Mutant reverse transcriptase and its application

Abstract

The present invention belongs to the field of molecular biology technology, and specifically discloses a mutant reverse transcriptase and its application. The present invention improves the heat resistance of the enzyme and the tolerance to mismatched primers by introducing the E233R mutation in the DNA polymerase active domain of the wild-type MMLV reverse transcriptase. At the same time, by introducing the H204R mutation and the D524G mutation in the RNase H active domain, the thermostability of the enzyme is further improved, and the RNase H activity is reduced or removed. Experiments show that the reverse transcriptase mutant M4 has a reverse transcription activity at 50-55°C and tolerance to mismatched primers (mismatch at the 1st or 3rd position of the 3' end) that is significantly better than other mutants and commercial enzymes, and is suitable for one-step RT-PCR detection, which is conducive to improving the accuracy, sensitivity and specificity of the detection, and has a wide range of application prospects in the fields of molecular diagnosis and precision medicine testing.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a mutant reverse transcriptase and an application thereof. Background Art

[0002] MMLV reverse transcriptase is composed of 671 amino acids and is a functional monomer. It consists of four subdomains (finger domain, palm domain, thumb domain and connecting domain) and ribonuclease H (RNase H) active domain. The finger domain consists of amino acids 41-124 and 160-192, and the palm domain consists of amino acids 1-40, 125-159 and 193-275. In other words, amino acids 1-275 together constitute the finger / palm domain. The thumb domain, connecting domain and RNase H active domain are composed of amino acids 276-361, 362-496 and 497-671, respectively. Studies have shown that the finger domain, palm domain and thumb domain located at the N-terminus constitute the RNA- and DNA-dependent DNA polymerase active domain, in which the palm domain contains the catalytic residues required for ribonucleotide or deoxyribonucleotide transfer and forms a template-primer binding site, the finger domain and thumb domain contain residues that interact with the template-primer complex and deoxyribonucleotides, forming the side of the nucleic acid binding site; the RNase H active domain located at the C-terminus specifically degrades the RNA chain in the RNA-DNA hybrid chain.

[0003] Among the many enzymatic properties of MMLV reverse transcriptase, thermal stability is an important characteristic that affects its application. In the process of synthesizing cDNA using RNA as a template, increasing the reaction temperature can reduce the secondary structure of RNA and the nonspecific binding of primers, but the optimal reaction temperature of MMLV enzyme is 37°C, so it is necessary to improve the thermal stability of the enzyme. At the same time, the inhibitor tolerance of the wild-type MMLV enzyme is low. When 42.9mM guanidine salt, 2.6mM EDTA, 8.7% v / v formamide, 13.1% v / v ethanol, 0.035% w / v xylan or 0.27% w / v pectin are present in the system, the activity of MMLV enzyme decreases by 50%, and improving inhibitor tolerance is crucial for establishing an RT-PCR direct amplification system. To solve the above problems, Chinese patent CN113817707B (Beijing Union, Hangzhou Yousida) provides a recombinant mutant reverse transcriptase, which is obtained by site-directed mutation of the amino acid sequence of the wild-type MMLV reverse transcriptase and codon optimization. The site-directed mutation is: the 67th amino acid of the wild-type MMLV enzyme is mutated from S to T, the 303rd amino acid is mutated from F to R, the 312th amino acid is mutated from L to S, the 432nd amino acid is mutated from L to G, the 479th amino acid is mutated from N to F, and the 524th amino acid is mutated from D to Q. The resulting mutant enzyme has high thermal stability and can synthesize cDNA chains at a maximum temperature of 65°C, and the reverse transcription activity and tolerance to common inhibitors are significantly improved. Chinese patent CN114480329B (Guangzhou Daan Gene) discloses a MMLV enzyme mutant that is resistant to high temperatures and has high reverse transcription efficiency. Compared with the wild-type MMLV enzyme, the 313th amino acid is mutated to Q, the 583rd amino acid is mutated to N, and the 446th amino acid is mutated to C. Chinese patent CN110291196B (Takara) also discloses a thermostable reverse transcriptase mutant, which stabilizes the cyclic stereostructure in the sequence by replacing the 55th amino acid of the wild-type MMLV enzyme (glycine replaces threonine), thereby improving the heat resistance of the enzyme. However, the above patents do not focus on the mismatch tolerance of the MMLV enzyme, including reverse transcriptase products on the market such as Invitrogen's SuperScript series, NEB's M-MuLV, Qiagen's Omniscript and Sensiscript, Agilent's Thermostable M-MLV-RT, etc., which do not involve the improvement of enzyme mismatch tolerance. However, pathogenic microorganisms such as viruses and bacteria are of various types and mutate rapidly, and mismatches between the target and the designed primer are prone to occur during detection, resulting in inaccurate test results and false negatives or false positives. At the same time, the RNase H activity of the MMLV reverse transcriptase will cut the RNA template in the RNA:cDNA hybrid chain produced by cDNA synthesis, thereby reducing the yield of cDNA. Therefore, improving enzyme performance requires reducing or eliminating RNase H activity to prevent template degradation before full-length reverse transcription is completed.

