Mutant recombinant reverse transcriptase as well as preparation method and application thereof

Abstract

The invention relates to the field of gene engineering and enzyme engineering, in particular to mutant recombinant reverse transcriptase as well as a preparation method and application thereof. The invention discloses a mutant recombinant reverse transcriptase and a preparation method and application thereof, the mutant recombinant reverse transcriptase comprises an amino acid sequence as shown in any one of SEQ ID NO: 1-6, and the mutant recombinant reverse transcriptase is obtained by artificially synthesizing an expression vector, utilizing a preferred host cell and carrying out IPTG (isopropyl-beta-d-thiogalactoside) induced expression; the activity temperature of the modified recombinant mutant reverse transcriptase is 37-67 DEG C, the reverse transcription efficiency of the modified recombinant mutant reverse transcriptase is 65.2 times that of a wild type reverse transcriptase, and the endurance capacity of the modified recombinant mutant reverse transcriptase to common interferents is improved; the performance of the M-MLV is obviously superior to that of a wild type M-MLV, and the M-MLV shows advantages in comparison with the performance of a marketized M-MLV.

Description

Technical Field

[0001] The invention relates to the fields of genetic engineering and enzyme engineering, and in particular to a mutant recombinant reverse transcriptase and a preparation method and application thereof. Background Art

[0002] Reverse transcriptase is a widely used tool enzyme in molecular biology research and applications. It uses RNA as a template to synthesize DNA and is an important means of RNA analysis and detection. It is widely used in many fields such as gene expression analysis, gene function research, pathogen detection, genetic disease screening, etc.

[0003] Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase is one of the commonly used reverse transcriptases. The enzyme is a polypeptide chain, including an N-terminal polymerization domain and a C-terminal RNaseH domain. However, the enzyme still has many limitations in its application.

[0004] First of all, the suitable reaction temperature of M-MLV reverse transcriptase is 37-42°C. Too low or too high reaction temperature will significantly reduce its reaction efficiency or lose its activity. The biggest challenge in the RNA reverse transcription and cDNA synthesis process is that the RNA double-strand complementary structure is complex. At relatively low temperatures, RNA will form a secondary structure, which will affect the efficiency of transcription and synthesis of cDNA. Generally speaking, the generation of RNA secondary structure can be reduced by increasing the reaction temperature of RNA reverse transcription. However, ordinary M-MLV is difficult to work well at temperatures above 55°C. In addition, the low temperature tolerance also limits its integration with nucleic acid amplification technologies such as cross-primer isothermal amplification.

[0005] Secondly, the reverse transcription efficiency of ordinary M-MLV is still not ideal, and it is difficult to meet the needs of applications such as rapid nucleic acid testing.

[0006] Finally, in the actual application of reverse transcriptase, the interference substances contained in the sample are often affected, and the efficiency of cDNA synthesis is reduced. Ordinary M-MLV has a low tolerance concentration to common interference substances such as ethanol, heparin, and guanidine salts, which limits its application.

[0007] Therefore, optimizing and improving ordinary M-MLV, expanding its applicable reaction temperature range, improving its synthesis efficiency, and optimizing its anti-interference performance are of great significance for molecular biology research and detection, and have broad market prospects. Summary of the invention

[0008] The purpose of the present invention is to provide a recombinant reverse transcriptase and its expression gene sequence that are resistant to high temperatures, interference and have high reverse transcription efficiency; the second purpose of the present invention is to provide its corresponding recombinant vector and host cell; the third purpose of the present invention is to provide a method for preparing the aforementioned reverse transcriptase and its application in RT-PCR or isothermal amplification reactions.

[0009] In a first aspect, the present application provides a mutant recombinant reverse transcriptase:

[0010] A mutant recombinant reverse transcriptase, comprising an amino acid sequence shown in any one of SEQ ID NOs: 1 to 6.

