Heat-resistant MMLV reverse transcriptase and application thereof

Abstract

The invention belongs to the technical field of molecular biology, and particularly discloses heat-resistant MMLV reverse transcriptase and application thereof. According to the heat-resistant MMLV reverse transcriptase provided by the invention, T246K mutation is introduced into a DNA polymerase active structural domain of a wild type MMLV reverse transcriptase, so that the heat stability and reverse transcription activity of the enzyme are remarkably improved. E286R, E302K and / or L435R mutations are further introduced into a thumb domain and a connecting structural domain, and the heat resistance of the enzyme can be improved by cooperating with T246K mutations. Meanwhile, D524G, E562Q and / or D583N mutations are introduced into an RNase H active structural domain, so that the RNase H activity of the enzyme can be reduced or removed. Compared with a commercial enzyme, the heat-resistant MMLV reverse transcriptase has the optimal reaction temperature higher than 50 DEG C, is suitable for high-temperature reverse transcription reaction, and has wide application prospects and higher economic value in the fields of molecular diagnosis and medical examination.

Description

Technical Field

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

[0002] Moloney murine leukemia virus reverse transcriptase (MMLV RT) is the most commonly used reverse transcriptase in biomedical tool enzymes. Among the many enzymatic properties of MMLV reverse transcriptase, thermal stability is an important property 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, thereby improving the efficiency of reverse transcription. However, the optimal reaction temperature of wild-type MMLV reverse transcriptase is only 37°C, which is not suitable for high-temperature reverse transcription. Therefore, it is necessary to develop a new heat-resistant MMLV reverse transcriptase to improve the stability of the enzyme at 50-60°C and make it suitable for high-temperature reverse transcription.

[0003] In order to solve this problem, US Patent No. 8900814B2 (Kyoto University) provides a mutant MMLV reverse transcriptase, selected from mutations at the following sites and combinations thereof: E69, D108, E117, D124, E286, E302, W313, L435, N454. Among them, the mutants E286R / E302K / L435R and E286R / E302K / L435R / D524A have higher affinity for template / primer than the wild-type MMLV reverse transcriptase, and have cDNA synthesis activity at 60°C, while the maximum temperature for cDNA synthesis of the wild-type enzyme is 54°C. In addition, Chinese patent CN110709513B (Roche) also discloses a mutant MMLV reverse transcriptase with increased thermal stability, which adds three mutations, A32V, L72R and W388R, to E286R / E302K / L435R. The resulting reverse transcriptase mutant can still synthesize cDNA at 65°C, and its thermal stability is better than that of the E286R / E302K / L435R mutant. However, the above two patents do not disclose the optimal reaction temperature of the mutant MMLV reverse transcriptase, and too many mutations will affect the growth of the host Escherichia coli and the expression and purification of the enzyme, which is not conducive to obtaining a high-yield, high-purity raw enzyme product.

[0004] Therefore, it is urgent to develop a new type of heat-resistant MMLV reverse transcriptase to improve the stability and reverse transcription efficiency of the enzyme at high temperatures and make it suitable for high-temperature reverse transcription and RT-PCR. Summary of the invention

[0005] The main technical problem solved by the present invention is to provide a heat-resistant MMLV reverse transcriptase, which improves the stability and reverse transcription efficiency of the enzyme at high temperature compared with the wild-type MMLV reverse transcriptase.

[0006] Secondly, the present invention provides a gene encoding a heat-resistant MMLV reverse transcriptase.

[0007] Thirdly, the present invention provides a biological material comprising a heat-resistant MMLV reverse transcriptase or a gene encoding the same.

[0008] Finally, the present invention provides a heat-resistant MMLV reverse transcriptase or its encoding gene, and biological materials for use in reverse transcription synthesis of cDNA, RT-PCR detection, RNA sequencing library construction, RNA sequencing, or preparation of related reagents.

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

[0010] A thermostable MMLV reverse transcriptase, comprising the following mutation sites compared to the amino acid sequence of a wild-type MMLV reverse transcriptase:

[0011] T246K, mutating the threonine at position 246 to lysine;

[0012] The amino acid sequence of the wild-type MMLV reverse transcriptase is shown in SEQ ID NO:1.