[0004] In summary, it is necessary to develop a new type of reverse transcriptase that improves the thermal stability of the enzyme, reduces or eliminates RNase H activity, and improves the mismatch tolerance of MMLV reverse transcriptase. Summary of the invention

[0005] The main technical problem solved by the present invention is to provide a mutant reverse transcriptase, improve the heat resistance of the enzyme and the tolerance to primer mismatch, and improve the accuracy, sensitivity and specificity in the detection of pathogenic microorganisms.

[0006] Secondly, the present invention provides a gene encoding a mutant reverse transcriptase.

[0007] Thirdly, the present invention provides a biological material comprising a mutant reverse transcriptase or a gene encoding the mutant reverse transcriptase.

[0008] Finally, the present invention provides a mutant reverse transcriptase or its encoding gene, biological material for use in preparing reverse transcription reagents, conventional RT-PCR detection reagents, multiplex RT-PCR detection reagents, RNA sequencing library construction reagents, RNA sequencing universal reagents, etc.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] A mutant MMLV reverse transcriptase, compared with the amino acid sequence of a wild-type MMLV reverse transcriptase (as shown in SEQ ID NO: 1), comprises the following mutations:

[0011] Mutation 1: E233R, i.e., glutamic acid at position 233 mutates to serine.

[0012] As a preferred embodiment of the present invention, the mutant MMLV reverse transcriptase has a sequence homology of more than 90% with the wild-type MMLV reverse transcriptase, and more preferably has a sequence homology of more than 95%, more than 96%, more than 97%, more than 98% or more than 99%.

[0013] As a preferred embodiment of the present invention, the mutant MMLV reverse transcriptase comprises mutation 1 compared with the amino acid sequence of the wild-type MMLV reverse transcriptase, and further comprises one or two of the following mutations:

[0014] Mutation 2: D524G, i.e., aspartic acid at position 524 mutated to glycine;

[0015] Mutation 3: H204R, i.e. the histidine at position 204 mutates to arginine.

[0016] As a preferred embodiment of the present invention, the mutant MMLV reverse transcriptase can also be fused with Sso7d, PCNA, or with a protein tag (such as His, GST) to obtain a fusion protein with improved activity.

[0017] A gene encoding a mutant MMLV reverse transcriptase.

[0018] As a preferred embodiment of the present invention, without changing the amino acid sequence of the encoded mutant MMLV reverse transcriptase, the sequence can be optimized according to the codon preference of the host cell.

[0019] A biological material comprising a mutant MMLV reverse transcriptase or a gene encoding the mutant MMLV reverse transcriptase.

[0020] As a preferred embodiment of the present invention, the biological material includes but is not limited to a recombinant expression vector (such as a plasmid), a gene expression cassette, a recombinant bacterium, and the like.

[0021] As a preferred embodiment of the present invention, the method for preparing the recombinant expression vector comprises: inserting the coding gene of the mutant MMLV reverse transcriptase into the expression vector to obtain the recombinant expression vector.

[0022] As a preferred embodiment of the present invention, the method for preparing the recombinant bacteria comprises: transforming a recombinant expression vector containing a gene encoding a mutant MMLV reverse transcriptase into a host bacterium (such as Escherichia coli) to obtain the recombinant bacteria.

