CN115011578A

CN115011578A - Enhanced M-MLV reverse transcriptase mutant and application thereof

Info

Publication number: CN115011578A
Application number: CN202210434762.7A
Authority: CN
Inventors: 朱升龙; 姜旋; 陈永泉; 王振; 叶贤龙; 潘珍珍
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-04-24
Filing date: 2022-04-24
Publication date: 2022-09-06
Anticipated expiration: 2042-04-24
Also published as: CN115011578B

Abstract

The invention discloses an enhanced M-MLV reverse transcriptase mutant and application thereof, belonging to the technical field of biology. The M-MLV reverse transcriptase mutant is formed by artificial modification on the basis of wild-type Moloney murine leukemia virus reverse transcriptase, and the amino acid sequence is shown as Seq ID No. 2. The enhanced M-MLV reverse transcriptase has high amplification efficiency, strong inhibition resistance and thermal stability, the heat resistance can reach 70 ℃, the reaction sensitivity is high, the reaction time is fast, the interference of high-concentration ethanol or isopropanol mixed in an RNA sample can be eliminated, and the reverse transcription of RNA is realized. The enhanced reverse transcriptase mutant can be applied to the fields of trace virus RNA reverse transcription and detection, rapid synthesis of common and long-chain cDNA, construction of various transcriptome libraries and the like, and has wide application prospect and market value.

Description

Enhanced M-MLV reverse transcriptase mutant and application thereof

Technical Field

The invention relates to an enhanced M-MLV reverse transcriptase mutant and application thereof, belonging to the technical field of biology.

Background

In recent years, novel coronavirus wraps the world, and the RT-PCR technology is applied to detect nucleic acid of the novel coronavirus to find an infection source, block a disease transmission chain and further prevent and control epidemic situations. The template used in RT-PCR technology is single-stranded cDNA or double-stranded DNA, and when the detection template is RNA, reverse transcriptase is needed to firstly reverse transcribe the RNA into cDNA, and then PCR amplification is carried out by taking the cDNA as the template. Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase is a DNA polymerase commonly used by taking RNA as a template, has RNase H activity and 3'-5' exonuclease activity, can be used for synthesizing cDNA by reverse transcription, but the MMLV reverse transcriptase still has the problems of lower yield, poor thermal stability, low capability of tolerating inhibitors, low synthesis speed and sensitivity, and the like at present, and limits the application of the MMLV reverse transcriptase in the fields of trace RNA virus detection and the like. At present, the new coronary pneumonia epidemic situation is not resolved in the global scope, and the improvement of the efficiency and the accuracy of the nucleic acid diagnosis method is the key point of the current research, so that the requirements of high sensitivity, high specificity, low cost, easy operation and short time are met by modifying reverse transcriptase.

Disclosure of Invention

Based on the above-mentioned prior art problems, the present invention aims to provide an enhanced M-MLV reverse transcriptase, which is an enhanced reverse transcriptase that is artificially modified on the basis of a wild-type M-MLV reverse transcriptase and has strong stress resistance, higher thermal stability and faster catalytic activity.

The first purpose of the invention is to provide an enhanced M-MLV reverse transcriptase, and the amino acid sequence of the enhanced M-MLV reverse transcriptase is shown as SEQ ID No. 2.

It is a second object of the present invention to provide a nucleotide sequence encoding the enhanced M-MLV reverse transcriptase.

In one embodiment, the nucleotide sequence is set forth in SEQ ID No. 1.

It is a third object of the present invention to provide a recombinant expression vector comprising said nucleotide sequence encoding said enhanced M-MLV reverse transcriptase.

In one embodiment, the starting vector of the recombinant expression vector includes, but is not limited to, pET series, Duet series, pGEX series, pHY300PLK, pPIC3K, pPIC9K series vectors, or pPSUMO.

Preferably, the starting vector is pPSUMO or pET-30a (+).

It is a fourth object of the present invention to provide a recombinant microorganism expressing the enhanced M-MLV reverse transcriptase or comprising the recombinant expression vector.

In one embodiment, the recombinant microorganism is a host cell of a strain of Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, or the like.

The fifth purpose of the invention is to provide a reverse transcription kit, which contains the reinforced reverse transcriptase with the amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the formulation and further comprises RNase H ₂ O, reverse transcription reaction buffer solution, dNTPs and reverse transcription reaction primers.