[0011] Furthermore, the amino acid sequences shown in SEQ ID NOs: 1 to 6 are respectively represented as Mut-32 / 302, Mut-72 / 432, Mut-282 / 518, Mut-32 / 282 / 432, Mut-72 / 302 / 518, and M-6m.

[0012] Furthermore, the amino acid sequence of the mutant recombinant reverse transcriptase is selected from any one of SEQ ID NO.4-6.

[0013] Furthermore, the Ct value of the mutant recombinant reverse transcriptase at 55-67°C is less than 19.3.

[0014] The recombinant mutant reverse transcriptase obtained by the present invention has high thermal stability and can synthesize cDNA chains at a maximum temperature of 67° C. Its reverse transcription rate and tolerance to common interferents are also significantly improved compared to wild-type M-MLV, and are superior to mainstream products on the market.

[0015] Among them, the amino acid sequence shown in SEQ ID NO.6 is obtained by performing site-directed mutagenesis at positions 32, 72, 282, 302, 432 and 518 of the wild-type M-MLV reverse transcriptase amino acid sequence, and the mutant reverse transcriptase amino acid sequence is codon optimized to obtain the expression gene sequence.

[0016] In a second aspect, the present application provides a gene fragment encoding the mutant recombinant reverse transcriptase described in the present application.

[0017] In a third aspect, the present application provides a recombinant vector:

[0018] A recombinant vector comprises the gene fragment of the mutant recombinant reverse transcriptase described in the present application.

[0019] Furthermore, the expression vector of the recombinant vector is selected from any one of pET-30a(+), pET-28a, pPICZαA, pHT43, and pFastBac1.

[0020] The recombinant vector comprising the aforementioned gene sequence provided by the present invention comprises a nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO.1-6, such as the amino acid sequence shown in SEQ ID NO.6 corresponding to the nucleotide sequence SEQ ID NO.8. The expression vector of the recombinant vector is selected from any one of pET-30a(+), pET-28a, pPICZαA, pHT43, and pFastBac1 as a vector, preferably pET-30a(+) or pET-28a, which has NdeI and HindIII double restriction sites, good host cell compatibility, and an efficient transcription initiation system.

[0021] In a fourth aspect, the present application provides a host cell:

[0022] A host cell comprising the recombinant vector described in the present application.

[0023] Furthermore, the host cell is derived from any one of Escherichia coli, Pichia pastoris, Bacillus subtilis, and insect cells for transformation.

[0024] The host cell of the present application, including the aforementioned recombinant vector, is obtained by transforming competent cells. The host cell is selected to include but is not limited to any one of Escherichia coli, Pichia pastoris, Bacillus subtilis, and insect cells. Preferably, Escherichia coli BL21 is used as the host cell, which has a higher expression amount under codon optimization design.

[0025] In a fifth aspect, the present application provides a method for preparing a mutant recombinant reverse transcriptase:

[0026] A method for preparing the mutant recombinant reverse transcriptase: on the basis of the wild-type reverse transcriptase shown in SEQ ID NO: 7, a gene fragment encoding the recombinant reverse transcriptase is obtained after site-directed mutagenesis, the gene fragment is connected to an expression vector to obtain a recombinant vector, and the recombinant vector is transferred into a competent host cell to obtain a cloning strain, and the strain is obtained after induction culture, fragmentation, and purification;

[0027] Wherein, the site-directed mutation comprises any one or more combinations of A32G, L72K, E282H, E302H, L432K, and D518A.

[0028] Furthermore, the induction culture conditions are 16-40 degrees Celsius and IPTG concentration is 0.2-5mM.

[0029] The reverse transcriptase of the present invention is obtained by connecting the aforementioned gene sequence to an expression vector having NdeI and HindIII double restriction sites to obtain a recombinant vector; the recombinant vector is transferred into a competent host cell to obtain a cloning strain, which is obtained after induction culture, fragmentation and purification.