[0023] Specifically, the preparation method of the recombinant bacteria comprises the following steps: transforming the recombinant expression vector into Escherichia coli, screening positive clones after culturing for fermentation, specifically inoculating the colony into a fermentation medium (LB or 2×YT medium), culturing at 37° C. until OD600 is 0.6-0.8, adding IPTG to induce expression and culturing at 16° C. overnight; collecting the bacterial cells and crushing them (physical crushing methods such as ultrasonic crushing, French press crushing, glass bead crushing, etc. can be used, or lysozyme can be used for lysis), adding PEI to the clear liquid to remove nucleic acids, then adding ammonium sulfate to precipitate proteins, and obtaining a crude enzyme solution through column purification and dialysis.

[0024] A use of a mutant MMLV reverse transcriptase or a gene encoding it, or a biological material, the use comprising one or more of the following:

[0025] a) Application in reverse transcription synthesis of cDNA;

[0026] b) Application in conventional RT-PCR detection;

[0027] c) Application in multiplex RT-PCR detection;

[0028] d) Application in RNA sequencing library construction;

[0029] e) Application in RNA sequencing;

[0030] Or, include one or more of the following:

[0031] 1) Application in the preparation of reverse transcription reagents;

[0032] 2) Application in the preparation of conventional RT-PCR detection reagents;

[0033] 3) Application in the preparation of multiplex RT-PCR detection reagents;

[0034] 4) Application in the preparation of RNA sequencing library construction reagents;

[0035] 5) Application in the preparation of universal reagents for RNA sequencing.

[0036] A reverse transcription reagent, a conventional RT-PCR detection reagent, a multiplex RT-PCR detection reagent, an RNA sequencing library construction reagent or an RNA sequencing universal reagent comprises at least one container containing a mutant MMLV reverse transcriptase.

[0037] As a preferred embodiment of the present invention, the mutant MMLV reverse transcriptase and the reaction buffer together constitute a reaction premix, and when used, it is only necessary to mix the reaction premix with primers, RNA template, etc. to prepare a reverse transcription reaction system.

[0038] As a preferred embodiment of the present invention, the reagent further comprises another container containing a reaction buffer, that is, the mutant MMLV reverse transcriptase and the reaction buffer are provided separately and taken separately when used and prepared into a reverse transcription reaction system.

[0039] As a preferred embodiment of the present invention, the reaction buffer may contain the necessary components required for the reverse transcription reaction, including but not limited to dNTPs, Mg 2+ One or more of activators, buffer substances, RNase inhibitors, cosolvents (such as betaine, DMSO), additives (such as DTT, KCl, non-ionic surfactants), etc.

[0040] Specifically, the necessary components in the reaction buffer are as follows:

[0041] (1)Mg 2+ Activators, such as MgCl2, MgSO4;

[0042] (2) Buffer substances, such as Tris-HCl, Tris-H2SO4;

[0043] (3) cosolvents, such as formamide, dimethyl sulfoxide (DMSO), ethylene glycol, 1,2-propylene glycol, and polyethylene glycol (PEG);

[0044] (4) Additives, such as NaCl, KCl, EDTA, betaine, tetramethylammonium chloride (TMAC), ammonium chloride, ammonium sulfate, glycerol, bovine serum albumin (BSA), gelatin, dithiothreitol (DTT), cationic or non-ionic detergents (such as Triton X-100, NP-40).

[0045] As a preferred embodiment of the present invention, the primers can be designed according to the target (specificity), or oligo(dT) or random primers can be used, the latter of which can bind to all mRNAs.

[0046] Specifically, in the application of preparing RNA sequencing library construction reagents, the primer is preferably oligo(dT) or a random primer (such as a random hexa-oligonucleotide primer) for synthesizing the first strand of cDNA, and the reverse transcription reaction system is preferably prepared at room temperature.