In one embodiment, the reverse transcription reaction buffer is a 5 × RT buffer; the concentration of the dNTPs is 10 mM; the RNase inhibitor concentration was 40U/. mu.L.

In one embodiment, the kit further comprises an rnase inhibitor.

It is a fifth object of the present invention to provide a method for synthesizing cDNA by reverse transcribing RNA using the enhanced M-MLV reverse transcriptase having the amino acid sequence shown in SEQ ID No.2 or the reverse transcription kit to produce cDNA.

In one embodiment, the reaction is carried out at 37-70 ℃ for not less than 1min, and then the reaction is denatured at 80-90 ℃ for 10-20 s.

In one embodiment, the reaction time is preferably 5 to 30 min.

In one embodiment, the concentration of RNA in the reaction system is 5X 10 ^-5 mg/L to 50mg/L, that is, 20. mu.L, 1pg to 1. mu.g of RNA is added to the system.

The sixth object of the present invention is to provide a method for preparing the enhanced M-MLV reverse transcriptase, which comprises the following steps:

(1) inoculating the recombinant microorganism of claim 4 to LB medium, culturing to OD ₆₀₀ To 0.4-0.6;

(2) adding an inducer IPTG (isopropyl thiogalactoside) into the bacterial liquid until the final concentration is 0.1-0.25 mmol/L, inducing cells to express proteins, and carrying out high-speed centrifugation to collect thalli sediment, wherein the obtained thalli sediment contains the enhanced M-MLV reverse transcriptase;

(3) adding a lysis buffer solution into the obtained thallus precipitate, carrying out ultrasonic oscillation crushing treatment, centrifuging to remove the precipitate, and filtering to obtain a supernatant;

(4) collecting the sample eluent containing the target protein by nickel ion affinity chromatography of the supernatant, and then carrying out ion exchange chromatography and SUMO protein enzyme digestion to obtain the enhanced M-MLV reverse transcriptase.

The seventh purpose of the invention is to provide the enhanced M-MLV reverse transcriptase or the nucleotide sequence or the recombinant microorganism cell, or the application of the kit in RNA reverse transcription and quantitative detection.

The invention has the beneficial effects that:

(1) the invention optimizes the expression vector of the intensified MMLV reverse transcriptase, selects the pPSUMO vector to improve the soluble expression of the target protein, and greatly facilitates the subsequent protein purification, thereby improving the protein yield and yield.

(2) The enhanced MMLV reverse transcriptase mutant has stronger reverse transcription activity and rapid and continuous synthesis capability, has higher cDNA yield and shorter time compared with the wild type under the same condition, can complete cDNA synthesis within 5min or even shorter time, can be applied to trace RNA virus detection and in-vitro molecular diagnosis, and plays an important role in reducing the dependence on imported products.

(3) The enhanced M-MLV reverse transcriptase provided by the invention can resist a high temperature of 70 ℃, has good thermal stability, high reaction sensitivity and accuracy, and has a very strong application prospect and a market popularization value.

(4) The invention carries out artificial modification on the basis of wild type M-MLV reverse transcriptase to obtain the enhanced M-MLV reverse transcriptase. The reverse transcriptase has strong stress resistance, can well resist the interference of an inhibitor on cDNA synthesis, has no influence on the activity of mutant reverse transcriptase in the sample reaction process of adding ethanol or isopropanol, can normally synthesize cDNA, and is suitable for RNA reverse transcription in various complex environments.

(5) The invention provides a kit method, which is particularly suitable for RT-PCR detection. The whole reaction process of reverse transcription and subsequent fluorescent quantitative PCR reaction is operated in one step, a tube cover does not need to be opened for many times, reagents are not added, possible pollution is avoided, the detection sensitivity is improved, the time is saved, the cost is reduced, and the requirement for research and application in multiple fields is better met.

Drawings

FIG. 1 is a graph showing the effect of comparing the expression of induced proteins using two expression vectors, pET-30a (+) and pPSUMO, in example 1 of the present invention.

FIG. 2 is a graph showing the results of comparing the concentrations of cDNA synthesized by wild-type and enhanced M-MLV reverse transcriptase in example 2 of the present invention.

FIG. 3 is a graph comparing reverse transcription effects of wild-type and enhanced M-MLV at different synthesis times in example 2 of the present invention.

FIG. 4 is a graph comparing reverse transcription effects of wild-type and enhanced M-MLV at different temperatures in example 2 of the present invention.

