CN117106746A - Reverse transcription mutant and application thereof - Google Patents

Reverse transcription mutant and application thereof Download PDF

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CN117106746A
CN117106746A CN202310944992.2A CN202310944992A CN117106746A CN 117106746 A CN117106746 A CN 117106746A CN 202310944992 A CN202310944992 A CN 202310944992A CN 117106746 A CN117106746 A CN 117106746A
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mutant
reverse transcriptase
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mmlv reverse
reverse transcription
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肖晓文
王文朋
李妍
李可欣
张雪静
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Tsingke Biotechnology Co Ltd
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Abstract

The application belongs to the technical field of biology, and discloses an MMLV reverse transcription mutant which is obtained by replacing an RNaseH domain of a wild MMLV reverse transcriptase with a domain for enhancing the binding with an RNA-DNA hybrid chain. The application also relates to mutant designs, wherein the preferred combination of mutations is E69K, D200K, P326K, P401K, T P and T306K. Compared with the wild type, the MMLV reverse transcription mutant has higher amplification efficiency. The application also provides the sequence of the MMLV reverse transcriptase mutant and the application thereof in reverse transcription reaction.

Description

Reverse transcription mutant and application thereof
The present application is a divisional application of patent application 202210621426.3 entitled "reverse transcription mutant and its use" filed by applicant at 1/6/2022.
Technical Field
The application belongs to the technical field of biology, and particularly relates to a reverse transcription mutant and application thereof.
Background
Reverse transcriptase is a polymerase which synthesizes DNA by taking RNA as a template and doping substrates dNTPs, and has wide application in the field of molecular biology. Reverse transcriptase derived from Murine Moroni leukemia virus (Murine-Moloney leukemia virus, MMLV) (hereinafter referred to as MMLV reverse transcriptase) is an important tool enzyme widely used in the field of basic research, and MMLV reverse transcriptase has high activity in a relatively broad temperature range of 37-60 ℃, and is particularly suitable for amplification of RNA templates with longer fragments and complex secondary structures.
Wild-type MMLV reverse transcriptase comprises a reverse transcription catalytic domain and an RNaseH domain, which itself has a binding site for small chemical molecules, which may include animal derived heparin and cholate, plant derived polysaccharides and polyphenols and alcohols used in RNA extraction, and binding of these small chemical molecules to the RNaseH domain results in a decrease in the sustained reverse transcription capacity of MMLV reverse transcriptase. In addition, the wild-type MMLV catalytic domain has a weak binding capacity to template strand RNA, which also results in a low sustained reverse transcription capacity of MMLV reverse transcriptase.
Accordingly, there is a need in the art for an improved reverse transcriptase having enhanced amplification efficiency to meet the needs of reverse transcription reaction applications in the art.
Disclosure of Invention
In view of the problems of the prior art, the present application will improve MMLV reverse transcriptase to obtain MMLV reverse transcriptase mutants with enhanced amplification efficiency, comprising replacing the RNaseH domain with other domains enhancing binding to RNA-DNA hybrid, and further mutating the site near the interface of catalytic domain interaction with RNA template to enhance interaction force of MMLV reverse transcriptase with RNA template.
The application provides an MMLV reverse transcription mutant, which has higher amplification efficiency compared with a wild type.
The present application provides an MMLV reverse transcription mutant obtained by replacing the RNaseH domain of wild type MMLV reverse transcriptase (SEQ ID NO. 1) with a sso7d-like family protein from Sulfurisphaera tokodaii, wherein lysine (K) at position 13 of the sso7d-like family protein is mutated to leucine (L) with the sequence shown in SEQ ID NO. 2.
In one embodiment, the RNaseH domain substitution is performed after amino acid residue 474, preferably from 474 to 495, more preferably at 495, of the wild-type MMLV reverse transcriptase, the sequence of the substituted MMLV reverse transcriptase mutant being shown in SEQ ID No. 3.
In one embodiment, the application also contemplates mutating the MMLV reverse transcriptase with an RNaseH domain, wherein the mutation occurs at a site comprising glutamic acid (H) at position 69, histidine (H) at position 126, asparagine (N) at position 131, aspartic acid (D) at position 200, histidine (H) at position 204, threonine (T) at position 306, proline (P) at position 326, threonine (T) at position 330, proline (P) at position 401, glutamine (Q) at position 374, glutamine (Q) at position 430.
In one embodiment, the MMLV reverse transcriptase mutation of the application is selected from E69K, P326K, P401K, H204R, T306K, H126Y, N131M, D200K, T330P, P K, P401K, Q374K, Q K or a combination thereof; preferably, the MMLV reverse transcriptase mutant comprises an amino acid substitution selected from (1) - (5):
(1) P326K, P401K, H Y and N131M;
(2) E69K, H126Y, N M, H R and T306K;
(3) E69K, D K and T330P;
(4) E69K, P326K, P401K, Q K and Q430K;
(5) E69K, D200K, P K, P401K, T P and T306K.
The application also provides a polynucleotide sequence of the MMLV reverse transcriptase mutant.
The application also provides an expression vector comprising the polynucleotide sequence of the MMLV reverse transcriptase mutant.
The application also provides a host cell comprising the polynucleotide sequence of the MMLV reverse transcriptase mutant or an expression vector as described above.
The application also provides application of the MMLV reverse transcriptase mutant in reverse transcription reaction.
The application also provides a kit for performing a reverse transcription reaction comprising an MMLV reverse transcriptase mutant as described above. Preferably, the kit further comprises a reverse transcription reaction buffer, dNTPs.
