149] SEQ ID NO.20:

[0150] 5'-CGTGGTTGGAATCTAGACTTCGAT-3';

[0151] SEQ ID NO.21:

[0152] 5'-TAAGATCGAAGTCTAGATTCCAACCACG-3';

[0153] (2) The nucleic acid sequences of the upstream amplification primer SEQ ID NO.22 and the downstream amplification primer SEQ ID NO.23 of the mutation site D280E are as follows:

[0154] SEQ ID NO.22:

[0155] 5'-TATCAGCTTGAACAACAGGCGCAAAAAG-3';

[0156] SEQ ID NO.23:

[0157] 5'-GCGCCTGTTGTTCAAGCTGATACACGAGA-3';

[0158] (3) The nucleic acid sequences of the upstream amplification primer SEQ ID NO.24 and the downstream amplification primer SEQ ID NO.25 of the mutation site F431Y are as follows:

[0159] SEQ ID NO.24:

[0160] 5'-TGCAGACTACCCCCAGATTGAACTTC-3';

[0161] SEQ ID NO.25:

[0162] 5'-CTGGGGGGTAGTCTGCAGTTATCA-3';

[0163] (4) The nucleic acid sequences of the upstream amplification primer SEQ ID NO.26 and the downstream amplification primer SEQ ID NO.27 of the mutation site S396T are as follows:

[0164] SEQ ID NO.26:

[0165] 5'-CCGACTGGGCGCATGAGCT-3';

[0166] SEQ ID NO.27:

[0167] 5'-CCAGTCGGGGCGGTGGTAGTGA-3';

[0168] The amplification conditions were as follows: amplification at 95℃ 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 the reverse transcriptase UCRT vL single-point mutant.

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

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

[0171] 5.2. Construction of a combinatorial mutant of the reverse transcriptase UCRT vL protein

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

[0173] (1) By selecting two mutation sites for combination, six thermostability-enhanced reverse transcriptase UCRT vL mutants can be constructed. The combined mutation sites are: G167N / D280E, G167N / F431Y, G167N / S396T, D280E / F431Y, D280E / S396T, and F431Y / S396T. The amino acid sequences of these six thermostability-enhanced reverse transcriptase UCRT vL combined mutants are SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively.

[0174] (2) By selecting three mutation sites for combination, four thermostability-enhanced reverse transcriptase UCRT vL combination mutants can be constructed. The combination mutation sites are: G167N / D280E / F431Y, G167N / D280E / S396T, D280E / F431Y / S396T, and G167N / F431Y / S396T. The amino acid sequences of these four thermostability-enhanced reverse transcriptase UCRT vL 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 thermo-stable reverse transcriptase UCRT vL combined mutant can be constructed. The combined mutation sites are: G167N / D280E / F431Y / S396T. The amino acid sequence of this thermo-stable reverse transcriptase UCRT vL combined mutant is SEQ ID NO.17.

[0176] Example 3:

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

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

[0179] SEQ ID NO.28

[0180]

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

[0182] SEQ ID NO.29

[0183]

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

[0185] SEQ ID NO.30

[0186]

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

[0188] SEQ ID NO.31

[0189]

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

[0191] SEQ ID NO.32

[0192]

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

[0194] SEQ ID NO.33

[0195]

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

[0197] SEQ ID NO.34

[0198]

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

[0200] SEQ ID NO.35

[0201]

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

[0203] SEQ ID NO.36

[0204]

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

[0206] SEQ ID NO.37

[0207]

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

[0209] SEQ ID NO.38

[0210]

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

[0212] SEQ ID NO.39

[0213]

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

[0215] SEQ ID NO.40

[0216]

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

[0218] SEQ ID NO.41

[0219]

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

[0221] SEQ ID NO.42

[0222]

[0223] Example 4:

[0224] This embodiment investigates the enzymatic characterization of the reverse transcriptase UCRT vL mutant.