[0047] Beneficial effects of the present invention:

[0048] The mutant MMLV reverse transcriptase provided by the present invention improves the heat resistance of the enzyme and the tolerance to mismatched primers by introducing the E233R mutation in the DNA polymerase active domain of the wild-type MMLV reverse transcriptase. Further introduction of the H204R mutation or introduction of the D524G mutation in the RNase H active domain is more conducive to improving the thermal stability of the enzyme and reducing or removing the RNase H activity. The experimental results show that the reverse transcriptase mutants M7 (H204R, E233R) and M4 (H204R, E233R, D524G) are significantly better than other mutants and commercial enzymes in reverse transcription activity at 50-55°C and tolerance to mismatched primers (especially mismatches at the 1st or 3rd position of the 3' end of the primer), are suitable for one-step RT-PCR detection, and have broad application prospects in the fields of molecular diagnosis and precision medicine testing.

[0049] The present invention also provides the use of mutant MMLV reverse transcriptase or its encoding gene and biological materials in the preparation of reverse transcription reagents, conventional RT-PCR detection reagents, multiple RT-PCR detection reagents, RNA sequencing library construction reagents, and RNA sequencing universal reagents, so as to improve the accuracy, sensitivity and specificity of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is the SDS-PAGE result of the crude enzyme solution in the example.

[0051] In order to more clearly illustrate the technical solution to be protected by the present invention, the above drawings obtained in the experimental examples are briefly introduced. It should be understood that the above drawings only show the technical solutions and technical effects of certain experimental examples of the present invention, and should not be regarded as any limitation on the protection scope of the present invention. For ordinary technicians in this field, other related drawings can also be obtained based on these drawings without paying creative work. DETAILED DESCRIPTION

[0052] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments and experimental examples. Those skilled in the art should understand that the following embodiments and experimental examples are only used to illustrate the technical solution of the present invention and should not be regarded as any limitation on the scope of protection of the claims. Based on the following embodiments and experimental examples, other technical solutions obtained by ordinary technicians in the field without paying creative work, such as technical solutions obtained after modification, denaturation or simple replacement, should all belong to the scope of protection of the present invention. In the absence of conflict, the technical features in different embodiments can be combined and replaced with each other.

[0053] The conventional methods of molecular biology and nucleic acid chemistry used in the following embodiments and experimental examples are all prior arts and are described in detail in the literature, see Molecular Cloning Laboratory Guide, Cold Spring Harbor Laboratory Press; Oligonucleotide Synthesis, MJ Gait et al., 1984; Nudeic Acid Hybridization, BD Hames and SJ Higgins, 1984; Basic Methods in Molecular Biology, Elsevier; Current Protocols in Molecular Biology, John Wiley and Sons; Methods in Enzymology, Academic Press, USA; the raw materials (including biological materials), reagents, culture media, instruments, etc. used are all commonly used in the art, easily available to the public or available through commercial channels unless otherwise specified; the terms and abbreviations involved have the conventional meanings in the art, such as PBS is phosphate buffered saline.

[0054] The term "gene" refers to a DNA sequence containing the control and coding sequences necessary for the production of a recoverable biologically active polypeptide or precursor.

[0055] The term "wild type" refers to a gene or gene product isolated from a natural source. The term also refers to a recombinant form of a natural protein prepared by molecular biology techniques, whose amino acid sequence is identical to that of the natural protein.

[0056] The term "mutant" refers to a gene whose nucleotide sequence has been altered or a gene product whose amino acid sequence has been altered, resulting in a gene product having functional properties different from those of the native or wild-type gene or gene product.

[0057] The term "mutation" refers to a single amino acid substitution. A point mutation is introduced into an amino acid sequence by changing the appropriate codon in the encoding DNA. The amino acid position is numbered according to the full-length sequence of the protein. Nucleotides and point mutations in DNA sequences are represented similarly.

[0058] Example 1

[0059] This embodiment provides a mutant MMLV reverse transcriptase (amino acid sequence as shown in SEQ ID NO: 3), which comprises the following mutations compared to the amino acid sequence of the wild-type MMLV reverse transcriptase (as shown in SEQ ID NO: 1):

[0060] Mutation 1: E233R, i.e., glutamic acid at position 233 mutates to serine.