Drawings
Fig. 1: expression of wild-type MMLV reverse transcriptase and mutants M1 to M5 purified western blots are shown, where WT is wild-type, M1 to M5 are 5 mutants, and Mw is protein molecular weight marker.
Fig. 2: A-C are fluorescent quantitative detection plots for 3 different genes (SCD 1, cyp7a1 and GADPH) using RNA from mice, with the Y-axis indicating the amplification signal and the X-axis indicating the number of amplification cycles.
Fig. 3: a-C are fluorescent quantitative detection plots for 3 different genes (60S, E and FAD 8) using RNA from peanuts, with the Y-axis indicating the amplification signal and the X-axis indicating the number of amplification cycles.
Detailed Description
Without wishing to be bound by a particular theory, the inventors believe that the RNaseH domain has activity to digest RNA in the RNA-DNA hybrid strand, which is detrimental to multiple cycle iterative reverse transcription of RNA, and thus require inactivation of the RNaseH activity. The RNaseH domain can be combined with RNA-DNA hybrid chains, plays an important role in the continuous reverse transcription capacity of MMLV reverse transcriptase, and deletion of the domain can cause the continuous reverse transcription capacity of the MMLV reverse transcriptase to be reduced. Thus, it is conventional to retain the RNaseH domain and mutate the key sites of the RNaseH domain associated with digestion activity under conditions that ensure that they still bind to RNA-DNA hybrid strands.
The MMLV reverse transcription mutant provided by the application is obtained by improvement on the basis of wild MMLV reverse transcriptase, wherein the sequence of the wild MMLV reverse transcriptase is shown as SEQ ID NO. 1.
The specific scheme of the application is as follows:
(1) The RNaseH domain is replaced.
In one embodiment of the application, the RNaseH domain of MMLV reverse transcriptase is replaced with sso7d-like family proteins from Sulfurisphaera tokodaii.
Because of the RNase activity of the sso7d-like family protein, in one embodiment of the present application, the RNaseH domain of MMLV reverse transcriptase is replaced with a sso7d-like family protein from Sulfurisphaera tokodaii, wherein lysine (K) at position 13 of the sso7d-like family protein from Sulfurisphaera tokodaii is replaced with leucine (L) and the sequence is shown in SEQ ID NO. 2.
Amino acid residues 362-474 of MMLV reverse transcriptase are a linking domain (connection domain) for linking the thumb domain (thumb domain) itself located between amino acid residues 276-361 and the RNaseH domain located between amino acid residues 474-672. The linking domain has an important regulatory function for the overall activity of MMLV reverse transcriptase, and thus the substitution of the RNaseH domain according to the present application is performed after amino acid residue 474 of MMLV reverse transcriptase, and may be from position 474 to position 495, preferably the substitution of the RNaseH domain is selected at position 495.
In one embodiment of the application, the MMLV reverse transcriptase sequence after substitution of the RNaseH domain is shown in SEQ ID NO. 3.
(2) MMLV reverse transcriptase mutant design.
In order to further enhance the activity of MMLV reverse transcriptase, the present inventors have analyzed the structure of MMLV to obtain amino acid sites that may affect the template binding ability, including glutamic acid (H) at position 69, histidine (H) at position 126, asparagine (N) at position 131, aspartic acid (D) at position 200, histidine (H) at position 204, threonine (T) at position 306, proline (P) at position 326, threonine (T) at position 330, glutamine (Q) at position 374, proline (P) at position 401, glutamine (Q) at position 430.
In the present application, the following sites may be specifically designed by mutation:
(1) Glutamic acid at position 69, threonine at position 306, proline at position 326, glutamine at position 374, proline at position 401 and glutamine at position 430, which are located near the template strand and can be bound to the phosphate backbone and thus can be mutated to histidine, lysine or arginine, preferably to lysine (K);
(2) Histidine at position 126, which faces the phosphate backbone of the template, can be mutated to tyrosine (Y) of the same genus and containing only one benzene ring side chain in order to enhance its interaction with the template chain, while ensuring that the secondary structure is not affected;
(3) Asparagine at position 131, which is closer to the base, presumably is bound to the template strand primarily by hydrophobic interactions and thus may be mutated to methionine, leucine or isoleucine, preferably to methionine (M);
(4) Aspartic acid at position 200 and histidine at position 204, which are remote from the template strand but close to the β -sheet involved in catalysis, indirectly modulating the interaction of the β -sheet with the template strand, may be mutated to lysine, arginine or asparagine, preferably to lysine (K);
(5) Threonine at position 330, adjacent to the alpha-helix bound to the template, can affect the interaction of the reverse transcriptase with the template, preferably by mutation to proline (P).
For the above mutation sites, the specific combinations selected for use in the present application are shown in table 1 below:
TABLE 1 mutation site combinations
(3) Obtaining MMLV reverse transcriptase mutant.
After designing the nucleic acid sequence of MMLV reverse transcriptase (shown as SEQ ID No.10 to SEQ ID No. 14), a commercial expression vector pET series based on the T7 promoter was ligated, and mutants were obtained using a commercial site-directed mutagenesis kit. The expression vector contains an operator sequence responding to IPTG induction expression, and simultaneously ensures the high-efficiency expression of the protein in escherichia coli. The specific culture temperature of the escherichia coli is 16 ℃, 25 ℃,30 ℃ or 37 ℃, and the culture time can be 20 hours, 16 hours, 12 hours or 4 hours. Preferably, the present application uses 16 ℃ for induction of expression, the induction time is 20h.
(4) Isolation and purification of MMLV reverse transcriptase mutants.
