Ucrt thermostable reverse transcriptase mutant with improved thermal stability, and construction method and application thereof

Abstract

The application belongs to the technical field of biology, and specifically provides a UCRT heat-resistant reverse transcriptase mutant with improved thermal stability, the mutant including four single-point mutants, six double mutants and four triple mutants, compared with the wild-type UCRT III heat-resistant reverse transcriptase, the mutant has a longer half-life at 65 DEG C; the double mutant has a better effect, and the half-life thereof 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 through the construction method has better thermal stability, and when reverse transcription and DNA synthesis are performed at a higher temperature, the mutant has higher thermal stability, and has great application potential.

Description

TECHNICAL FIELD

[0001] UCRT thermostable reverse transcriptase is composed of different structural enzymes with different biochemical activities. Although there are more or less differences in the functions of reverse transcriptases from different organisms, including DNA-dependent DNA polymerase activity, the main function of reverse transcriptase still depends on RNA-dependent DNA polymerase and RNase H enzyme activity. The ability of reverse transcriptase to tolerate high temperature is an important factor affecting cDNA synthesis. Increasing the reaction temperature helps to denature RNA with strong secondary structure and / or high GC content, so that the reverse transcriptase can read the sequence. Therefore, reverse transcription at a higher reaction temperature can achieve full-length cDNA synthesis with higher yield, thereby enabling RNA to be better reverse transcribed into cDNA. Therefore, it is of great significance to further direct the modification of the thermal stability of UCRT thermostable reverse transcriptase.

[0002] Directed evolution, by establishing a mutant library and high-throughput screening method, rapidly improves the specific properties of proteins, and is the most commonly used protein design and modification strategy in protein engineering. In the past decade, with the significant improvement in computer computing power and the continuous emergence of advanced algorithms, computer-aided protein design and modification have received great attention and development, becoming an important direction in protein engineering. Protein computational design based on structure simulation and energy calculation not only can modify the substrate specificity and thermal stability of enzymes, but also can design artificial enzymes with specific functions from scratch. The present application is guided by the Consensus Concept theory, assisted by bioinformatics and crystallography methods, and uses computer-aided molecular modeling combined with site-directed mutagenesis to realize the optimization of protein function, integrates and analyzes the sequences of the reverse transcriptase family, and obtains a new reverse transcriptase mutant with high stability. SUMMARY

[0003] The purpose of the present application is to improve the thermal stability of the existing UCRT III thermostable reverse transcriptase.

[0004] To this end, the present application provides a UCRT thermostable reverse transcriptase mutant with improved thermal stability, which is as follows (a1) or (a2):

[0005] (a1) a derivative protein in which the amino acid sequence shown in SEQ ID NO. 2 is substituted, deleted or added with one or more amino acids and has the same function as the amino acid sequence shown in SEQ ID NO. 2;

[0006] (a2) a derivative protein in which the amino acid sequence shown in SEQ ID NO. 2 is substituted, deleted or added with one or more amino acids and has at least 90% homology with the amino acid sequence shown in SEQ ID NO. 2.

[0007] Specifically, the amino acid sequence of the above-mentioned UCRT heat-resistant reverse transcriptase mutant is mutated to the amino acid sequence after mutation of one or more of the mutation sites D280E, D169K, I580M, and G335F on SEQ ID NO. 2.

[0008] Specifically, the above-mentioned mutation sites are D280E, D169K, I580M, G335F, D280E / D169K, D280E / I580M, D280E / G335F, D169K / I580M, D169K / G335F, I580M / G335F, D280E / D169K / I580M, D280E / D169K / G335F, D280E / I580M / G335F, D169K / I580M / G335F, D280E / D169K / I580M / G335F.

[0009] Specifically, the amino acid sequence of the single-point mutant corresponding to D280E is SEQ ID NO. 3;

[0010] The amino acid sequence of the single-point mutant corresponding to D169K is SEQ ID NO. 4;

[0011] The amino acid sequence of the single-point mutant corresponding to I580M is SEQ ID NO. 5;

[0012] The amino acid sequence of the single-point mutant corresponding to G335F is SEQ ID NO. 6;

[0013] The amino acid sequence of the combination mutant corresponding to D280E / D169K is SEQ ID NO. 7;

[0014] The amino acid sequence of the combination mutant corresponding to D280E / I580M is SEQ ID NO. 8;

[0015] The amino acid sequence of the combination mutant corresponding to D280E / G335F is SEQ ID NO. 9;

[0016] The amino acid sequence of the combination mutant corresponding to D169K / I580M is SEQ ID NO. 10;

[0017] The amino acid sequence of the combination mutant corresponding to D169K / G335F is SEQ ID NO. 11;

[0018] The amino acid sequence of the combination mutant corresponding to I580M / G335F is SEQ ID NO. 12;

[0019] The amino acid sequence of the combination mutant corresponding to D280E / D169K / I580M is SEQ ID NO. 13.