[0061] The method for preparing the mutant MMLV reverse transcriptase provided in this embodiment comprises the following steps:

[0062] 1. Construction of mutant strains

[0063] (1) Circle expansion PCR

[0064] The coding gene of wild-type MMLV reverse transcriptase (as shown in SEQ ID NO: 2) was commissioned to be synthesized by Bio-Industry, and constructed on the pET-30a plasmid. The plasmid was used as a template, and the primer pairs shown in Table 5 were used for expansion PCR amplification. The amplification reaction system was shown in Table 1, and the amplification reaction procedure was shown in Table 2 to obtain a linear plasmid fragment with a mutation site. 5 μL of PCR product was taken for agarose gel electrophoresis to confirm that the target band size was correct and single, and the remaining product was used for subsequent experiments.

[0065] Table 1 Amplification reaction system

[0066]

[0067]

[0068] Table 2 Amplification reaction program

[0069]

[0070] (2) Dpn1 digestion

[0071] 1 μL DpnI (NEB, R0176S) was added to 20 μL PCR product and incubated at 37° C. in a PCR instrument for 40 min.

[0072] (3) Conversion

[0073] 5 μL of DpnI digestion product was added to BL21 (DE3), ice bathed for 30 min, heat shocked at 42 °C for 90 s, ice bathed for 2 min, 800 μL of LB medium was added, shaken on a shaker for 1 h, 100 μL of the mixture was spread, cultured at 37 °C overnight, and then positive clones were screened by colony PCR, and recombinant strains containing corresponding mutation sites were obtained by sequencing. The PCR screening system is shown in Table 3, which was divided into 14 μL / tube, and 1 μL of bacterial solution was added for detection. The reaction procedure was: 95 °C pre-reaction for 5 min, followed by 95 °C for 30 s, 65 °C for 30 s, and 72 °C for 3 min, 28 cycles. After the reaction, agarose gel electrophoresis was performed to screen positive clones.

[0074] Table 3 PCR screening system

[0075] Components Dosage μL water 22 10 μM Primer T7 1 10 μM Primer T7 ter 1 Bacterial liquid 1 2×Taq Mix (Novozyme) 25

[0076] 2. Expression and purification of recombinant proteins

[0077] The correctly sequenced recombinant strain was inoculated and fermented to prepare bacteria. It was inoculated into LB medium at a 2% inoculation rate and cultured at 37°C with shaking until OD600 was 0.6-0.8. IPTG (final concentration of 0.5 mM) was added for induction. The culture was carried out at 16°C overnight for about 16 hours, and the bacteria were collected. The cells were resuspended in a buffer solution (composed of 50 mM Tris-HCl, pH 7.5, 0.3 M NaCl, 1 mM EDTA, 1 mM DTT) at a ratio of 1:10, the cells were broken by ultrasonic treatment, and the supernatant was collected by centrifugation; PEI was added to the supernatant at a final concentration of 0.2%, and the supernatant was collected by centrifugation; 40% ammonium sulfate was added to the supernatant to precipitate the protein, and the collected protein was purified by SP chromatography column to obtain a crude sample from which most of the impurities were removed, which was stored in a storage buffer (composed of 20 mM Tris-HCl, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.01% NP-40Alternative, 50% glycerol, pH 7.5 at 25°C) and diluted to 0.71 mg / mL, which can be used for mutant screening later (the results of SDS-PAGE of the crude enzyme solution are shown in Figure 2). Figure 1 ).

[0078] Example 2

[0079] This embodiment provides a mutant MMLV reverse transcriptase (amino acid sequence as shown in SEQ ID NO: 4), which comprises the following mutations compared to the amino acid sequence of the wild-type MMLV reverse transcriptase (as shown in SEQ ID NO: 1):

[0080] Mutation 1: E233R, i.e., glutamic acid at position 233 mutates to serine;

[0081] Mutation 2: D524G, i.e., aspartic acid at position 524 mutated to glycine;

[0082] Mutation 3: H204R, i.e. the histidine at position 204 mutates to arginine.

[0083] The preparation method of the mutant MMLV reverse transcriptase provided in this example is basically the same as that in Example 1.

[0084] In other embodiments of the present invention, mutants shown in Table 4 were constructed according to the same method as in Example 1, and the primers corresponding to each mutation site were shown in Table 5.