After the set induction expression culture time was reached, the cultured cells were collected by centrifugation and resuspended by shaking with a buffer. The pH of the buffer is 7.0-8.0, which contains 10-50 mM Trima, HEPES or Tricine, etc. as pH buffer and 200-500 mM NaCl or KCl as salt. The buffer used for cleavage and purification contains 0.5 mM-2 mM reducing agent, and can prevent erroneous disulfide bond folding inside the enzyme. The buffer solution is prepared by using water without nuclease, and is subjected to filtration and high-temperature high-pressure sterilization treatment. The re-suspended thallus suspension is cracked and broken by ultrasonic mode, and then the MMLV reverse transcriptase mutant with higher purity is obtained by using an affinity chromatography method. Furthermore, ion exchange chromatography is used to remove more impurity proteins, so that the purity of the mutant target protein reaches more than 90%. The mutant protein of interest was finally dialyzed into storage buffer.
(5) Detection of amplification efficiency of MMLV reverse transcriptase mutant.
Reverse transcription was performed using RNA from 2 different species with a template amount of 500ng. The necessary components for reverse transcription and the obtained MMLV reverse transcriptase mutant are added, primers of different sites are designed to synthesize cDNA fragments, and then qPCR fluorescence quantitative detection is carried out on the amount of the product.
Compared with the prior art, the technical scheme of the application has the following beneficial effects: the mutant has higher amplification efficiency.
Examples
Example 1: expression and purification of reverse transcriptase.
Wild type MMLV reverse transcriptase and its mutant plasmid were added to BL21 (DE 3) competent, ice bath 30min,42℃heat shock 90s, ice bath 2min. 900. Mu.L of LB was added, incubated at 37℃at 220rpm/1h, plated on LB plates containing kana resistance, and incubated at 37℃overnight with inversion. The above-mentioned monoclonal was inoculated into 10mL of LB (Kan+), and the mixture was shaken at 220rpm at 37℃until it became turbid, and the bacterial solution was inoculated into 1000mL of LB (Kan+), the total amount of the starting bacteria was 8L, and the mixture was shaken at 220rpm at 37℃until OD=0.6, and 0.5mM IPTG 500. Mu.L was added and the mixture was induced at 16℃overnight at 120rpm for 18 hours. The induced bacterial solution was centrifuged at 3500rpm at 4℃for 10min, the bacterial cells were collected and resuspended in 100mL buffer (20 mM Tris-Cl7.6, 300mM NaCl,10% glycerol), sonicated for 15min, and then centrifuged at 9500rpm at 4℃for 30min, and the supernatant was collected. The supernatant was applied to an affinity column, and after loading, the Ni-NTA chromatography column was equilibrated for 5 column volumes with an affinity buffer, and 5 column volumes were washed with a washing buffer (20 mM Tris-Cl7.6, 300mM NaCl,50mM imidazole, 10% glycerol), and finally the target protein was eluted with an elution buffer (20 mM Tris-Cl7.6, 150mM NaCl,300mM imidazole, 10% glycerol). Further purification was then performed by anion exchange chromatography, the column was equilibrated with buffer (20 mM Tris-Cl7.6, 300mM NaCl,10% glycerol), followed by direct loading for purification, and the flow samples were collected and subjected to SDS-PAGE analysis. Protein samples were dialyzed overnight against dialysis buffer (20 mM Tris-Cl7.6, 300mM NaCl,1mM DTT,1mM EDTA,0.2% IPGAL-CA630, 50% glycerol), and the target proteins were collected and stored at-20 ℃.
The results of the obtained protein are shown in figure 1, and the wild MMLV reverse transcriptase and each mutant can be well expressed in a soluble way.
Example 2: and (5) detecting amplification efficiency.
1. Mu.g of RNA was used as template, which was derived from mice and peanuts. Three different site genes (mice: SCD1, cyp7a1 and GADPH; peanuts: 60S, E and FAD 8) of each species were selected for reverse transcription, and the specific reaction system is shown in Table 2. The reaction conditions are as follows: incubate at 25℃for 10min, incubate at 50℃for 5min, and react at 85℃for 5min.
Subsequently, detection was performed using fluorescent quantitative PCR, and after the reverse transcription product was diluted 3-fold, 1. Mu.L was taken for qRT-PCR reaction. The reaction system is shown in Table 3, the reaction conditions are shown in Table 4, and the primer sequences used are shown in Table 5.
TABLE 2 reverse transcription reaction system
TABLE 3qRT-PCR reaction System
TABLE 4qRT-PCR reaction conditions
TABLE 5 primer sequences
The number of the products is judged by comparing Ct values, and the amplification efficiency is further judged. In short, the smaller the Ct value, the lower the number of cycles required to reach the threshold of the fluorescent quantitative PCR apparatus, i.e., the higher the initial amount of reverse transcription product, the higher the reverse transcriptase efficiency.
As shown in FIG. 2 (mouse gene fluorescent quantitative detection) and FIG. 3 (peanut gene fluorescent quantitative detection), the amplification efficiency of the reverse transcriptase mutants M1 to M5 is better than that of the wild type reverse transcriptase, wherein the mutant M5 performs better, the highest value of the mutant M5 can be better than that of the wild type 4 Cts in the mouse gene fluorescent quantitative detection, and the highest value of the mutant M5 can be better than that of the wild type 2 Cts in the peanut gene fluorescent quantitative detection.