FIG. 5 is a graph comparing reverse transcription effects of wild-type and enhanced M-MLV at different temperatures for a short period of time in example 2 of the present invention.

FIG. 6 is a graph comparing the reverse transcription effects of wild-type and enhanced M-MLV at different ethanol addition ratios in example 3 of the present invention.

FIG. 7 is a graph comparing the reverse transcription effects of wild-type and enhanced M-MLV at different isopropanol addition ratios in example 3 of the present invention.

FIG. 8 is a graph comparing reverse transcription effects of wild-type and enhanced M-MLV under different initial amounts of RNA added in example 4 of the present invention.

FIG. 9 is a graph showing the amplification curves of the wild-type and enhanced M-MLV reverse transcription qPCR detection of the expression of two genes in example 5 of the present invention.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1: preparation of M-MLV reverse transcription mutants

The method comprises the following steps:

1. construction of enhanced M-MLV reverse transcriptase

The method is characterized in that the method is implemented by carrying out artificial modification on the basis of a wild type M-MLV reverse transcriptase protein sequence (the modified protein sequence is shown as SEQ ID NO. 2), and inserting the modified protein sequence into two expression vectors, namely pET-30a (+) and pPSUMO through NdeI/XhoI and BamHI/EcoRI enzyme cutting sites respectively according to the codon optimized nucleotide sequence (the sequence is shown as SEQ ID NO. 1) of escherichia coli. Mixing the above plasmid with Escherichia coli Rosetta (DE3) competent cells, ice-cooling for 30min, heat-shocking at 42 deg.C for 90 s, and labeling M-MLV respectively; pET-30a (+) and M-MLV; pPSUMO, and screening of the monoclonal strains by LB plate containing kanamycin.

2. Protein induction expression and purification

The single colonies on the plate were inoculated into 10ml of LB liquid medium containing 50. mu.g/ml kanamycin and cultured at 37 ℃ on a shaker at 200rpmConversion to OD ₆₀₀ The value is 0.4-0.6, adding inducer IPTG with final concentration of 0.1-0.25mM, and culturing at 16 deg.C and 70rpm to induce protein expression. And centrifuging after 12 hours, collecting thalli precipitates, and carrying out low-temperature ultrasonic disruption to obtain M-MLV reverse transcriptase crude extracts of two different expression vectors. Crude fluid was separated by SDS-PAGE and stained with Coomassie Brilliant blue R-250 to identify protein bands, as shown in FIG. 1, M-MLV; pET-30a (+) and M-MLV; the target protein can be successfully induced by the pPSUMO two expression vectors under the same condition, wherein M-MLV; the content of target protein in supernatant and sediment after the induction of the pPSUMO expression vector is higher than that of M-MLV; pET-30a (+) is high, the use of pPSUMO vector promotes the soluble expression of protein, more protein is detected in supernatant, and the subsequent protein purification is facilitated while the yield is improved.

3. Protein purification

After the precipitated bacteria are fully crushed by a cell ultrasonication instrument, the precipitated bacteria are subjected to affinity purification by a nickel column, subjected to ion exchange chromatography, then subjected to enzyme digestion by adding SUMO protease, and finally dialyzed and concentrated to obtain purified protein, and the protein is dialyzed and stored in an enzyme storage solution (consisting of 20mM Tris-HCl (pH 7.5), 100mM NaCl, 0.1mM EDTA, 1mM DTT, 0.01% (v/v) NP-40 and 50% (v/v) glycerol) and is stored at the temperature of-80 ℃ for later experiments.

4. Reverse transcription Activity detection

Using 1. mu.g of total mouse liver RNA as a template, reverse transcription was performed using wild-type and purified enhanced M-MLV reverse transcriptase. The reaction system is as follows:

TABLE 1

5 XRT buffer from 250mM Tris-HCl (pH 8.3), 375mM KCl, 15mM MgCl ₂ 50mM DTT.

Reaction procedure: 20min at 37 ℃ and 15sec at 85 ℃. After the reaction is finished, the concentration of the cDNA synthesized by the wild type and the enhanced M-MLV reverse transcriptase is measured and compared by a Nano drop instrument, and the result is shown in figure 2, under the condition that the addition amount of the RNA template is the same, the concentration of the cDNA synthesized by the enhanced M-MLV reverse transcriptase is obviously higher than that of the wild type, which shows that the modified reverse transcriptase improves the capability of continuously synthesizing the cDNA and the utilization rate of the RNA template.