[0020] The amino acid sequence of the combination mutant corresponding to D280E / D169K / G335F is SEQ ID NO. 14.

[0021] The amino acid sequence of the combination mutant corresponding to D280E / I580M / G335F is SEQ ID NO. 15.

[0022] The amino acid sequence of the combination mutant corresponding to D169K / I580M / G335F is SEQ ID NO. 16.

[0023] The amino acid sequence of the combination mutant corresponding to D280E / D169K / I580M / G335F is SEQ ID NO. 17.

[0024] The application also provides a construction method of the UCRT heat-resistant reverse transcriptase mutant with improved thermal stability.

[0025] Search and select an amino acid sequence with more than 50% consistency with the amino acid sequence shown in SEQ ID NO. 2 in the database, then perform multiple sequence alignment, and generate a consensus sequence that can be edited later through software;

[0026] Perform protein three-dimensional structure prediction on SEQ ID NO. 2, and screen mutation sites related to stability: D280E, D169K, I580M, and G335F.

[0027] Specifically, the amplification primer sequence of the mutation site D280E is SEQ ID NO. 20 and SEQ ID NO. 21.

[0028] The amplification primer sequence of the mutation site D169K is SEQ ID NO. 22 and SEQ ID NO. 23.

[0029] The amplification primer sequence of the mutation site I580M is SEQ ID NO. 24 and SEQ ID NO. 25.

[0030] The amplification primer sequence of the mutation site G335F is SEQ ID NO. 26 and SEQ ID NO. 27.

[0031] The application also provides a gene of the UCRT heat-resistant reverse transcriptase mutant with improved thermal stability.

[0032] The application also provides a recombinant plasmid comprising the gene.

[0033] The application also provides a soluble protein, an immobilized enzyme or an engineered bacterium comprising the UCRT heat-resistant reverse transcriptase mutant with improved thermal stability.

[0034] The UCRT heat-resistant reverse transcriptase mutant with improved thermal stability provided by the application can be used for reverse transcription catalytic DNA synthesis.

[0035] Compared with the prior art, the application has the following advantages and beneficial effects:

[0036] 1. The UCRT III heat-resistant reverse transcriptase mutant with improved thermal stability provided by the application includes single-point mutants and combined mutants, and the half-life of the single-point mutants and the combined mutants at 65 DEG C is longer than that of the wild-type UCRT III heat-resistant reverse transcriptase; in particular, the combined mutants exhibit the superposition effect of the thermal stability of the single-point mutants, and the half-life is about 3 times that of the wild type. The UCRT III heat-resistant reverse transcriptase mutant with improved thermal stability has excellent catalytic activity and good application prospect.

[0037] 2. The construction method of the UCRT III heat-resistant reverse transcriptase mutant with improved thermal stability provided by the application is different from the rational design based on the precise structure-function relationship of the protein. The application is guided by the Consensus Concept theory, analyzes the information capable of improving the thermal stability of the enzyme from the evolutionary point of view, integrates and analyzes the sequences of the heat-resistant reverse transcriptase family, and obtains the new UCRT III heat-resistant reverse transcriptase mutant with high stability by combining the bioinformatics and crystallography methods.

[0038] The application will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is a schematic diagram of the UCRT III heat-resistant reverse transcriptase protein mimetic crystal structure provided by the embodiment 2 of the application. DETAILED DESCRIPTION

[0040] The technical solutions in the application will be described clearly and completely below with reference to the embodiments. Obviously, the described embodiments are only a part of the embodiments of the application, but not all the embodiments. Although the representative embodiments of the application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the application without departing from the scope of the application. Therefore, the scope of the application should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0041] The present application provides a UCRT heat-resistant reverse transcriptase mutant with improved thermal stability, which is as follows (a1) or (a2):

[0042] (a1) a derivative protein in which one or more amino acids of the amino acid sequence shown in SEQ ID NO. 2 are substituted, deleted, or added, and which has the same function as the amino acid sequence shown in SEQ ID NO. 2;

[0043] (a2) a derivative protein in which one or more amino acids of the amino acid sequence shown in SEQ ID NO. 2 are substituted, deleted, or added, and which has at least 90% homology with the amino acid sequence shown in SEQ ID NO. 2.

[0044] The amino acid sequence of the UCRT heat-resistant reverse transcriptase mutant is configured as the amino acid sequence after mutation of one or more combinations of mutation sites D280E, D169K, I580M, and G335F on SEQ ID NO. 2.

[0045] The mutation sites are D280E, D169K, I580M, G335F, D280E / D169K, D280E / I580M, D280E / G335F, D169K / I580M, D169K / G335F, I580M / G335F, D280E / D169K / I580M, D280E / D169K / G335F, D280E / I580M / G335F, D169K / I580M / G335F, and D280E / D169K / I580M / G335F.