The sequence is as follows:
SEQ ID NO.1 (sequence of wild type MMLV):
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVS
IKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEM
GISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDC
QQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQP
TPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQAL
LTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPC
LRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLD
TDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTD
GSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVY
TDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGH
SAEARGNRMADQAARKAAITETPDTSTLL
SEQ ID NO.2 (mutated Sulfurisphaera tokodaii sequence): MVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQM LEKSGKK
SEQ ID NO.3 (sequence after substitution of RNaseH domain):
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVS
IKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN
KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEM
GISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDC
QQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQP
TPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQAL
LTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPC
LRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLD
TDRVQFGPVVALNPATLLPLPEEGLQHNGTGGGGMVTVKFKYKGEELEVDISKIKKVW
RVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKKSEQ ID NO.4 (mutant M1)
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVS
IKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN
KRVEDIYPTVPMPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEM
GISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDC
QQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQP
TPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYKLTKTGTLFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDKVAAGWPP
CLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLL
DTDRVQFGPVVALNPATLLPLPEEGLQHNGTGGGGMVTVKFKYKGEELEVDISKIKKV
WRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK
SEQ ID NO.5 (mutant M2)
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVS
IKQYPMSQKARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREV
NKRVEDIYPTVPMPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPE
MGISGQLTWTRLPQGFKNSPTLFDEALRRDLADFRIQHPDLILLQYVDDLLLAATSELD
CQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
PTPKTPRQLREFLGKAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQ
ALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWP
PCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLL
DTDRVQFGPVVALNPATLLPLPEEGLQHNGTGGGGMVTVKFKYKGEELEVDISKIKKV
WRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK
SEQ ID NO.6 (mutant M3)
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVS
IKQYPMSQKARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREV
NKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPE
MGISGQLTWTRLPQGFKNSPTLFKEALHRDLADFRIQHPDLILLQYVDDLLLAATSELD
CQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
PTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQA
LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPP
CLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLL
DTDRVQFGPVVALNPATLLPLPEEGLQHNGTGGGGMVTVKFKYKGEELEVDISKIKKV
WRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK
SEQ ID NO.7 (mutant M4)
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVS
IKQYPMSQKARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREV
NKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPE
MGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELD
CQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQ
PTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYKLTKTGTLFNWGPDQQKAYQEIKQ
ALLTAPALGLPDLTKPFELFVDEKKGYAKGVLTQKLGPWRRPVAYLSKKLDKVAAGWP
PCLRMVAAIAVLTKDAGKLTMGKPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLL
DTDRVQFGPVVALNPATLLPLPEEGLQHNGGGTGGGGMVTVKFKYKGEELEVDISKIK
KVWRVGKMISFTYDDNGKTGRGAVSEKDAPKELLQMLEKSGKK
SEQ ID NO.8 (mutant M5)
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATST
PVSIKQYPMSQKARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQ
DLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFA
FEWRDPEMGISGQLTWTRLPQGFKNSPTLFKEALHRDLADFRIQHPDLILLQYVDD
LLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRW
LTEARKETVMGQPTPKTPRQLREFLGKAGFCRLWIPGFAEMAAPLYKLTKPGTLFN
WGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWR
RPVAYLSKKLDKVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEAL
VKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNGTG
GGGMVTVKFKYKGEELEVDISKIKKVWRVGKMISFTYDDNGKTGRGAVSEKDAP
KELLQMLEKSGKK
SEQ ID NO.9 (nucleic acid sequence after substitution of the RNaseH Domain)
ACCCTGAACATTGAAGATGAACATCGTCTGCATGAAACCAGCAAAGAACCGGA
TGTGAGCCTGGGCAGCACCTGGCTGAGCGATTTTCCGCAGGCGTGGGCGGAAA
CCGGCGGCATGGGCCTGGCGGTGCGTCAGGCGCCGCTGATTATTCCGCTGAAA
GCGACCAGCACCCCGGTGAGCATTAAACAGTATCCGATGAGCCAGGAAGCGCG
TCTGGGCATTAAACCGCATATTCAGCGTCTGCTGGATCAGGGCATTCTGGTGCC
GTGCCAGAGCCCGTGGAACACCCCGCTGCTGCCGGTGAAAAAACCGGGCACCA
ACGATTATCGTCCGGTGCAGGATCTGCGTGAAGTGAACAAACGTGTGGAAGAT
ATTCATCCGACCGTGCCGAACCCGTATAACCTGCTGAGCGGCCTGCCGCCGAGC
CATCAGTGGTATACCGTGCTGGATCTGAAAGATGCGTTTTTTTGCCTGCGTCTG
CATCCGACCAGCCAGCCGCTGTTTGCGTTTGAATGGCGTGATCCGGAAATGGGC
ATTAGCGGCCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTTAAAAACAGCCC
GACCCTGTTTGATGAAGCGCTGCATCGTGATCTGGCGGATTTTCGTATTCAGCA
TCCGGATCTGATTCTGCTGCAGTATGTGGATGATCTGCTGCTGGCGGCGACCAG
CGAACTGGATTGCCAGCAGGGCACCCGTGCGCTGCTGCAGACCCTGGGCAACC
TGGGCTATCGTGCGAGCGCGAAAAAAGCGCAGATTTGCCAGAAACAGGTGAAA
TATCTGGGCTATCTGCTGAAAGAAGGCCAGCGTTGGCTGACCGAAGCGCGTAA
AGAAACCGTGATGGGCCAGCCGACCCCGAAAACCCCGCGTCAGCTGCGTGAAT
TTCTGGGCACCGCGGGCTTTTGCCGTCTGTGGATTCCGGGCTTTGCGGAAATGG
CGGCGCCGCTGTATCCGCTGACCAAAACCGGCACCCTGTTTAACTGGGGCCCGG
ATCAGCAGAAAGCGTATCAGGAAATTAAACAGGCGCTGCTGACCGCGCCGGCG
CTGGGCCTGCCGGATCTGACCAAACCGTTTGAACTGTTTGTGGATGAAAAACAG
GGCTATGCGAAAGGCGTGCTGACCCAGAAACTGGGCCCGTGGCGTCGTCCGGT
GGCGTATCTGAGCAAAAAACTGGATCCGGTGGCGGCGGGCTGGCCGCCGTGCC
TGCGTATGGTGGCGGCGATTGCGGTGCTGACCAAAGATGCGGGCAAACTGACC
ATGGGCCAGCCGCTGGTGATTCTGGCGCCGCATGCGGTGGAAGCGCTGGTGAA
ACAGCCGCCGGATCGTTGGCTGAGCAACGCGCGTATGACCCATTATCAGGCGC
TGCTGCTGGATACCGATCGTGTGCAGTTTGGCCCGGTGGTGGCGCTGAACCCGG
CGACCCTGCTGCCGCTGCCGGAAGAAGGCCTGCAGCATAACGGCACCGGCGGC
GGCGGCATGGTGACCGTGAAATTTAAATATAAAGGCGAAGAACTGGAAGTGGA
TATTAGCAAAATTAAAAAAGTGTGGCGTGTGGGCAAAATGATTAGCTTTACCTA
TGATGATAACGGCAAAACCGGCCGTGGCGCGGTGAGCGAAAAAGATGCGCCGA
AAGAACTGCTGCAGATGCTGGAAAAAAGCGGCAAAAAATAA
SEQ ID NO.10 (mutant M1 nucleic acid sequence)
ACCCTGAACATTGAAGATGAACATCGTCTGCATGAAACCAGCAAAGAACCGGA