[0085] Table 4 Constructed MMLV reverse transcriptase mutants

[0086] Mutants Mutation site M1 D524G M2 D524G、H204R M3 D524G、E233R M4 D524G, H204R, E233R M5 H204R M6 E233R M7 H204R、E233R

[0087] Table 5 Primers corresponding to each mutation site and other primers and probes

[0088]

[0089]

[0090] Experimental example

[0091] 1. Optimal temperature and reverse transcription activity of different mutants

[0092] Test the reverse transcription activity and optimal temperature of different reverse transcriptase mutants at different temperatures. Prepare the reverse transcription reaction system with different reverse transcriptase mutant crude enzyme solutions according to Table 6. After incubation at 25°C for 2 minutes, react at 42°C, 50°C, and 55°C for 30 minutes, and then react at 70°C for 15 minutes to heat-inactivate the reverse transcriptase. Take 0.5 μL of the reverse transcription product for PCR amplification. The amplification reaction system is shown in Table 7. The amplification reaction procedure is: 95°C pre-reaction for 5 minutes; 95°C for 15 seconds, 60°C for 30 seconds (collecting fluorescence), a total of 40 cycles, and judge the activity of the reverse transcriptase by the Ct value. The results are shown in Figure 8.

[0093] Table 6 Reverse transcription reaction system

[0094]

[0095] Note:

[0096] The composition of 5×RT buffer is: 250mM Tris-HCl, pH 8.3 at room temperature, 375mM KCl, 15mM MgCl2.

[0097] Table 7 PCR amplification reaction system

[0098] Components Dosage μL 2×Taq Mix (Novogene) 10 10 μM Primer RT1-F 0.3 10 μM Primer RT1-R 0.3 RT1 probe 0.2 Reverse transcription product 0.5 <![CDATA[H2O]]> 8.7

[0099] Table 8 Experimental results

[0100]

[0101] As can be seen from Table 8, the heat resistance of the reverse transcriptase mutant M1 (D524G) is basically the same as that of the wild-type MMLV, while the heat resistance of the mutants M5 (H204R) and M6 (E233R) is significantly improved compared with the wild-type MMLV, and the reverse transcription activity of the mutant M5 at 50°C and 55°C is significantly better than that of M6. The heat resistance of M2 (D524G, H204R) and M3 (D524G, E233R), which added H204R or E233R mutations on the basis of D524G, was significantly improved compared with the wild-type MMLV, but the heat resistance of mutant M2 was significantly better than that of M3, but slightly inferior to that of mutants M4 (D524G, H204R, E233R) and M7 (H204R, E233R), indicating that the simultaneous addition of H204R and E233R mutations can significantly improve the heat resistance of reverse transcriptase and improve the reverse transcription activity of reverse transcriptase at 50℃-55℃, and the effect of H204R and E233R double mutations on enzyme activity is better than that of single point mutations to a certain extent.

[0102] 2. Tolerance of different mutants to primer mismatches

[0103] One-tube RT-PCR was used to evaluate the tolerance of different reverse transcriptase mutants to primer mismatches. Reverse transcriptase and KAPA2G Hot Start were added to the reaction system (as shown in Table 9) at the same time, and amplification was performed using MS2 RNA as a template. The primers and probes were shown in Table 5, the reaction procedure was shown in Table 10, and the results were shown in Table 11.

[0104] Table 9 Verification reaction system

[0105]

[0106]

[0107] Table 10 Verification reaction procedure

[0108]

[0109] Table 11 Verification results

[0110]

[0111] As can be seen from Table 11, the reverse transcription activity of wild-type MMLV reverse transcriptase and mutants M1, M3 and M6 (none of which contains the H204R mutation and has a low optimal reaction temperature) at 50°C is relatively low, but it does not affect the horizontal comparison of the use of different reverse transcriptases for matched primers and mismatched primers (mismatch at the first or third position at the 3' end). When using mismatched primers for amplification, the amplification efficiency of mutants M1 and M5 is significantly reduced, similar to the performance of wild-type MMLV, while the amplification efficiency of mutant M6 is basically the same as that of using matched primers, indicating that adding the E233R mutation can improve the mismatch tolerance of reverse transcriptase to primers. Mutants M3 and M7 are also better than mutant M2 in tolerance to mismatched primers (especially the first mismatch at the 3' end) due to the E233R mutation. At the same time, mutants M7 and mutant M4 not only tolerate 50°C high temperature, but also have higher tolerance to mismatched primers, which makes these two mutants have greater application potential in laboratory testing.