[0013] As a preferred embodiment of the present invention, the amino acid sequence of the heat-resistant MMLV reverse transcriptase has more than 90% sequence homology with the wild-type MMLV reverse transcriptase.

[0014] More preferably, the sequence homology is 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0015] As a preferred embodiment of the present invention, the thermostable MMLV reverse transcriptase further comprises one or more of the following mutation sites:

[0016] E286R, glutamic acid at position 286 mutated to arginine;

[0017] E302K, i.e., glutamic acid at position 302 is mutated to lysine;

[0018] L435R, i.e. the leucine at position 435 is mutated to arginine.

[0019] As a preferred embodiment of the present invention, the thermostable MMLV reverse transcriptase further comprises one or more of the following mutation sites:

[0020] D524G, i.e., the aspartic acid at position 524 was mutated to glycine;

[0021] E562Q, i.e., glutamic acid at position 562 mutated to glutamine;

[0022] D583N, i.e., the aspartic acid at position 583 is mutated to asparagine.

[0023] More preferably, the thermostable MMLV reverse transcriptase comprises the following mutations:

[0024] T246K, D524G, E562Q and D583N.

[0025] More preferably, the thermostable MMLV reverse transcriptase comprises the following mutations:

[0026] T246K, E286R, E302K, L435R, D524G, E562Q, and D583N.

[0027] As a preferred embodiment of the present invention, the heat-resistant MMLV reverse transcriptase is fused with Sso7d or PCNA, or fused with a protein tag (such as His, GST) to obtain a fusion protein with improved activity.

[0028] A gene encoding a thermostable MMLV reverse transcriptase.

[0029] As a preferred embodiment of the present invention, without changing the amino acid sequence of the encoded heat-resistant MMLV reverse transcriptase, the sequence can be optimized according to the codon preference of the host cell.

[0030] A biological material comprising a heat-resistant MMLV reverse transcriptase or a gene encoding the same.

[0031] 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 (such as a recombinant Escherichia coli), and the like.

[0032] As a preferred embodiment of the present invention, the method for preparing the recombinant expression vector comprises:

[0033] The gene encoding the heat-resistant MMLV reverse transcriptase is inserted into an expression vector to obtain a recombinant expression vector.

[0034] As a preferred embodiment of the present invention, the method for preparing the recombinant bacteria comprises:

[0035] The recombinant expression vector containing the gene encoding the heat-resistant MMLV reverse transcriptase is transformed into a host bacterium (such as Escherichia coli) to obtain a recombinant bacterium.

[0036] A use of a heat-resistant MMLV reverse transcriptase or a gene encoding it, or a biological material, comprising one or more of the following:

[0037] (1) Application in reverse transcription and cDNA synthesis;

[0038] (2) Application in RT-PCR detection;

[0039] (3) Application in RNA sequencing library construction;

[0040] (4) Application in RNA sequencing.

[0041] A use of a heat-resistant MMLV reverse transcriptase or a gene encoding it, or a biological material, comprising one or more of the following:

[0042] (1) Application in the preparation of reverse transcription reagents;

[0043] (2) Application in the preparation of RT-PCR detection reagents;

[0044] (3) Application in the preparation of RNA sequencing library construction reagents;

[0045] (4) Application in the preparation of universal reagents for RNA sequencing.

[0046] A reverse transcription reagent, an RT-PCR detection reagent, an RNA sequencing library construction reagent or an RNA sequencing universal reagent comprises at least one container containing a heat-resistant MMLV reverse transcriptase.

[0047] As a preferred embodiment of the present invention, the heat-resistant MMLV reverse transcriptase is mixed with other components required for the reverse transcription reaction to form a reaction premix. The reaction premix is ​​contained in the above container, and when used, it only needs to be mixed with primers, RNA templates, etc. to form a reverse transcription reaction system.

[0048] As a preferred embodiment of the present invention, the reagent further comprises another container, wherein the container comprises a reaction buffer, wherein the reaction buffer comprises other components required for the reverse transcription reaction in addition to the reverse transcriptase. When used, the heat-resistant MMLV reverse transcriptase and the reaction buffer are taken separately, and the two are mixed with primers, RNA templates, etc. to prepare a reverse transcription reaction system.