TGTGAGCCTGGGCAGCACCTGGCTGAGCGATTTTCCGCAGGCGTGGGCGGAAA
CCGGCGGCATGGGCCTGGCGGTGCGTCAGGCGCCGCTGATTATTCCGCTGAAA
GCGACCAGCACCCCGGTGAGCATTAAACAGTATCCGATGAGCCAGGAAGCGCG
TCTGGGCATTAAACCGCATATTCAGCGTCTGCTGGATCAGGGCATTCTGGTGCC
GTGCCAGAGCCCGTGGAACACCCCGCTGCTGCCGGTGAAAAAACCGGGCACCA
ACGATTATCGTCCGGTGCAGGATCTGCGTGAAGTGAACAAACGTGTGGAAGAT
ATTTATCCGACCGTGCCGATGCCGTATAACCTGCTGAGCGGCCTGCCGCCGAGC
CATCAGTGGTATACCGTGCTGGATCTGAAAGATGCGTTTTTTTGCCTGCGTCTG
CATCCGACCAGCCAGCCGCTGTTTGCGTTTGAATGGCGTGATCCGGAAATGGGC
ATTAGCGGCCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTTAAAAACAGCCC
GACCCTGTTTGATGAAGCGCTGCATCGTGATCTGGCGGATTTTCGTATTCAGCA
TCCGGATCTGATTCTGCTGCAGTATGTGGATGATCTGCTGCTGGCGGCGACCAG
CGAACTGGATTGCCAGCAGGGCACCCGTGCGCTGCTGCAGACCCTGGGCAACC
TGGGCTATCGTGCGAGCGCGAAAAAAGCGCAGATTTGCCAGAAACAGGTGAAA
TATCTGGGCTATCTGCTGAAAGAAGGCCAGCGTTGGCTGACCGAAGCGCGTAA
AGAAACCGTGATGGGCCAGCCGACCCCGAAAACCCCGCGTCAGCTGCGTGAAT
TTCTGGGCACCGCGGGCTTTTGCCGTCTGTGGATTCCGGGCTTTGCGGAAATGG
CGGCGCCGCTGTATAAACTGACCAAAACCGGCACCCTGTTTAACTGGGGCCCG
GATCAGCAGAAAGCGTATCAGGAAATTAAACAGGCGCTGCTGACCGCGCCGGC
GCTGGGCCTGCCGGATCTGACCAAACCGTTTGAACTGTTTGTGGATGAAAAACA
GGGCTATGCGAAAGGCGTGCTGACCCAGAAACTGGGCCCGTGGCGTCGTCCGG
TGGCGTATCTGAGCAAAAAACTGGATAAAGTGGCGGCGGGCTGGCCGCCGTGC
CTGCGTATGGTGGCGGCGATTGCGGTGCTGACCAAAGATGCGGGCAAACTGAC
CATGGGCCAGCCGCTGGTGATTCTGGCGCCGCATGCGGTGGAAGCGCTGGTGA
AACAGCCGCCGGATCGTTGGCTGAGCAACGCGCGTATGACCCATTATCAGGCG
CTGCTGCTGGATACCGATCGTGTGCAGTTTGGCCCGGTGGTGGCGCTGAACCCG
GCGACCCTGCTGCCGCTGCCGGAAGAAGGCCTGCAGCATAACGGCACCGGCGG
CGGCGGCATGGTGACCGTGAAATTTAAATATAAAGGCGAAGAACTGGAAGTGG
ATATTAGCAAAATTAAAAAAGTGTGGCGTGTGGGCAAAATGATTAGCTTTACCT
ATGATGATAACGGCAAAACCGGCCGTGGCGCGGTGAGCGAAAAAGATGCGCCG
AAAGAACTGCTGCAGATGCTGGAAAAAAGCGGCAAAAAATAA
SEQ ID NO.11 (mutant M2 nucleic acid sequence)
ACCCTGAACATTGAAGATGAACATCGTCTGCATGAAACCAGCAAAGAACCGGA
TGTGAGCCTGGGCAGCACCTGGCTGAGCGATTTTCCGCAGGCGTGGGCGGAAA
CCGGCGGCATGGGCCTGGCGGTGCGTCAGGCGCCGCTGATTATTCCGCTGAAA
GCGACCAGCACCCCGGTGAGCATTAAACAGTATCCGATGAGCCAGAAAGCGCG
TCTGGGCATTAAACCGCATATTCAGCGTCTGCTGGATCAGGGCATTCTGGTGCC
GTGCCAGAGCCCGTGGAACACCCCGCTGCTGCCGGTGAAAAAACCGGGCACCA
ACGATTATCGTCCGGTGCAGGATCTGCGTGAAGTGAACAAACGTGTGGAAGAT
ATTTATCCGACCGTGCCGATGCCGTATAACCTGCTGAGCGGCCTGCCGCCGAGC
CATCAGTGGTATACCGTGCTGGATCTGAAAGATGCGTTTTTTTGCCTGCGTCTG
CATCCGACCAGCCAGCCGCTGTTTGCGTTTGAATGGCGTGATCCGGAAATGGGC
ATTAGCGGCCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTTAAAAACAGCCC
GACCCTGTTTGATGAAGCGCTGCGTCGTGATCTGGCGGATTTTCGTATTCAGCA
TCCGGATCTGATTCTGCTGCAGTATGTGGATGATCTGCTGCTGGCGGCGACCAG
CGAACTGGATTGCCAGCAGGGCACCCGTGCGCTGCTGCAGACCCTGGGCAACC
TGGGCTATCGTGCGAGCGCGAAAAAAGCGCAGATTTGCCAGAAACAGGTGAAA
TATCTGGGCTATCTGCTGAAAGAAGGCCAGCGTTGGCTGACCGAAGCGCGTAA
AGAAACCGTGATGGGCCAGCCGACCCCGAAAACCCCGCGTCAGCTGCGTGAAT
TTCTGGGCAAAGCGGGCTTTTGCCGTCTGTGGATTCCGGGCTTTGCGGAAATGG
CGGCGCCGCTGTATCCGCTGACCAAAACCGGCACCCTGTTTAACTGGGGCCCGG
ATCAGCAGAAAGCGTATCAGGAAATTAAACAGGCGCTGCTGACCGCGCCGGCG
CTGGGCCTGCCGGATCTGACCAAACCGTTTGAACTGTTTGTGGATGAAAAACAG
GGCTATGCGAAAGGCGTGCTGACCCAGAAACTGGGCCCGTGGCGTCGTCCGGT
GGCGTATCTGAGCAAAAAACTGGATCCGGTGGCGGCGGGCTGGCCGCCGTGCC
TGCGTATGGTGGCGGCGATTGCGGTGCTGACCAAAGATGCGGGCAAACTGACC
ATGGGCCAGCCGCTGGTGATTCTGGCGCCGCATGCGGTGGAAGCGCTGGTGAA
ACAGCCGCCGGATCGTTGGCTGAGCAACGCGCGTATGACCCATTATCAGGCGC
TGCTGCTGGATACCGATCGTGTGCAGTTTGGCCCGGTGGTGGCGCTGAACCCGG
CGACCCTGCTGCCGCTGCCGGAAGAAGGCCTGCAGCATAACGGCACCGGCGGC