[0046] The amino acid sequence of the single-point mutant corresponding to D280E is SEQ ID NO. 3;

[0047] The amino acid sequence of the single-point mutant corresponding to D169K is SEQ ID NO. 4;

[0048] The amino acid sequence of the single-point mutant corresponding to I580M is SEQ ID NO. 5;

[0049] The amino acid sequence of the single-point mutant corresponding to G335F is SEQ ID NO. 6;

[0050] The amino acid sequence of the combination mutant corresponding to D280E / D169K is SEQ ID NO. 7;

[0051] The amino acid sequence of the combination mutant corresponding to D280E / I580M is SEQ ID NO. 8;

[0052] The amino acid sequence of the corresponding combination mutant of D280E / G335F is SEQ ID NO. 9;

[0053] The amino acid sequence of the corresponding combination mutant of D169K / I580M is SEQ ID NO. 10;

[0054] The amino acid sequence of the corresponding combination mutant of D169K / G335F is SEQ ID NO. 11;

[0055] The amino acid sequence of the corresponding combination mutant of I580M / G335F is SEQ ID NO. 12;

[0056] The amino acid sequence of the corresponding combination mutant of D280E / D169K / I580M is SEQ ID NO. 13;

[0057] The amino acid sequence of the corresponding combination mutant of D280E / D169K / G335F is SEQ ID NO. 14;

[0058] The amino acid sequence of the corresponding combination mutant of D280E / I580M / G335F is SEQ ID NO. 15;

[0059] The amino acid sequence of the corresponding combination mutant of D169K / I580M / G335F is SEQ ID NO. 16;

[0060] The amino acid sequence of the corresponding combination mutant of D280E / D169K / I580M / G335F is SEQ ID NO. 17.

[0061] The application also provides a construction method of the above-mentioned UCRT heat-resistant type reverse transcriptase mutant with improved thermal stability, comprising the following steps:

[0062] By searching the amino acid sequence shown in SEQ ID NO. 2 in the Pfam database and the NCBI database, removing the same sequence that appears repeatedly, selecting the amino acid sequence with more than 30% consistency with the amino acid sequence shown in SEQ ID NO. 2, then performing multiple sequence alignment by Clustalx1.83 software, arranging the remaining amino acid sequence into a fasta. file uploaded to the Consensus Maker v2.0.0 server, and modifying the parameters as needed, the online software will generate a consensus sequence that can be edited later;

[0063] The three-dimensional structure of the protein represented by SEQ ID NO. 2 was predicted by the Swissmodel online tool, and the protein crystal structure represented by SEQ ID NO. 2 was observed by PyMOL, and the mutation sites related to thermal stability were screened as: D280E, D169K, I580M, G335F.

[0064] The amplification primer sequence of the mutation site D280E is SEQ ID NO. 20 and SEQ ID NO. 21.

[0065] The amplification primer sequence of the mutation site D169K is SEQ ID NO. 22 and SEQ ID NO. 23.

[0066] The amplification primer sequence of the mutation site I580M is SEQ ID NO. 24 and SEQ ID NO. 25.

[0067] The amplification primer sequence of the mutation site G335F is SEQ ID NO. 26 and SEQ ID NO. 27.

[0068] The effect of the UCRT III heat-resistant reverse transcriptase mutant with improved thermal stability of the application is studied through specific examples.

[0069] Example 1:

[0070] The example provides a UCRT III heat-resistant reverse transcriptase mutant with improved thermal stability, wherein the UCRT III heat-resistant reverse transcriptase is a wild-type UCRT III heat-resistant reverse transcriptase derived from Yellowstone Park heat-resistant bacteria, named protein UCRT III heat-resistant reverse transcriptase, and the nucleic acid sequence of the UCRT III heat-resistant reverse transcriptase polymerase protein is SEQ ID NO. 1 and the amino acid sequence is SEQ ID NO. 2.

[0071] SEQ ID NO. 1

[0072]

[0073] SEQ ID NO. 2

[0074] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0075] The UCRT III heat-resistant type reverse transcriptase mutant with improved thermal stability provided in the embodiment includes: one or more amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO. 2 to form a derivative protein having the same function as the amino acid sequence shown in SEQ ID NO. 2 (i.e. the UCRT III heat-resistant type reverse transcriptase protein), or one or more amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO. 2 to form a derivative protein having at least 90% homology with the amino acid sequence shown in SEQ ID NO. 2 (i.e. the UCRT III heat-resistant type reverse transcriptase protein).

[0076] Specifically, one site in the amino acid sequence shown in SEQ ID NO. 2 is selected for single-point mutation, and four single-point mutants of UCRT III heat-resistant reverse transcriptase are obtained, respectively, and the mutation sites are: D280E, D169K, I580M, G335F. The activity of the four single-point mutants of UCRT III heat-resistant reverse transcriptase is determined, and the amino acid sequences are SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively.