[0049] Specifically, the other components required for the reverse transcription reaction include one or more of the following:

[0050] (1)Mg 2+ Activator;

[0051] (2) dNTPs;

[0052] (3) KCl;

[0053] (4) a buffer substance, such as Tris-HCl, Tris-H2SO4, etc.;

[0054] (5) Additives, including one or more of glycerol, Triton X-100, BSA, DTT, RNase inhibitors, etc.

[0055] Specifically, the primer is a random primer (such as a random hexa-oligonucleotide primer), oligo(dT) or a specific primer designed according to the target.

[0056] Beneficial effects of the present invention:

[0057] The heat-resistant MMLV reverse transcriptase provided by the present invention significantly improves the thermostability and reverse transcription activity of the enzyme by introducing the T246K mutation in the DNA polymerase active domain of the wild-type MMLV reverse transcriptase. Further introduction of E286R, E302K and / or L435R mutations in the thumb domain and the connecting domain can cooperate with the T246K mutation to improve the heat resistance of the enzyme. At the same time, the introduction of D524G, E562Q and / or D583N mutations in the RNase H active domain can reduce or remove the RNase H activity of the enzyme, avoid non-specific degradation of RNA in the reaction, and improve the specificity and fidelity of cDNA synthesis. The experimental results show that the thermal stability of MMLV reverse transcriptase mutants T7 and T11 is significantly better than that of other mutants or commercial enzymes, and the optimal reaction temperature is about 55°C, which is suitable for high-temperature reverse transcription reactions, and has broad application prospects and high economic value in the fields of molecular diagnosis and medical testing.

[0058] The present invention also provides the use of heat-resistant MMLV reverse transcriptase or its encoding gene and biological materials in reverse transcription synthesis of cDNA, RT-PCR detection, RNA sequencing library construction, RNA sequencing or preparation of related reagents to improve the accuracy, specificity and sensitivity of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The figure shows the SDS-PAGE result of crude enzyme solution of MMLV reverse transcriptase mutant.

[0060] Figure 2 This is the standard curve of enzyme amount and fluorescence value in the experimental example.

[0061] 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

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] Example 1

[0069] This example provides a thermostable MMLV reverse transcriptase (as shown in SEQ ID NO: 3), which has only the following mutations compared to the amino acid sequence of the wild-type MMLV reverse transcriptase (as shown in SEQ ID NO: 1):

[0070] T246K, mutating the threonine at position 246 to lysine;

[0071] D524G, i.e., the aspartic acid at position 524 was mutated to glycine;

[0072] E562Q, i.e., glutamic acid at position 562 mutated to glutamine;

[0073] D583N, i.e., the aspartic acid at position 583 is mutated to asparagine.

[0074] This embodiment also provides a method for preparing the above-mentioned heat-resistant MMLV reverse transcriptase, comprising the following steps:

[0075] 1. Construction of recombinant strains

[0076] (1) Circle expansion PCR

[0077] The coding gene sequence of MMLV reverse transcriptase mutant T1 (as shown in SEQ ID NO: 2) was synthesized outsourced and constructed on the pET-30a plasmid. Using the plasmid as a template, the primer pairs shown in Table 5 were used for expansion PCR, the amplification reaction system was shown in Table 1, and the reaction procedure was shown in Table 2 to obtain the PCR product. 5 μL of the PCR product was taken for agarose gel electrophoresis to confirm that the target band size was correct and single.

[0078] Table 1 Amplification reaction system

[0079] Components Dosage Template DNA (100 ng / μL) 0.5μL 10 μM Primer T246K-F 0.3μL 10 μM Primer T246K-R 0.3μL PrimeSTAR 0.3μL dNTPs(10mM) 0.5μL 5×PS buffer 5μL <![CDATA[H2O]]> Up to 25 μL

[0080] Table 2 Amplification reaction program

[0081]

[0082]

[0083] (2) Dpn1 digestion

[0084] 1 μL DpnI (NEB, R0176S) was added to 20 μL PCR product and incubated at 37°C in a PCR instrument for 40 min.