GGCGGCATGGTGACCGTGAAATTTAAATATAAAGGCGAAGAACTGGAAGTGGA
TATTAGCAAAATTAAAAAAGTGTGGCGTGTGGGCAAAATGATTAGCTTTACCTA
TGATGATAACGGCAAAACCGGCCGTGGCGCGGTGAGCGAAAAAGATGCGCCGA
AAGAACTGCTGCAGATGCTGGAAAAAAGCGGCAAAAAATAA
SEQ ID NO.12 (mutant M3 nucleic acid sequence)
ACCCTGAACATTGAAGATGAACATCGTCTGCATGAAACCAGCAAAGAACCGGA
TGTGAGCCTGGGCAGCACCTGGCTGAGCGATTTTCCGCAGGCGTGGGCGGAAA
CCGGCGGCATGGGCCTGGCGGTGCGTCAGGCGCCGCTGATTATTCCGCTGAAA
GCGACCAGCACCCCGGTGAGCATTAAACAGTATCCGATGAGCCAGAAAGCGCG
TCTGGGCATTAAACCGCATATTCAGCGTCTGCTGGATCAGGGCATTCTGGTGCC
GTGCCAGAGCCCGTGGAACACCCCGCTGCTGCCGGTGAAAAAACCGGGCACCA
ACGATTATCGTCCGGTGCAGGATCTGCGTGAAGTGAACAAACGTGTGGAAGAT
ATTCATCCGACCGTGCCGAACCCGTATAACCTGCTGAGCGGCCTGCCGCCGAGC
CATCAGTGGTATACCGTGCTGGATCTGAAAGATGCGTTTTTTTGCCTGCGTCTG
CATCCGACCAGCCAGCCGCTGTTTGCGTTTGAATGGCGTGATCCGGAAATGGGC
ATTAGCGGCCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTTAAAAACAGCCC
GACCCTGTTTAAAGAAGCGCTGCATCGTGATCTGGCGGATTTTCGTATTCAGCA
TCCGGATCTGATTCTGCTGCAGTATGTGGATGATCTGCTGCTGGCGGCGACCAG
CGAACTGGATTGCCAGCAGGGCACCCGTGCGCTGCTGCAGACCCTGGGCAACC
TGGGCTATCGTGCGAGCGCGAAAAAAGCGCAGATTTGCCAGAAACAGGTGAAA
TATCTGGGCTATCTGCTGAAAGAAGGCCAGCGTTGGCTGACCGAAGCGCGTAA
AGAAACCGTGATGGGCCAGCCGACCCCGAAAACCCCGCGTCAGCTGCGTGAAT
TTCTGGGCACCGCGGGCTTTTGCCGTCTGTGGATTCCGGGCTTTGCGGAAATGG
CGGCGCCGCTGTATCCGCTGACCAAACCGGGCACCCTGTTTAACTGGGGCCCGG
ATCAGCAGAAAGCGTATCAGGAAATTAAACAGGCGCTGCTGACCGCGCCGGCG
CTGGGCCTGCCGGATCTGACCAAACCGTTTGAACTGTTTGTGGATGAAAAACAG
GGCTATGCGAAAGGCGTGCTGACCCAGAAACTGGGCCCGTGGCGTCGTCCGGT
GGCGTATCTGAGCAAAAAACTGGATCCGGTGGCGGCGGGCTGGCCGCCGTGCC
TGCGTATGGTGGCGGCGATTGCGGTGCTGACCAAAGATGCGGGCAAACTGACC
ATGGGCCAGCCGCTGGTGATTCTGGCGCCGCATGCGGTGGAAGCGCTGGTGAA
ACAGCCGCCGGATCGTTGGCTGAGCAACGCGCGTATGACCCATTATCAGGCGC
TGCTGCTGGATACCGATCGTGTGCAGTTTGGCCCGGTGGTGGCGCTGAACCCGG
CGACCCTGCTGCCGCTGCCGGAAGAAGGCCTGCAGCATAACGGCACCGGCGGC
GGCGGCATGGTGACCGTGAAATTTAAATATAAAGGCGAAGAACTGGAAGTGGA
TATTAGCAAAATTAAAAAAGTGTGGCGTGTGGGCAAAATGATTAGCTTTACCTA
TGATGATAACGGCAAAACCGGCCGTGGCGCGGTGAGCGAAAAAGATGCGCCGA
AAGAACTGCTGCAGATGCTGGAAAAAAGCGGCAAAAAATAA
SEQ ID NO.13 (mutant M4 nucleic acid sequence)
ACCCTGAACATTGAAGATGAACATCGTCTGCATGAAACCAGCAAAGAACCGGA
TGTGAGCCTGGGCAGCACCTGGCTGAGCGATTTTCCGCAGGCGTGGGCGGAAA
CCGGCGGCATGGGCCTGGCGGTGCGTCAGGCGCCGCTGATTATTCCGCTGAAA
GCGACCAGCACCCCGGTGAGCATTAAACAGTATCCGATGAGCCAGAAAGCGCG
TCTGGGCATTAAACCGCATATTCAGCGTCTGCTGGATCAGGGCATTCTGGTGCC
GTGCCAGAGCCCGTGGAACACCCCGCTGCTGCCGGTGAAAAAACCGGGCACCA
ACGATTATCGTCCGGTGCAGGATCTGCGTGAAGTGAACAAACGTGTGGAAGAT
ATTCATCCGACCGTGCCGAACCCGTATAACCTGCTGAGCGGCCTGCCGCCGAGC
CATCAGTGGTATACCGTGCTGGATCTGAAAGATGCGTTTTTTTGCCTGCGTCTG
CATCCGACCAGCCAGCCGCTGTTTGCGTTTGAATGGCGTGATCCGGAAATGGGC
ATTAGCGGCCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTTAAAAACAGCCC
GACCCTGTTTGATGAAGCGCTGCATCGTGATCTGGCGGATTTTCGTATTCAGCA
TCCGGATCTGATTCTGCTGCAGTATGTGGATGATCTGCTGCTGGCGGCGACCAG
CGAACTGGATTGCCAGCAGGGCACCCGTGCGCTGCTGCAGACCCTGGGCAACC
TGGGCTATCGTGCGAGCGCGAAAAAAGCGCAGATTTGCCAGAAACAGGTGAAA
TATCTGGGCTATCTGCTGAAAGAAGGCCAGCGTTGGCTGACCGAAGCGCGTAA
AGAAACCGTGATGGGCCAGCCGACCCCGAAAACCCCGCGTCAGCTGCGTGAAT
TTCTGGGCAAAGCGGGCTTTTGCCGTCTGTGGATTCCGGGCTTTGCGGAAATGG
CGGCGCCGCTGTATAAACTGACCAAAACCGGCACCCTGTTTAACTGGGGCCCG
GATCAGCAGAAAGCGTATCAGGAAATTAAACAGGCGCTGCTGACCGCGCCGGC
GCTGGGCCTGCCGGATCTGACCAAACCGTTTGAACTGTTTGTGGATGAAAAAA
AAGGCTATGCGAAAGGCGTGCTGACCCAGAAACTGGGCCCGTGGCGTCGTCCG