[0077] SEQ ID NO. 3

[0078] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0079] SEQ ID NO. 4

[0080] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLKFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0081] SEQ ID NO. 5

[0082] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAMVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0083] SEQ ID NO. 6

[0084] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFFVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0085] Or in the amino acid sequence represented by SEQ ID NO. 2, a plurality of mutation sites are selected and combined, such as 2 mutation sites selected from the above 4 mutation sites are combined, to obtain the following 6 heat-stable UCRT III heat-resistant reverse transcriptase mutants, and the combination of the mutation sites is: D280E / D169K, D280E / I580M, D280E / G335F, D169K / I580M, D169K / G335F, I580M / G335F, and the amino acid sequences are SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12, respectively.

[0086] SEQ ID NO. 7

[0087] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLKFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0088] SEQ ID NO. 8

[0089] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAMVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0090] SEQ ID NO. 9

[0091] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFFVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0092] SEQ ID NO. 10

[0093] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLKFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAMVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0094] SEQ ID NO. 11

[0095] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLKFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFFVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0096] SEQ ID NO. 12

[0097] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFFVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAMVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0098] As 3 mutation sites are selected from the above 4 mutation sites for combination, 4 heat-stable UCRT III heat-resistant reverse transcriptase mutants with improved heat stability are obtained, and the combination mutation sites are: D280E / D169K / I580M, D280E / D169K / G335F, D280E / I580M / G335F, and D169K / I580M / G335F, and the amino acid sequences are SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, and SEQ ID NO. 16, respectively.

[0099] SEQ ID NO. 13

[0100] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLKFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFGVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAMVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0101] SEQ ID NO. 14

[0102] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLKFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFFVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAIVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0103] SEQ ID NO. 15

[0104] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLDFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFFVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAMVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0105] SEQ ID NO. 16

[0106] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLKFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLDQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFFVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAMVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0107] As four mutation sites are selected from the above-mentioned four mutation sites for combination, one heat-stable UCRTIII heat-resistant reverse transcriptase is obtained, and the combined mutation sites are: D280E / D169K / I580M / G335F, and the amino acid sequence is SEQ ID NO. 17.

[0108] SEQ ID NO. 17

[0109] MVKVKFKYKGEELQVDTSKIKKVWRVGKAISFTYDQGKTGRGAVSEKDAPKELLDMLARAEREKKGSAGMGEDGLSLPKMMNTPKPILKPQPKALVEPVLCDSIDEIPAKYNEPVYFDLATDEDRPVLASIYQPHFERKVYCLNLLKEKVARFKDWLLKFSEIRGWGLKFDLRVLGYTYEQLRNKKIVDVQLAIKVQHYERFKQGGTKGEGFRLDDVARDLLGIEYPMNKTKIRETFKNNMFHSFSNEQLLYASLDAYIPHLLYEQLTSSTLNSLVYQLEQQAQKVVIETSQHGMPVKLKALEEEIHRLTQLRSEMQKQIPFNYNSPKQTAKFFFVNSSSKDVLMDLALQGNEMAKKVLEARQIEKSLAFAKDLYDIAKRSGGRIYGNFFTTTAPSGRMSCSDINLQQIPRRLRSFIGFDTEDKKLITADFPQIELRLAGVIWNEPKFIEAFRQGIDLHKLTASILFDKNIEEVSKEERQIGKSANFGLIYGIAPKGFAEYCIANGINMTEEQAYEIVRKWKKYYTKIAEQHQVAYERFKYNEYVDNETWLNRTYRAWKPQDLLNYQIQGSGAELFKKAMVLLKETKPDLKIVNLVHDEIVVEADSKEAQDLAKLIKEKMEEAWDWCLEKAEEFGNRVAKIKLEVEEPHVGNTWEKP

[0110] Example 2

[0111] The present embodiment provides a method for constructing a UCRT III thermostable reverse transcriptase mutant with improved thermal stability, comprising the following steps:

[0112] 1. Cloning of wild-type UCRT III thermostable reverse transcriptase gene

[0113] The wild type UCRT III thermostable reverse transcriptase gene is codon-optimized with E. coli as a host cell to obtain an optimized UCRT III thermostable reverse transcriptase gene, the nucleic acid sequence of which is SEQ ID NO. 1, and the expressed amino acid sequence is SEQ ID NO. 2; taking SEQ ID NO. 1 as a target gene, the target gene is amplified by using an upstream amplification primer SEQ ID NO. 18 and a downstream amplification primer SEQ ID NO. 19;

[0114] The nucleic acid sequence of SEQ ID NO. 18 is:

[0115] 5'-ACTGCT CATATG ATGGTAAAAGTTAAGTTTAAGTATAAA-3' (where the underlined part is a restriction endonuclease Nde I recognition site);

[0116] The nucleic acid sequence of SEQ ID NO. 19 is:

[0117] 5'-TCAGCT CTCGAG GGGCTTCTCCCATGTGTTACCCAC-3' (where the underlined part is a restriction endonuclease Xho I recognition site).