Example 2: enhanced M-MLV reverse transcriptase reverse transcription capability test

The reverse transcription time and temperature were varied to test the reverse transcription ability of the reverse transcriptase under different conditions.

Taking 1 mug of mouse liver total RNA as a template, wherein a reverse transcription system is shown as an example 1, the reinforced M-MLV reverse transcriptase and the wild type reverse transcriptase are adopted to carry out PCR reaction under the same reverse transcription condition of 37 ℃ and 20min by changing reverse transcription time (1min, 5min, 15min, 20min and 30min), the length of a PCR amplification fragment is 154bp, a PCR amplification gene is fibroblast growth factor 21(FGF21), and the used primers are as follows: FGF 21-F: 5'-CTGCTGGGGGTCTACCAAG-3' and FGF 21-F: 5'-CTGCGCCTACCACTGTTCC-3', PCR, 2 xTaq Plus Master Mix (Nanjing Nodezam Biotechnology Co., Ltd., product number P212-01), the reaction system and the program refer to the instruction for preparation and reaction, the PCR amplification product is subjected to agarose gel electrophoresis, the experimental result is shown in figure 3, compared with the wild type, the enhanced M-MLV reverse transcriptase of the invention shows obvious reverse transcription activity within 1min and 5min, the intensity of the PCR product band is high, the modified enhanced reverse transcriptase reaction speed is shown, and the method is better suitable for rapid detection by an RT-PCR method. Then only changing the temperature conditions (37 ℃, 42 ℃, 50 ℃, 55 ℃, 65 ℃ and 70 ℃), the reverse transcription reaction time is 20min, the result is shown in figure 4, the application range of the enhanced M-MLV reverse transcriptase temperature is obviously higher than that of the wild type, the reverse transcription reaction can still be normally carried out at the high temperature of 65 ℃ and 70 ℃, the capability of the wild type for synthesizing cDNA by reverse transcription is obviously reduced along with the increase of the temperature, at the temperature, the secondary structure of the RNA sample is completely opened, the binding capacity of the primer and the sample is improved, the non-specific binding is reduced, the cDNA synthesis efficiency and quality are finally greatly improved, the comparison result shows that the thermal activity and the thermal stability of the modified reverse transcriptase are obviously improved compared with the wild type, the reverse transcriptase is suitable for the reverse transcription reaction of the RNA template with a complex structure, and the cDNA can be efficiently synthesized with high quality at the same time. Meanwhile, the invention also compares the reverse transcription reaction of the enhanced M-MLV reverse transcriptase and the wild type under the conditions of different temperatures (37 ℃, 42 ℃, 50 ℃, 65 ℃ and 70 ℃) and short time (5min), and the result is shown in figure 5.

Example 3: inhibitor tolerance validation of enhanced M-MLV reverse transcriptase mutants

In some cases, some inhibitors may exist in the RNA template to reduce the actual effect of the reverse transcription reaction, in order to detect the tolerance of the enhanced M-MLV reverse transcriptase to the inhibitors, 5-25% (v/v) of ethanol or isopropanol is respectively added into the two groups of reaction systems, the reverse transcription reaction is carried out at 37 ℃ for 20min, the PCR reaction is utilized, and the reaction product is subjected to agarose gel electrophoresis to detect the experimental result, the results are shown in FIGS. 6 and 7, the experimental result shows that the enhanced M-MLV reverse transcriptase has stronger anti-reverse capability than the wild type, the bioactivity of the enhanced M-MLV reverse transcriptase is still not influenced in the presence of ethanol and isopropanol, the interference of the inhibition components on the cDNA synthesis can be resisted, and the yield and the integrity are improved at the same time, can be suitable for RNA reverse transcription reaction under various complex and extreme environments.

Example 4: reaction sensitivity verification of enhanced M-MLV reverse transcriptase mutant

In order to detect the sensitivity of the enhanced M-MLV reverse transcriptase mutant to a sample with low RNA concentration, 1 mu g of RNA template is subjected to gradient dilution to 100ng, 10ng, 1ng and 1pg respectively, a reaction system is subjected to reverse transcription reaction at 37 ℃ as described above, and the result of the experiment is detected by using a PCR reaction and agarose gel electrophoresis method of the reaction product, as shown in figure 8.