GTGGCGTATCTGAGCAAAAAACTGGATAAAGTGGCGGCGGGCTGGCCGCCGTG
CCTGCGTATGGTGGCGGCGATTGCGGTGCTGACCAAAGATGCGGGCAAACTGA
CCATGGGCAAACCGCTGGTGATTCTGGCGCCGCATGCGGTGGAAGCGCTGGTG
AAACAGCCGCCGGATCGTTGGCTGAGCAACGCGCGTATGACCCATTATCAGGC
GCTGCTGCTGGATACCGATCGTGTGCAGTTTGGCCCGGTGGTGGCGCTGAACCC
GGCGACCCTGCTGCCGCTGCCGGAAGAAGGCCTGCAGCATAACGGCGGCGGCA
CCGGCGGCGGCGGCATGGTGACCGTGAAATTTAAATATAAAGGCGAAGAACTG
GAAGTGGATATTAGCAAAATTAAAAAAGTGTGGCGTGTGGGCAAAATGATTAG
CTTTACCTATGATGATAACGGCAAAACCGGCCGTGGCGCGGTGAGCGAAAAAG
ATGCGCCGAAAGAACTGCTGCAGATGCTGGAAAAAAGCGGCAAAAAATAA
SEQ ID NO.14 (mutant M5 nucleic acid sequence)
ACCCTGAACATTGAAGATGAACATCGTCTGCATGAAACCAGCAAAGAACCGGA
TGTGAGCCTGGGCAGCACCTGGCTGAGCGATTTTCCGCAGGCGTGGGCGGAAA
CCGGCGGCATGGGCCTGGCGGTGCGTCAGGCGCCGCTGATTATTCCGCTGAAA
GCGACCAGCACCCCGGTGAGCATTAAACAGTATCCGATGAGCCAGAAAGCGCG
TCTGGGCATTAAACCGCATATTCAGCGTCTGCTGGATCAGGGCATTCTGGTGCC
GTGCCAGAGCCCGTGGAACACCCCGCTGCTGCCGGTGAAAAAACCGGGCACCA
ACGATTATCGTCCGGTGCAGGATCTGCGTGAAGTGAACAAACGTGTGGAAGAT
ATTCATCCGACCGTGCCGAACCCGTATAACCTGCTGAGCGGCCTGCCGCCGAGC
CATCAGTGGTATACCGTGCTGGATCTGAAAGATGCGTTTTTTTGCCTGCGTCTG
CATCCGACCAGCCAGCCGCTGTTTGCGTTTGAATGGCGTGATCCGGAAATGGGC
ATTAGCGGCCAGCTGACCTGGACCCGTCTGCCGCAGGGCTTTAAAAACAGCCC
GACCCTGTTTAAAGAAGCGCTGCATCGTGATCTGGCGGATTTTCGTATTCAGCA
TCCGGATCTGATTCTGCTGCAGTATGTGGATGATCTGCTGCTGGCGGCGACCAG
CGAACTGGATTGCCAGCAGGGCACCCGTGCGCTGCTGCAGACCCTGGGCAACC
TGGGCTATCGTGCGAGCGCGAAAAAAGCGCAGATTTGCCAGAAACAGGTGAAA
TATCTGGGCTATCTGCTGAAAGAAGGCCAGCGTTGGCTGACCGAAGCGCGTAA
AGAAACCGTGATGGGCCAGCCGACCCCGAAAACCCCGCGTCAGCTGCGTGAAT
TTCTGGGCAAAGCGGGCTTTTGCCGTCTGTGGATTCCGGGCTTTGCGGAAATGG
CGGCGCCGCTGTATAAACTGACCAAACCGGGCACCCTGTTTAACTGGGGCCCG
GATCAGCAGAAAGCGTATCAGGAAATTAAACAGGCGCTGCTGACCGCGCCGGC
GCTGGGCCTGCCGGATCTGACCAAACCGTTTGAACTGTTTGTGGATGAAAAACA
GGGCTATGCGAAAGGCGTGCTGACCCAGAAACTGGGCCCGTGGCGTCGTCCGG
TGGCGTATCTGAGCAAAAAACTGGATAAAGTGGCGGCGGGCTGGCCGCCGTGC
CTGCGTATGGTGGCGGCGATTGCGGTGCTGACCAAAGATGCGGGCAAACTGAC
CATGGGCCAGCCGCTGGTGATTCTGGCGCCGCATGCGGTGGAAGCGCTGGTGA
AACAGCCGCCGGATCGTTGGCTGAGCAACGCGCGTATGACCCATTATCAGGCG
CTGCTGCTGGATACCGATCGTGTGCAGTTTGGCCCGGTGGTGGCGCTGAACCCG
GCGACCCTGCTGCCGCTGCCGGAAGAAGGCCTGCAGCATAACGGCACCGGCGG
CGGCGGCATGGTGACCGTGAAATTTAAATATAAAGGCGAAGAACTGGAAGTGG
ATATTAGCAAAATTAAAAAAGTGTGGCGTGTGGGCAAAATGATTAGCTTTACCT
ATGATGATAACGGCAAAACCGGCCGTGGCGCGGTGAGCGAAAAAGATGCGCCG
AAAGAACTGCTGCAGATGCTGGAAAAAAGCGGCAAAAAATAA。

Claims (7)

1. An MMLV reverse transcriptase mutant, which is characterized in that the amino acid sequence of the mutant is shown as SEQ ID NO. 4.
2. A polynucleotide sequence encoding an MMLV reverse transcriptase mutant as claimed in claim 1.
3. An expression vector comprising the polynucleotide sequence of claim 2.
4. A host cell comprising the polynucleotide sequence of claim 2 or the expression vector of claim 3.
5. Use of an MMLV reverse transcriptase mutant according to claim 1 in a reverse transcription reaction.
6. A kit for performing a reverse transcription reaction comprising the MMLV reverse transcriptase mutant of claim 1.
7. The kit for performing a reverse transcription reaction according to claim 6, further comprising: reverse transcription reaction buffer, dNTP.
CN202310944992.2A 2022-06-01 2022-06-01 Reverse transcription mutant and application thereof Pending CN117106746A (en)

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