[0030] The induction culture conditions are 16-40°C and IPTG concentration is 0.2-5mM. The crushing process is selected from a method including but not limited to ultrasonic crushing, and the crude enzyme solution after crushing and centrifugal filtration is purified by a process including but not limited to nickel ion affinity chromatography column elution, and the purified enzyme solution is collected and stored. During elution, Tris-HCl buffer with different imidazole concentrations is used for elution, and the enzyme solution is collected by selecting an eluent with higher purity.

[0031] Further, the site-directed mutagenesis is any of the following:

[0032] A32G, E282H, L432K;

[0033] L72K, E302H, D518;

[0034] A32G, L72K, E282H, E302H, L432K, D518A.

[0035] In a sixth aspect, the present application provides an application of a mutant recombinant reverse transcriptase in RT-PCR or cross-primer isothermal amplification.

[0036] Beneficial effects:

[0037] The present invention discloses a mutant recombinant reverse transcriptase and a preparation method and application thereof. The mutant recombinant reverse transcriptase comprises an amino acid sequence shown in any one of SEQ ID NOs: 1 to 6. The enzyme is obtained by artificially synthesizing an expression vector, utilizing a preferred host cell, and inducing expression through IPTG. The activity temperature of the modified recombinant mutant reverse transcriptase is 37-67°C, the reverse transcription efficiency is 65.2 times that of the wild-type reverse transcriptase, and the tolerance to common interferents is improved. The performance thereof is significantly better than that of the wild-type M-MLV, and the performance thereof is superior to that of the commercialized M-MLV. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a data table showing the efficiency of reverse transcription reactions in each test group in Example 4 of the present invention;

[0039] Figure 2 is a bar graph of the reverse transcription reaction efficiency of each test group in Example 4 of the present invention;

[0040] Figure 3 This is the amplification curve of the cross primer isothermal amplification fluorescence quantitative PCR in Example 6 of the present invention. The horizontal axis represents the number of amplification cycles, and the vertical axis represents the fluorescence intensity. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0042] Example 1

[0043] The mutant recombinant reverse transcriptase (M-6m) provided in this embodiment has an amino acid sequence shown in SEQ ID NO.6, which is based on the amino acid sequence of wild-type Moloney murine leukemia virus reverse transcriptase M-MLV (GenBank: AAC82568.2) and site-directed mutagenesis (A32G, L72K, E282H, E302H, L432K, D518A), and the nucleotide sequence of its expressed gene is obtained after codon optimization, and the nucleotide sequence is shown in SEQ ID NO.8. The preparation method of the mutant recombinant reverse transcriptase provided in this embodiment comprises the following steps:

[0044] (1) The vector pET-30a(+) double-digested with NdeI and HindIII was ligated with SEQ ID NO. 8 using T4 DNA ligase at a reaction temperature of 16°C overnight to obtain a recombinant vector.

[0045] (2) The recombinant vector was mixed with competent E. coli BL21 (DE3) cells, placed on ice for 30 minutes, heat-shocked at 42°C for 60 seconds, placed on ice for 5 minutes, added with 500 μl of liquid LB medium, cultured at 37°C for 45 minutes, spread on a plate containing kanamycin, and cultured at 37°C overnight.

[0046] (3) Select positive clones and culture them in liquid culture medium overnight. Screen positive clones by bacterial liquid PCR and sequencing. Inoculate the screened positive clone strains into liquid LB culture medium and culture at 37°C and 200 rpm. When the culture medium OD 600 When the value reaches 0.6, IPTG is added to a final concentration of 1 mM for induction, and the induction is carried out overnight at 20°C. After the induction, the cells are collected by centrifugation.