Example 5: application of enhanced M-MLV reverse transcriptase in RT-PCR detection

The reverse transcription method is as shown in example 1, and the reagents used in RT-PCR are

qPCR SYBR Green Master Mix (san Biotech Co., Ltd, Shanghai, cat # 11201ES03) the reaction system was as follows:

TABLE 2

The CFX96 Bio-Rad fluorescence quantitative PCR instrument is used for detecting the target gene,

FGF21 F:5’-CTGCTGGGGGTCTACCAAG-3’，

FGF21 R:5'-CTGCGCCTACCACTGTTCC-3’；

β-actin F:5’-GGCTGTATTCCCCTCCATCG-3’，

β-actin R:5'-CCAGTTGGTAACAATGCCATGT-3’。

the two-step reaction conditions were 95 ℃ for 5min, 95 ℃ for 10sec, 60 ℃ for 30sec, and 40 cycles. The results of the detection (Ct values) are shown in the following table:

TABLE 3

The Ct value result and the amplification curve in the table are shown in figure 9, the Ct value of the same gene reinforced type is lower than that of the wild type, and the modified reinforced reverse transcriptase is applied to RT-PCR reaction, so that the detection sensitivity is improved, the whole detection reaction can be completed more quickly, the time cost is saved, the new crown epidemic situation problem can be solved, the rapid detection of a large number of nucleic acid samples can be better applied, and powerful support is provided for the prevention and control screening of the epidemic situation.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> enhanced M-MLV reverse transcriptase mutant and application thereof

<130> BAA220428A

<160> 2

<170> PatentIn version 3.3

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gcgccgctta ttatcccgct gaaggctacg agtactccgg tgagcattaa acaatacccg 180

atgagccagg aggcccgtct tggcatcaag ccgcacattc agcgcctgct ggaccaaggc 240

atcctggtgc cgtgtcagag cccgtggaat accccgttgc tcccggtgaa aaagccgggt 300

acgaacgatt atcgtccggt tcaagatctg cgtgaagtca ataaacgtgt tgaagatatt 360

cacccgaccg ttccgaatcc gtataacctg ttgtcaggct tgccgccgtc ccatcagtgg 420

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ccgttgttcg cgttcgagtg gcgtgatccg gaaatgggca tctctggtca gctgacttgg 540

acccgtttgc cgcagggatt caaaaacagc ccgaccctgt ttgatgaagc tcttcatcgt 600

gacctggcgg attttcgtat ccagcatccg gacctgattc tgttgcaata cgtggatgat 660

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accctgggca acctgggtta ccgcgcatct gcgaagaagg ctcaaatttg ccaaaaacaa 780

gttaaatatc tgggctacct gcttaaagag ggtcaacgct ggctgaccga ggcccgcaaa 840

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actgctggct tttgccgtct gtggattccg ggtttcgcgg agatggcggc tccgctgtat 960

ccgctaacga agaccggtac cctcttcaac tggggtccgg accagcagaa agcctaccaa 1020

gaaatcaagc aggcgctgct cacggcgcct gcactgggtc ttcccgactt gaccaagccg 1080

tttgaactgt ttgttgatga gaagcagggg tacgcaaaag gcgttctgac ccaaaaactg 1140

ggtccgtggc gtcgtccggt ggcgtacttg tccaaaaagc tggacccggt cgcagccggc 1200

tggccgccat gcctgcgcat ggttgctgcc atcgcagtgc tgaccaagga cgccggtaag 1260

ctcaccatgg gtcagccgtt ggtgattctc gcgccgcatg cagttgaagc actggtgaag 1320

caaccgccgt tgtccaacgc acgcatgacc cattaccagg cgttgctgtt ggacaccgat 1380

cgtgtacagt ttggtcccgt cgtcgcgttg aatccggcga cgctgttgcc gttgccagag 1440

gaaggtctgc agctggccga ggcgcacggc acccgtccgg acctgaccga ccaaccgctg 1500

ccagacgccg accatacctg gtacaccgac ggcagcagcc tgctgcaaga aggtcagcgc 1560

aaagcaggcg cggcggtgac caccgagacc gaggtgatct gggcgaaggc gctgccagcg 1620

ggcacgagcg cgcagcgtgc ggagttgatc gctcttaccc aggcactcaa gatggcggag 1680

ggcaaaaaac tgaatgttta taccgatagc cgttatgcct tcgccaccgc tcacatccac 1740

ggtgaaattt atcgccgtcg cggtattaag aacaaagacg agatccttgc gttactgaaa 1800

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gagacaccgg ataccagcac cctgctgtaa 1950