[0118] The amplification conditions are: 2 min of amplification at 95℃, then 20 sec of amplification at 56℃, 90 sec of amplification at 72℃, a total of 30 cycles, and finally 10 min of amplification at 72℃.

[0119] After the reaction is completed, the PCR amplification product is detected by 1.5% agarose gel electrophoresis to obtain a 1.0 kb band, which is consistent with the expected result. According to the standard operation of the kit, the target fragment is recovered and purified, the target fragment and the pET28a plasmid are double-digested using restriction endonucleases Xho I and Nde I, and then T4 DNA ligase is used for ligation. The obtained ligation product is transformed into E. coli BL21 (DE3) competent cells, the transformed cells are spread on LB plates containing 50 μg / ml kanamycin, the positive clone plasmid is extracted, and sequencing is performed. The results show that the cloned UCRT III thermostable reverse transcriptase gene sequence is correct, and has been correctly inserted into the pET28a plasmid to obtain a recombinant plasmid pET28a-Bst;

[0120] The wild type UCRT III thermostable reverse transcriptase is derived from a virus isolated from Yellowstone Park thermostable bacteria;

[0121] The UCRT III thermostable reverse transcriptase gene is provided by Suzhou Jinyuzhi Biotechnology Co., Ltd.;

[0122] PCR amplification enzyme is KOD high-fidelity polymerase provided by TOYOBO.

[0123] 2. Expression and purification of UCRT III thermostable reverse transcriptase protein

[0124] The engineering bacteria in the glycerol tube were inoculated into 4 mL LB medium test tubes containing 100 μg / mL Kan at a volume ratio of 1%, and cultured at 37°C and 220 rpm for 12 h; 4 mL of the bacterial solution was transferred to a 1 L LB medium shaking flask containing 50 μg / mL Kan, and cultured at 37°C and 220 rpm for 2.5 h to make the OD600 reach about 0.9, and then 0.1 mM IPTG inducer was added, and the culture was induced at 25°C and 200 rpm for 14 h. The harvested E. coli cell suspension after fermentation was ultrasonically broken, and then subjected to one-step Ni-NTA affinity chromatography to obtain UCRT III thermostable reverse transcriptase protein with a purity of >95%, and the amino acid sequence was SEQ ID NO. 2.

[0125] 3. Multiple sequence alignment and consensus analysis of UCRT III thermostable reverse transcriptase homologous protein

[0126] 3.1. Enter the Pfam database homepage (http: / / pfam.xfam.org / ), and input the amino acid sequence of the UCRT III reverse transcriptase in the SEQUENCE SEARCH tool for searching. The server will directly feedback the alignment results of the amino acid sequences of the entire family of the protein, and the abundance of various amino acids at each mutation site will be displayed in the form of a column chart. The website can also automatically generate the consensus sequence of the protein family;

[0127] 3.2. Input the amino acid sequence shown in SEQ ID NO. 2 into the NCBI protein database and the Pfam database, and use the Blast tool to find all protein sequences with an identity of more than 30% to the amino acid sequence of the UCRT III reverse transcriptase protein (SEQ ID NO. 2). Delete the same sequences that appear repeatedly, arrange the remaining amino acid sequences into fasta. format, and input them into Clustalx1.83 software for multiple sequence alignment. The alignment results are output in aln., dnd. and fasta. formats, wherein the dnd. file is an evolutionary tree file, and the aln. and fasta. files are sequence files in different formats;

[0128] The above fasta. file is uploaded to the Consensus Maker v2.0.0 (http: / / www.hiv.lanl.gov / content / sequence / CONSENSUS / consensus.html) server, and the online software will generate a consensus sequence that can be edited later after modifying the setting parameters as needed.

[0129] 3.3. Compare the amino acid sequence of UCRT III thermostable reverse transcriptase protein (SEQ ID NO. 2) with the consensus sequence of the family and the amino acid abundance map of each site.

[0130] 4. Simulation of the three-dimensional structure of UCRT III thermostable reverse transcriptase protein and selection of mutation hotspots

[0131] 4.1. Predict the three-dimensional structure of UCRT III thermostable reverse transcriptase protein (amino acid sequence SEQ ID NO. 2) by Swissmodel online tool;

[0132] 4.2. Observe the crystal structure of UCRT III thermostable reverse transcriptase (amino acid sequence SEQ ID NO. 2) with PyMOL, review the above mutation sites and mutation forms according to the structure information, and screen the mutation sites that are most likely to improve the thermal stability of UCRT III thermostable reverse transcriptase, with the following screening conditions:

[0133] (1) The standard for judging a site as a candidate site is:

[0134] ① The overall amino acid abundance of most proteins in the family at this site is high;

[0135] ② The amino acid at this site is conserved;

[0136] ③ The amino acid with high frequency at this site has a large difference in physicochemical properties with the amino acid at this site of UCRT III thermostable reverse transcriptase, such as charge difference, polarity strength, steric hindrance size, etc.