[0047] (4) Weigh the collected bacteria, resuspend the bacteria at a ratio of wet weight of bacteria: lysis buffer = 1g: 20ml, and use an ultrasonic cell crusher to crush the bacterial suspension, with a power of 500W and a working time of 2 seconds and a rest time of 4 seconds. After crushing, centrifuge at 13000rpm for 30min and collect the supernatant. Place the supernatant in a 60℃ warm bath for 30min, then centrifuge at 13000rpm for 30min, collect the supernatant, and filter the supernatant with a 0.22μm filter membrane to obtain a crude enzyme solution.

[0048] (5) The crude enzyme solution after filtration is passed through a nickel affinity chromatography column, and the impurities are washed and the target protein is eluted with buffers of different imidazole concentrations. The amount and purity of the target protein in the collected solution are identified by SDS-PAGE gel, and the eluate with higher purity is selected for collection. The enzyme solution containing the target protein is placed in a 30KD dialysis bag and dialyzed overnight. The enzyme solution in the dialysis bag is filtered with a 0.22μm filter membrane and the concentration is determined.

[0049] (6) Determine the enzyme activity unit. The specific method is to determine the amount of enzyme required to incorporate 1 nmol of dTTP into the acidic precipitable material using Poly(A)-Oligo(dT) as a template / primer at 37°C within 10 minutes. This amount of enzyme is one activity unit (U).

[0050] (7) After measuring the enzyme activity units, the enzyme solution was diluted to 200 U / μl using reverse transcriptase storage solution. The components of the storage solution are: 50 mM Tris / HCl (pH 8.5), 300 mM NaCl, 0.1 mM EDTA, 50 v / v% Glycerol, 0.1 v / v% Tween 20, 0.3 M trehalose.

[0051] Example 2

[0052] This example compares the heat resistance of reverse transcriptases with different mutation types. Human β-actin gene mRNA was used as a template, and the human β-actin gene fragment was amplified by fluorescent quantitative PCR. The heat resistance of 13 reverse transcriptases was compared by detecting the difference in the Ct value of the signal. It includes wild-type M-MLV reverse transcriptase (WT), six reverse transcriptases with single-point mutations based on wild-type M-MLV (Mut-32, Mut-72, Mut-282, Mut-302, Mut-432, Mut-518, and the corresponding mutation sites are: A32G, L72K, E282H, E302H, L432K, D518A), and three reverse transcriptases with double-point mutations based on wild-type M-MLV (Mut-32 / 302, Mut-72 / 4 The reverse transcriptase (M-6m) provided in Example 1 was prepared by using the following methods:

[0053] The cDNA synthesis reaction system is:

[0054]

[0055] The reverse transcription reaction was carried out according to the above system, and the reaction was carried out at 37°C, 55°C, 61°C, and 67°C for 15 minutes, and then inactivated at 75°C for 5 minutes. The reverse transcription product was taken and subjected to fluorescence quantitative PCR detection according to the following system.

[0056] β-actin primers were designed as follows:

[0057] FB:5'-AGAAGGCTGGGGCTCATTTG-3'

[0058] RB:5'-AGGGGCCATCCATCCACAGTCTTC-3'

[0059] The fluorescence quantitative PCR reaction system is:

[0060]

[0061] The fluorescence quantitative PCR reaction procedure is step 1: 95°C, 3 min; step 2: 95°C, 15 seconds; 55°C, 15 seconds; 72°C, read the fluorescence signal; 30 cycles. The experimental instrument is BIO-RADCFX96.

[0062] As shown in Table 1, after reverse transcription reaction at different temperatures, the amplification reaction results of the 12 mutant enzyme groups in this embodiment were better than those of the wild type, indicating that the heat resistance of the mutant reverse transcriptases was improved to a certain extent compared with the wild type (WT). Among them, the M-6m mutant reverse transcriptase had the best experimental results, and the Ct value corresponding to the enzyme was the smallest at 67°C, indicating that the M-6m mutant reverse transcriptase had the strongest heat resistance.