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Gly Ser Met Gly Ser Ser Leu Glu Val Leu Phe Gln Gly Pro Ser Leu

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Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala Trp Ala Glu Thr Gly

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Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu Ile Ile Pro Leu Lys

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Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr Pro Met Ser Gln Glu

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Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg Leu Leu Asp Gln Gly

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Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr Pro Leu Leu Pro Val

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Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val Gln Asp Leu Arg Glu

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Val Asn Lys Arg Val Glu Asp Ile His Pro Thr Val Pro Asn Pro Tyr

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Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln Trp Tyr Thr Val Leu

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Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu His Pro Thr Ser Gln

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Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu Met Gly Ile Ser Gly

165 170 175

Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe Lys Asn Ser Pro Thr

180 185 190

Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala Asp Phe Arg Ile Gln

195 200 205

His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp Asp Leu Leu Leu Ala

210 215 220

Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr Arg Ala Leu Leu Gln

225 230 235 240

Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala Lys Lys Ala Gln Ile

245 250 255

Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu Leu Lys Glu Gly Gln

260 265 270

Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr Val Met Gly Gln Pro Thr

275 280 285

Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu Gly Thr Ala Gly Phe

290 295 300

Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met Ala Ala Pro Leu Tyr

305 310 315 320

Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp Gly Pro Asp Gln Gln

325 330 335

Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu Thr Ala Pro Ala Leu

340 345 350

Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu Phe Val Asp Glu Lys

355 360 365

Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys Leu Gly Pro Trp Arg

370 375 380

Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp Pro Val Ala Ala Gly

385 390 395 400

Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile Ala Val Leu Thr Lys

405 410 415

Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu Val Ile Leu Ala Pro

420 425 430

His Ala Val Glu Ala Leu Val Lys Gln Pro Pro Leu Ser Asn Ala Arg

435 440 445

Met Thr His Tyr Gln Ala Leu Leu Leu Asp Thr Asp Arg Val Gln Phe

450 455 460

Gly Pro Val Val Ala Leu Asn Pro Ala Thr Leu Leu Pro Leu Pro Glu

465 470 475 480

Glu Gly Leu Gln Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr

485 490 495

Asp Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser

500 505 510

Ser Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr

515 520 525

Glu Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala

530 535 540

Gln Arg Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu

545 550 555 560

Gly Lys Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr

565 570 575

Ala His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Ile Lys Asn Lys

580 585 590

Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu Phe Leu Pro Lys Arg Leu

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Ser Ile Ile His Cys Pro Gly His Gln Lys Gly His Ser Ala Glu Ala

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Arg Gly Asn Arg Met Ala Asp Gln Ala Ala Arg Lys Ala Ala Ile Thr

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Glu Thr Pro Asp Thr Ser Thr Leu Leu

645

Claims

1. An enhanced M-MLV reverse transcriptase, which is characterized in that the amino acid sequence of the enhanced M-MLV reverse transcriptase is shown as SEQ ID No. 2.

2. A nucleotide sequence encoding the enhanced M-MLV reverse transcriptase of claim 1.

3. A recombinant expression vector comprising the nucleotide sequence of claim 2.

4. A recombinant microorganism expressing the enhanced M-MLV reverse transcriptase of claim 1 or comprising the recombinant expression vector of claim 3.

5. A reverse transcription kit comprising the enhanced reverse transcriptase of claim 1.

6. The kit of claim 5, wherein the formulation further comprises RNase H ₂ O, reverse transcription reaction buffer solution, dNTPs and reverse transcription reaction primers.

7. A method for synthesizing cDNA by reverse transcription of RNA using the enhanced M-MLV reverse transcriptase of claim 1 or the reverse transcription kit of claim 5 or 6 to produce cDNA.

8. The method of claim 7, wherein the reaction is carried out at 37-70 ℃ for not less than 1min and then the denaturation is carried out at 80-90 ℃ for 10-20 s.

9. The method of claim 8, wherein the concentration of RNA in the reaction system is 5X 10 ^-5 mg/L～50mg/L。

10. Use of the reverse transcriptase of claim 1, the recombinant microorganism of claim 4 or the reverse transcription kit of claim 5 or 6 for reverse transcription of RNA and quantitative detection.