[0137] (2) Remove amino acid residues near the active center, i.e. within a distance of 5 A from the catalytic residues (Asp 110, Asp 112, and Lys 114), and remove amino acid residues in a buried or semi-buried state. After the above two steps of screening, a total of 10 difference sites remain, most of which are located on the surface of the UCRT III thermostable reverse transcriptase protein molecule, as shown in

[0138] , the arrow points to the mutation site. Figure 1

[0139] ​(3) According to the crystal structure of UCRT III thermostable reverse transcriptase protein, the above-mentioned 10 mutant forms are analyzed in detail one by one, and the mutant which can improve the thermal stability of UCRT III thermostable reverse transcriptase protein is screened out.

[0140] The main judgment criteria are: ① the mutation should eliminate the original unfavorable thermal stability force form, such as electrostatic repulsion, charge aggregation, etc.; ② the mutation should not destroy the existing thermal stability force form and stable protein structure; ③ the mutation should introduce new thermal stability force form, such as hydrogen bond, salt bridge, hydrophobic interaction, etc.

[0141] Four single-point mutant bodies are designed, and the mutation sites are D280E, D169K, I580M and G335F, respectively;

[0142] The activity of the four UCRT III thermostable reverse transcriptase mutants is determined, and four UCRT III thermostable reverse transcriptase mutants with improved thermal stability are screened out, and the mutation sites are D280E, D169K, I580M and G335F, and 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.

[0143] 5. Construction, expression and purification of mutants

[0144] 5.1. Construction of UCRT III thermostable reverse transcriptase single-point mutant

[0145] The recombinant plasmid with specific mutation site is obtained by using KOD high-fidelity enzyme for whole plasmid PCR amplification with the recombinant plasmid pET28a-Bst in step 1 as the template and a pair of complementary oligonucleotides with mutation sites as amplification primers;

[0146] The amplification primer pair used is:

[0147] (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 D280E are as follows:

[0148] SEQ ID NO. 20:

[0149] 5'-CAGCTTGAGCAACAGGCGCAAAAAGTG-3'

[0150] SEQ ID NO. 21:

[0151] 5'-CACTTTTTGCGCCTGTTGCTCAAGCTG-3'

[0152] (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 D169K are as follows, respectively:

[0153] SEQ ID NO. 22:

[0154] 5'-GGCCTAAAGTTCGATCTTAGAGTATTAGGG-3';

[0155] SEQ ID NO. 23:

[0156] 5'-CCCTAATACTCTAAGATCGAACTTTAGGCC-3';

[0157] (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 I580M are as follows, respectively:

[0158] SEQ ID NO. 24:

[0159] 5'-AAAGCCATGGTCCTACTAAAGGAGACT-3';

[0160] SEQ ID NO. 25:

[0161] 5'-AGTCTCCTTTAGTAGGACCATGGCTTT-3';

[0162] (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 G335F are as follows, respectively:

[0163] SEQ ID NO. 26:

[0164] 5'-TTTTTCTTTGTAAATTCGTCCAGCAAA-3';

[0165] SEQ ID NO. 27:

[0166] 5'-TTTGCTGGACGAATTTACAAAGAAAAA-3';

[0167] The amplification conditions are: 2 min at 95°C, then 20 sec at 56°C, 90 sec at 72°C, for 30 cycles, and finally 10 min at 72°C; the PCR amplification product is recovered by gel, the recovered product is digested by DpnI enzyme at 37°C for 2 h to degrade the original template; the digestion product is transformed into E. coli BL21(DE3) competent cells, spread on LB agar plates containing 50 μg / mL kanamycin, incubated at 37°C overnight, positive clones are screened, verified by sequencing, and the recombinant bacteria containing the single-point mutant of UCRT III thermostable reverse transcriptase are obtained;

[0168] The KOD high-fidelity enzyme is provided by TakaRa Company;

[0169] The DpnI enzyme is provided by Fermentas Company.

[0170] 5.2. Construction of UCRT III thermostable reverse transcriptase protein combined mutant

[0171] Using a similar construction method as the single-point mutant, the stability-improved single-point mutants are combined, and a plurality of mutation sites are selected for combination in the amino acid sequence shown in SEQ ID NO. 2, such as 2-4 mutation sites selected from the above 4 mutation sites for combination, to obtain different DUCRT III thermostable reverse transcriptase combined mutants:

[0172] (1) Selecting 2 mutation sites for combination can construct 6 UCRT III thermostable reverse transcriptase mutants with improved thermal stability, and the combined mutation sites are: D280E / D169K, D280E / I580M, D280E / G335F, D169K / I580M, D169K / G335F, and I580M / G335F. The amino acid sequences of the 6 UCRT III thermostable reverse transcriptase mutants with improved thermal stability are SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12, respectively;