[0063] Table 1. List of the results of the heat resistance test of M-MLV reverse transcriptase mutants

[0064]

[0065] Example 3

[0066] This experiment compares the heat resistance of the mutant recombinant reverse transcriptase described in Example 1 with that of the commercially available general reverse transcriptase. The wild-type M-MLV reverse transcriptase has poor thermal stability, weak ability to resist RNA with complex structures, and unsatisfactory reverse transcription efficiency. Control A is the M-MuLV reverse transcriptase produced by Company A, and Control B is the heat-resistant reverse transcriptase produced by Company B. Both Control A and Control B are new reverse transcriptases lacking RNaseH activity obtained by genetic engineering technology, which are different from the amino acid sequences of the mutant recombinant reverse transcriptase described in Example 1. Therefore, there may be differences in the thermal stability of the three enzymes. Four reverse transcriptases (wild type, control A, control B, M-6m) were evaluated using human β-actin gene mRNA as a template to synthesize cDNA by reverse transcription at 37°C, 55°C, 61°C, and 67°C, respectively.

[0067] The RT-PCR reaction system is:

[0068]

[0069] The reverse transcription reaction was carried out according to the above system, and the reaction was carried out at 37°C, 55°C, 61°C, and 67°C for 15 minutes, and then inactivated at 75°C for 5 minutes. The reverse transcription product was taken and subjected to fluorescence quantitative PCR detection according to the system described below.

[0070] β-actin primers were designed as follows:

[0071] FB:5'-AGAAGGCTGGGGCTCATTTG-3'

[0072] RB:5'-AGGGGCCATCCATCCACAGTCTTC-3'

[0073] The fluorescence quantitative PCR reaction system is:

[0074]

[0075] The fluorescence quantitative PCR reaction procedure is step 1: 95℃, 3 minutes; step 2: 95℃, 15 seconds; 55℃, 15 seconds; 72℃, read the fluorescence signal; 30 cycles. The experimental instrument is BIO-RADCFX96.

[0076] As shown in Table 2, under different temperature conditions, no product was detected for the wild-type reverse transcriptase at 61°C and 67°C. The Ct value of the M-6m mutant reverse transcriptase provided in Example 1 of the present invention was the smallest when tested at the highest temperature of 67°C and the higher temperature of 61°C, indicating that M-6m has better heat resistance than control group A and control group B (control A is the M-MuLV reverse transcriptase produced by Company A, and control B is the heat-resistant reverse transcriptase of Company B).

[0077] Table 2. List of heat resistance test results of mutant and wild-type reverse transcriptases and commercially available universal reverse transcriptases

[0078] Group 37℃ / Ct mean 55℃ / Ct mean 61℃ / Ct mean 67℃ / Ct mean WT 17.32 18.41 Not detected Not detected Comparison A 16.31 16.89 18.95 25.62 Control B 15.23 14.78 16.36 26.23 M-6m 14.45 12.31 12.54 15.91

[0079] Example 4

[0080] This example compares the reverse transcription synthesis efficiency of the mutant recombinant reverse transcriptase provided in Example 1 and the commercially available universal reverse transcriptase. Fluorescence quantitative PCR was used to amplify the human GAPDH gene and compare four reverse transcriptases, including the wild-type reverse transcriptase, the control A, which is the M-MuLV reverse transcriptase produced by Company A, the control B, which is the thermostable reverse transcriptase of Company B, and the reverse transcriptase (M-6m) provided in Example 1.

[0081] The RT-PCR reaction system is:

[0082]

[0083]

[0084] The reverse transcription reaction was carried out according to the above system, reacted at 55°C for 15 minutes, and then inactivated at 75°C for 5 minutes. The reverse transcription product was then taken for fluorescence quantitative PCR detection according to the following system.