[0112] It can be seen from the above experimental results that the introduction of the E233R mutation in the wild-type MMLV reverse transcriptase can improve the enzyme's tolerance to high temperatures of 50°C-55°C to a certain extent, and has a higher tolerance to primer mismatches. In one-step RT-PCR, the reverse transcriptase is required to have a certain heat resistance, and the reverse transcription reaction can be carried out at a higher temperature, which is conducive to the opening of the RNA secondary structure, thereby improving the amplification effect of one-step RT-PCR. The present invention introduces the E233R mutation on the basis of the D524G and H204R mutations, which can not only reduce or remove RNase H activity and improve the heat resistance of the enzyme, but also improve the enzyme's tolerance to mismatched primers, and develop a reverse transcriptase with both thermal stability and fault tolerance, which has a wider market application prospect.

[0113] Although the technical solution of the present invention has been described in detail above with general descriptions, specific embodiments and experimental examples, for ordinary technicians in this field, modifications, substitutions or improvements made without departing from the spirit of the present invention should all fall within the scope of protection of the present invention.

Claims

1. A mutant MMLV reverse transcriptase, characterized in that: Compared with the amino acid sequence of the wild-type MMLV reverse transcriptase, the mutant MMLV reverse transcriptase only has mutations in the following amino acid sites: Mutation 1: E233R; Alternatively, the mutant MMLV reverse transcriptase has only mutations at the following amino acid sites compared to the amino acid sequence of the wild-type MMLV reverse transcriptase: Mutation 1: E233R; Mutation 2: D524G; Alternatively, the mutant MMLV reverse transcriptase has only mutations at the following amino acid sites compared to the amino acid sequence of the wild-type MMLV reverse transcriptase: Mutation 1: E233R; Mutation 3: H204R; Alternatively, the mutant MMLV reverse transcriptase has only mutations at the following amino acid sites compared to the amino acid sequence of the wild-type MMLV reverse transcriptase: Mutation 1: E233R; Mutation 2: D524G; Mutation 3: H204R; The amino acid sequence of the wild-type MMLV reverse transcriptase is shown in SEQ ID NO:

1.

2. A gene encoding the mutant MMLV reverse transcriptase as claimed in claim 1.

3. A biological material comprising the mutant MMLV reverse transcriptase according to claim 1 or the encoding gene according to claim 2, characterized in that: The biological material is a recombinant expression vector, a gene expression cassette or a recombinant bacterium.

4. A use of the mutant MMLV reverse transcriptase according to claim 1, or the encoding gene according to claim 2, or the biomaterial according to claim 3, characterized in that: The uses include one or more of the following: a) Application in reverse transcription and cDNA synthesis; b) Application in conventional RT-PCR detection; c) Application in multiplex RT-PCR assays; d) Application in RNA sequencing library construction; e) Application in RNA sequencing.

5. The use according to claim 4, characterized in that: The uses include one or more of the following: 1) Application in the preparation of reverse transcription reagents; 2) Application in the preparation of conventional RT-PCR detection reagents; 3) Application in the preparation of multiplex RT-PCR detection reagents; 4) Application in the preparation of RNA sequencing library construction reagents; 5) Application in the preparation of universal reagents for RNA sequencing.

6. A reverse transcription reagent, a conventional RT-PCR detection reagent, a multiplex RT-PCR detection reagent, an RNA sequencing library construction reagent or an RNA sequencing universal reagent, characterized in that: The reagents include at least one container, wherein the container contains the mutant MMLV reverse transcriptase according to claim 1.

7. The reagent according to claim 6, characterized in that: The mutant MMLV reverse transcriptase and the reaction buffer in the reagent together constitute a reaction premix; Alternatively, the reagent further comprises another container, wherein the another container comprises a reaction buffer.

8. The reagent according to claim 7, characterized in that: The reaction buffer includes dNTPs, Mg 2+ , buffer substances, RNase inhibitors, and co-solvents.

Images