[0173] (2) Select 3 mutation sites for combination, 4 heat-stable UCRT III heat-resistant reverse transcriptase combination mutants can be constructed, the combination mutation sites are: D280E / D169K / I580M, D280E / D169K / G335F, D280E / I580M / G335F, D169K / I580M / G335F, the amino acid sequences of the 4 heat-stable UCRT III heat-resistant reverse transcriptase combination mutants are SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16 respectively;

[0174] (3) Select 4 mutation sites for combination, 1 heat-stable UCRT III heat-resistant reverse transcriptase combination mutant can be constructed, the combination mutation sites are: D280E / D169K / I580M / G335F, the amino acid sequence of the 1 heat-stable UCRT III heat-resistant reverse transcriptase combination mutant is SEQ ID NO. 17.

[0175] Example 3:

[0176] The embodiment provides a gene encoding the heat-stable UCRT III heat-resistant reverse transcriptase as described in the embodiment 1:

[0177] (1) The nucleic acid sequence of the UCRT III heat-resistant reverse transcriptase mutant encoding the mutation site D280E is SEQ ID NO. 28;

[0178] SEQ ID NO. 28

[0179]

[0180] (2) The nucleic acid sequence of the mutant of UCRT III heat-resistant reverse transcriptase with a mutation site of D169K is SEQ ID NO. 29;

[0181] SEQ ID NO. 29

[0182]

[0183] (3) The nucleic acid sequence of the mutant of UCRT III heat-resistant reverse transcriptase with a mutation site of I580M is SEQ ID NO. 30;

[0184] SEQ ID NO. 30

[0185]

[0186] (4) The nucleic acid sequence encoding the mutant of the UCRT III heat-resistant reverse transcriptase with a mutation site of G335F is SEQ ID NO. 31;

[0187] SEQ ID NO. 31

[0188]

[0189] (5) The nucleic acid sequence encoding the mutant of UCRT III heat-resistant reverse transcriptase with mutation site D280E / D169K is SEQ ID NO. 32;

[0190] SEQ ID NO. 32

[0191]

[0192] (6) The nucleic acid sequence encoding the mutant of UCRT III heat-resistant reverse transcriptase with mutation site D280E / I580M is SEQ ID NO. 33;

[0193] SEQ ID NO. 33

[0194]

[0195] (7) The nucleic acid sequence encoding the mutant of UCRT III heat-resistant reverse transcriptase with mutation site D280E / G335F is SEQ ID NO. 34;

[0196] SEQ ID NO. 34

[0197]

[0198] (8) The nucleic acid sequence of the mutant of UCRT III thermostable reverse transcriptase with mutation site D169K / I580M is SEQ ID NO. 35;

[0199] SEQ ID NO. 35

[0200]

[0201] (9) The nucleic acid sequence of the UCRT III thermostable reverse transcriptase mutant with mutation site D169K / G335F is SEQ ID NO. 36;

[0202] SEQ ID NO. 36

[0203]

[0204] (10) The nucleic acid sequence encoding the mutant of UCRT III heat-resistant reverse transcriptase with mutation site I580M / G335F is SEQ ID NO. 37;

[0205] SEQ ID NO. 37

[0206]

[0207] (11) The nucleic acid sequence encoding the mutant of UCRT III heat-resistant reverse transcriptase with mutation site D280E / D169K / I580M is SEQ ID NO. 38;

[0208] SEQ ID NO. 38

[0209]

[0210] (12) The nucleic acid sequence encoding the mutant of UCRT III thermostable reverse transcriptase with mutation site D280E / D169K / G335F is SEQ ID NO. 39;

[0211] SEQ ID NO. 39

[0212]

[0213] (13) The nucleic acid sequence encoding the UCRT III thermostable reverse transcriptase mutant with mutation site D169K / I580M / G335F is SEQ ID NO. 40;

[0214] SEQ ID NO. 40

[0215]

[0216] (14) The nucleic acid sequence encoding the UCRT III thermostable reverse transcriptase mutant with mutation site D169K / I580M / G335F is SEQ ID NO. 41;

[0217] SEQ ID NO. 41

[0218]

[0219] (15) The nucleic acid sequence encoding the mutant of UCRT III thermostable reverse transcriptase with mutation sites of D280E / D169K / I580M / G335F is SEQ ID NO. 42.

[0220] SEQ ID NO. 42

[0221]

[0222] Example 4:

[0223] This example studies the characterization of the enzymatic properties of the UCRT III thermostable reverse transcriptase mutant

[0224] The wild-type UCRT III thermostable reverse transcriptase and the various UCRT III thermostable reverse transcriptase mutants provided in Example 2 are subjected to thermal stability testing, according to the conventional UCRT III thermostable reverse transcriptase activity determination method, specifically:

[0225] The enzyme solution is incubated at a certain temperature, and samples are taken at different treatment times, and the residual activity percentage of the UCRT III thermostable reverse transcriptase or the UCRT III thermostable reverse transcriptase mutant is determined, and the ln value of the residual activity percentage is plotted against the time t (min), and the slope of the straight line is the inactivation constant kinact, and the half-life of the wild-type UCRT III thermostable reverse transcriptase or the UCRT III thermostable reverse transcriptase mutant at this temperature is obtained from t1 / 2 = ln2 / kinact.