[0085] The primers for human GAPDH gene were designed as follows: FB: 5'-CCTGCACCACCAACTGCTTAG-3'

[0086] RB:5'-AGGGGCCATCCACAGTCTTC-3'

[0087] The fluorescence quantitative PCR reaction system is:

[0088]

[0089] The fluorescence quantitative PCR reaction procedure is step 1: 95℃, 3 minutes; step 2: 95℃, 15 seconds; 55℃, 15 seconds; 72℃, read the fluorescence signal; 30 cycles. The experimental instrument is BIO-RADCFX96.

[0090] The wild-type reverse transcriptase amplification efficiency of 100% was used as a control to calculate the reaction efficiency of other reverse transcriptases. The calculation formula is as follows:

[0091] Reverse transcriptase reaction efficiency = 100% x 2 (A-B)

[0092] Among them, A is the Ct value of wild-type reverse transcriptase, and B is the Ct value of reverse transcriptase in the control group.

[0093] like Figure 1 and Figure 2 ( Figure 2 according to Figure 1 The experimental results of this example show that after the reaction at 55°C, the amplification efficiency of GAPDH by the wild-type reverse transcriptase is significantly lower than that of the enzyme produced by other companies. The amplification efficiency of M-6m reverse transcriptase is 65.2 times that of the wild-type reverse transcriptase, and is significantly higher than that of the reverse transcriptases in other groups.

[0094] Example 5

[0095] This example compares the anti-interference capabilities of different reverse transcriptases, and the method of the interferor tolerance test is as follows:

[0096] Interferors were added to the reverse transcription reaction system, and β-actin was amplified by fluorescent quantitative PCR using the cDNA obtained by reverse transcription. The obtained Ct value was calculated to obtain the half-inhibitory concentration (IC50) of each reverse transcriptase against the interferor, and the ability of each reverse transcriptase to resist the interferor was further obtained.

[0097] The RT-PCR reaction system is:

[0098]

[0099]

[0100] The reverse transcription reaction was carried out according to the above system, reacted at 37°C for 15 minutes, and then inactivated at 75°C for 5 minutes. The reverse transcription product was taken for fluorescence quantitative PCR detection.

[0101] Fluorescence quantitative PCR is as follows:

[0102] β-actin primers:

[0103] FB:5'-AGAAGGCTGGGGCTCATTTG-3'

[0104] RB:5'-AGGGGCCATCCATCCACAGTCTTC-3'

[0105] The fluorescence quantitative PCR reaction system is:

[0106]

[0107] The reaction procedure of fluorescence quantitative PCR was as follows: step 1: 95°C, 3 minutes; step 2: 95°C, 15 seconds; 5°C, 15 seconds; 72°C, read the fluorescence signal; 30 cycles. The experimental instrument was BIO-RADCFX96.

[0108] As shown in Table 3, the tolerance (IC50) of the M-6m mutant reverse transcriptase provided in Example 1 to different interfering substances is superior to that of the wild-type reverse transcriptase and other commercially available universal reverse transcriptases.

[0109] Table 3. List of test results of reverse transcriptase tolerance to different interfering substances

[0110]

[0111] Example 6

[0112] This example verifies the efficiency of each reverse transcriptase in cross-primer constant temperature amplification (CPA), compares the commercially available universal reverse transcriptase with the reverse transcriptase provided in Example 1, and detects the human β-actin gene using constant temperature amplification. The test includes the reverse transcriptase provided in Example 1 combined with the CPA amplification system (M-6m), and the control groups are: wild-type reverse transcriptase combined with the CPA amplification system, the M-MuLV reverse transcriptase CPA amplification system produced by Company A (control A), and the heat-resistant reverse transcriptase CPA amplification system produced by Company B (control B).

[0113] The CPA reaction assay uses a fixed copy number (1000 copies / test) of RNA as template.