[0225] The thermostability of wild-type reverse transcriptase UCRT vL and various reverse transcriptase UCRT vL mutants provided in Example 2 was tested according to the conventional method for determining the activity of reverse transcriptase UCRT vL.

[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 reverse transcriptase UCRT vL or reverse transcriptase UCRT vL mutant. The ln value of the residual activity percentage was plotted against time t (min), and the slope of the straight line was the inactivation constant kinact. The half-life of the wild-type reverse transcriptase UCRT vL or reverse transcriptase UCRT vL mutant at this temperature was obtained by t1 / 2=ln2 / kinact.

[0227] Experimental results show that among the various reverse transcriptase UCRT vL 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 reverse transcriptase UCRT vL, single-point mutants, and combinatorial mutants

[0229]

[0230]

[0231] As shown in Table 1, the reverse transcriptase UCRT vL mutants provided by this invention include single-point mutants and combined mutants. Compared with wild-type reverse transcriptase UCRT vL, 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 thermal stability of the single-point mutants, and their half-life is about 3 times that of the wild type.

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

Claims

1. A thermostable reverse transcriptase UCRT vL mutant, characterized in that, The amino acid sequence of the reverse transcriptase UCRT vL mutant is configured as the amino acid sequence after mutation at the mutation site on SEQ ID NO.2; the mutation site is G167N, F431Y, S396T, G167N / D280E, G167N / F431Y, G167N / S396T, D280E / F431Y, D280E / S396T, F431Y / S396T, G167N / D280E / F431Y, G167N / D280E / S396T, D280E / F431Y / S396T, G167N / F431Y / S396T or G167N / D280E / F431Y / S396T; The amino acid sequence of the single-point mutant corresponding to G167N is SEQ ID NO.3; The amino acid sequence of the single-point mutant corresponding to F431Y is SEQ ID NO.5; The amino acid sequence of the single-point mutant corresponding to S396T is SEQ ID NO.6; The amino acid sequence of the combined mutant corresponding to G167N / D280E is SEQ ID NO.7; The amino acid sequence of the combined mutant G167N / F431Y is SEQ ID NO.8; The amino acid sequence of the combined mutant G167N / S396T is SEQ ID NO.9; The amino acid sequence of the combined mutant D280E / F431Y is SEQ ID NO.10; The amino acid sequence of the combined mutant corresponding to D280E / S396T is SEQ ID NO.11; The amino acid sequence of the combined mutant F431Y / S396T is SEQ ID NO.12; The amino acid sequence of the combined mutant G167N / D280E / F431Y is SEQ ID NO.13; The amino acid sequence of the combined mutant G167N / D280E / S396T is SEQ ID NO.14; The amino acid sequence of the combined mutant D280E / F431Y / S396T is SEQ ID NO.15; The amino acid sequence of the combined mutant G167N / F431Y / S396T is SEQ ID NO.16; The amino acid sequence of the combined mutant G167N / D280E / F431Y / S396T is SEQ ID NO.

17.

2. The method for constructing the thermostable reverse transcriptase UCRT vL mutant as described in claim 1, characterized in that, Includes the following steps: Search the database for amino acid sequences that are more than 50% identical to the amino acid sequence shown in SEQ ID NO.2, then perform multiple sequence alignment, and 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: G167N, D280E, F431Y, and S396T.

3. The method for constructing the thermostable reverse transcriptase UCRT vL mutant as described in claim 2, characterized in that: The amplification primer sequences for the mutation site G167N are SEQ ID NO.20 and SEQ ID NO.21; The amplification primer sequences for the mutation site D280E are SEQ ID NO.22 and SEQ ID NO.23; The amplification primer sequences for the mutation site F431Y are SEQ ID NO.24 and SEQ ID NO.25; The amplification primer sequences for the mutation site S396T are SEQ ID NO.26 and SEQ ID NO.

27.

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

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

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

7. The application of the thermostable reverse transcriptase UCRT vL mutant as described in claim 1 in the reverse transcription of RNA into cDNA.

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