[0226] The experimental results show that among the above various UCRT III thermostable reverse transcriptase mutants, the thermal stability of 4 single-point mutants and 11 combined mutants is obviously improved, as shown in Table 1:

[0227] Table 1. Characterization of the enzymatic properties of the wild-type UCRT III thermostable reverse transcriptase, single-point mutants and combined mutants

[0228]

[0229]

[0230] As can be seen from Table 1, the UCRT III thermostable reverse transcriptase mutant provided by the present application includes single-point mutants and combined mutants, and compared with the wild-type UCRT III thermostable reverse transcriptase, the half-life of the single-point mutants and the combined mutants at 65℃ is longer; especially the combined mutants, which exhibit the superposition effect of the thermal stability of the single-point mutants, and the half-life is about 4 times that of the wild type.

[0231] The above examples are merely illustrative of the present application and do not constitute a limitation on the scope of protection of the present application, and any design identical or similar to the present application falls within the scope of protection of the present application.

Claims

1. A UCRT thermostable reverse transcriptase mutant with improved thermostability, characterized in that, The amino acid sequence of the UCRT heat-resistant reverse transcriptase mutant is mutated after the mutation of the mutation site on SEQ ID NO. 2; the mutation site is D169K, I580M, G335F, D280E / D169K, D280E / I580M, D280E / G335F, D169K / I580M, D169K / G335F, I580M / G335F, D280E / D169K / I580M, D280E / D169K / G335F, D280E / I580M / G335F, D169K / I580M / G335F, D280E / D169K / I580M / G335F; The amino acid sequence of the single-point mutant corresponding to D169K is SEQ ID NO. 4; The amino acid sequence of the single-point mutant corresponding to I580M is SEQ ID NO. 5; The amino acid sequence of the single-point mutant corresponding to G335F is SEQ ID NO. 6; The amino acid sequence of the combination mutant corresponding to D280E / D169K is SEQ ID NO. 7; The amino acid sequence of the combination mutant corresponding to D280E / I580M is SEQ ID NO. 8; The amino acid sequence of the combination mutant corresponding to D280E / G335F is SEQ ID NO. 9; The amino acid sequence of the combination mutant corresponding to D169K / I580M is SEQ ID NO. 10; The amino acid sequence of the combination mutant corresponding to D169K / G335F is SEQ ID NO. 11; The amino acid sequence of the combination mutant corresponding to I580M / G335F is SEQ ID NO. 12; The amino acid sequence of the combination mutant corresponding to D280E / D169K / I580M is SEQ ID NO. 13; The amino acid sequence of the combination mutant corresponding to D280E / D169K / G335F is SEQ ID NO. 14; The amino acid sequence of the combination mutant corresponding to D280E / I580M / G335F is SEQ ID NO. 15; The amino acid sequence of the combination mutant corresponding to D169K / I580M / G335F is SEQ ID NO. 16; The amino acid sequence of the combination mutant corresponding to D280E / D169K / I580M / G335F is SEQ ID NO.

17.

2. The method for constructing a heat-stable UCRT thermostable reverse transcriptase mutant according to claim 1, wherein The method comprises the following steps: Searching and selecting an amino acid sequence with more than 50% identity with the amino acid sequence shown in SEQ ID NO. 2 in the database, then performing multiple sequence alignment, and generating a consensus sequence that can be edited later by software; Protein three-dimensional structure prediction is performed on SEQ ID NO. 2, and mutation sites related to stability are screened: D280E, D169K, I580M, G335F. 3.The method for constructing a UCRT thermostable reverse transcriptase mutant with improved thermal stability according to claim 2, characterized in that: the amplification primer sequence of the mutation site D280E is SEQ ID NO. 20, SEQ ID NO. 21; the amplification primer sequence of the mutation site D169K is SEQ ID NO. 22, SEQ ID NO. 23; the amplification primer sequence of the mutation site I580M is SEQ ID NO. 24, SEQ ID NO. 25; the amplification primer sequence of the mutation site G335F is SEQ ID NO. 26, SEQ ID NO.

27. 4.A gene encoding the UCRT thermostable reverse transcriptase mutant with improved thermal stability according to claim 1. 5.A recombinant plasmid comprising the gene according to claim 4. 6.A soluble protein, immobilized enzyme or engineered bacteria comprising the UCRT thermostable reverse transcriptase mutant with improved thermal stability according to claim 1. 7.The use of the UCRT thermostable reverse transcriptase mutant with improved thermal stability according to claim 1 in reverse transcription catalyzing DNA synthesis.

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