[0114] CPA human β-actin gene primers:

[0115] Forward peripheral primer FB: 5'-AGTACCCCATCGAGCACG-3'

[0116] Reverse peripheral primer RB: 5'-AGCCTGGATAGCAACGTACA-3'

[0117] Forward cross-amplification primer CPF: 5'-GAGCCACACGCAGCTCATTGTATCACCAACTGGGAC GACA-3'

[0118] Reverse cross-amplification primer CPR: 5'-CTGAACCCCAAGGCCAACCGGCTGGGGTGTTGAAG GTC-3'

[0119] Enhanced primer IP1: 5'-GAGTGTGGGTGTTCCCTTTGTACGGGCCCG-3'

[0120] Detection probe IP2: 5'-(FAM)-GCGTCGGCCTACCCTCGTCCTAACACGGGAGCCTGCACTGACCCGACGC-(BHQ1)-3'

[0121] The CPA reaction system is as follows:

[0122]

[0123] Four reverse transcriptase samples were subjected to fluorescence amplification reaction according to the above system, with the reaction conditions being 58°C, 48 seconds per cycle, and 60 cycles in total. The experimental instrument was BIO-RADCFX96.

[0124] like Figure 3 As shown, M-6m reverse transcriptase corresponds to curve I, control A corresponds to curve III, control B corresponds to curve II, and wild type corresponds to curve IV. The fluorescence quantitative results of the CPA experiment in this example show that the M-6m reverse transcriptase detects the amplification product after about 12 cycles, and the amplification effect on β-actin is significantly stronger than that of other groups.

[0125] The present invention obtains a mutated recombinant reverse transcriptase with high temperature resistance and high reverse transcription efficiency by site-directed mutagenesis, and can be actually applied to clinical detection. Using the M-6m reverse transcriptase provided by the present invention and combining it with the CPA constant temperature amplification technology, the reverse transcription reaction and the amplification reaction can be completed in the same reaction system.

[0126] Table 4. List of reverse transcriptase CPA test results

[0127] Group curve Ct value WT IV N / A Comparison A III 19.11 Control B II 16.02 M-6m I 12.20

Claims

1. A mutant recombinant reverse transcriptase, characterized in that: The mutant recombinant reverse transcriptase comprises an amino acid sequence shown in any one of SEQ ID NOs: 1-6.

2. A mutant recombinant reverse transcriptase according to claim 1, characterized in that: The amino acid sequence of the mutant recombinant reverse transcriptase is selected from any one of SEQ ID NO.4-6.

3. A mutant recombinant reverse transcriptase according to claim 1 or 2, characterized in that: The Ct value of the mutant recombinant reverse transcriptase at 55-67° C. is less than 19.

3.

4. A gene fragment encoding a mutant recombinant reverse transcriptase as claimed in claim 1 or 2.

5. A recombinant vector, characterized in that: Comprising the gene fragment according to claim 4.

6. A host cell, characterized in that Comprising the recombinant vector according to claim 5.

7. A host cell according to claim 6, characterized in that The host cell is any one of Escherichia coli, Pichia pastoris, Bacillus subtilis, and insect cells for transformation.

8. A method for preparing a mutant recombinant reverse transcriptase according to any one of claims 1 to 3, characterized in that: Based on the wild-type reverse transcriptase shown in SEQ ID NO: 7, a gene fragment encoding a recombinant reverse transcriptase is obtained after site-directed mutagenesis, the gene fragment is connected to an expression vector to obtain a recombinant vector, and the recombinant vector is transferred into a competent host cell to obtain a cloning strain, which is obtained after induction culture, fragmentation, and purification; Wherein, the site-directed mutation comprises any one or more combinations of A32G, L72K, E282H, E302H, L432K, and D518A.

9. The method for preparing a mutant recombinant reverse transcriptase according to claim 8, characterized in that: The site-directed mutation is any of the following: A32G, E282H, L432K; L72K, E302H, D518; A32G, L72K, E282H, E302H, L432K, D518A.

10. Use of a mutant recombinant reverse transcriptase according to any one of claims 1 to 3 in RT-PCR or cross primer isothermal amplification.

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