[0085] (3) Conversion

[0086] Add 5 μL of DpnI digestion product to BL21 (DE3) and place on ice for 30 min, heat shock at 42°C for 90 s, place on ice for 2 min, add 800 μL of LB medium, and shake on a shaker for 1 h. Take 100 μL of the mixture and spread on a plate, culture at 37°C overnight, then screen positive clones by colony PCR and sequence to obtain recombinant strains containing the corresponding mutation sites.

[0087] The PCR screening system is shown in Table 3. 1 μL of bacterial solution was taken 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, for a total of 28 cycles. After the reaction, agarose gel electrophoresis was performed to screen positive clones.

[0088] Table 3 PCR screening system

[0089] Components Dosage water 22μL 10 μM screening primer-F 1μL 10 μM screening primer-R 1μL Bacterial liquid 1μL 2×Taq Mix (Yisheng) 25μL

[0090] 2. Expression and purification of recombinant proteins

[0091] The correctly sequenced recombinant strain was inoculated into LB medium at an inoculum of 2%, cultured at 37°C with shaking until OD600 was 0.6-0.8, IPTG (final concentration was 0.5mM) was added to induce expression, cultured overnight at 16°C for about 16 hours, and the bacteria were collected. The bacteria were resuspended in a buffer solution (composed of: 50mM Tris-HCl, pH 7.5, 0.3M NaCl, 1mM EDTA, 1mM DTT) at a ratio of 1:10, the cells were ultrasonically disrupted, and the supernatant was collected by centrifugation. PEI was added to the supernatant at a final concentration of 0.2%, and the supernatant was obtained 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 enzyme solution from which most of the impurities were removed. The crude enzyme solution was stored in storage buffer (composed of: 20 mM Tris-HCl, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.01% NP-40, 50% glycerol, pH 7.5@25°C) and diluted to 0.71 mg / mL for subsequent mutant screening.

[0092] Table 4 MMLV reverse transcriptase mutants

[0093]

[0094]

[0095] Table 5 Primers corresponding to each mutation site and other primers and probes (SEQ ID NO: 5-30)

[0096]

[0097]

[0098] Example 2

[0099] This example provides a thermostable MMLV reverse transcriptase (as shown in SEQ ID NO: 4), which has only the following mutations compared to the amino acid sequence of the wild-type MMLV reverse transcriptase (as shown in SEQ ID NO: 1):

[0100] T246K, mutating the threonine at position 246 to lysine;

[0101] E286R, glutamic acid at position 286 mutated to arginine;

[0102] E302K, i.e., glutamic acid at position 302 is mutated to lysine;

[0103] L435R, i.e., leucine at position 435 mutated to arginine;

[0104] D524G, i.e., the aspartic acid at position 524 was mutated to glycine;

[0105] E562Q, i.e., glutamic acid at position 562 mutated to glutamine;

[0106] D583N, i.e., the aspartic acid at position 583 is mutated to asparagine.

[0107] The preparation method of the heat-resistant MMLV reverse transcriptase in this example is basically the same as that in Example 1.

[0108] This embodiment also provides an application of a thermostable MMLV reverse transcriptase in reverse transcription synthesis of cDNA, comprising:

[0109] The total RNA extracted from the sample to be tested was used as a template, and heat-resistant MMLV reverse transcriptase, random primers and reaction buffer were added to synthesize cDNA by reverse transcription at 55°C for 15 minutes.

[0110] This embodiment also provides a use of a thermostable MMLV reverse transcriptase in preparing a reverse transcription reagent, comprising:

[0111] The thermostable MMLV reverse transcriptase is mixed with other components required for the reverse transcription reaction, such as MgCl, dNTPs, KCl, Tris-HCl, glycerol, and RNase inhibitor, to prepare a reverse transcription reaction premix.

[0112] This embodiment also provides a reverse transcription reagent, including a container, in which the reverse transcription reaction premix is ​​contained.

[0113] In other embodiments of the present invention, the heat-resistant MMLV reverse transcriptase has only the following mutations compared to the amino acid sequence of the wild-type MMLV reverse transcriptase (as shown in SEQ ID NO: 1): T246K, D524G. The heat stability of the enzyme is substantially equivalent to that of the heat-resistant MMLV reverse transcriptase in Example 1.

[0114] In other embodiments of the present invention, the thermostable MMLV reverse transcriptase has only the following mutations compared to the amino acid sequence of the wild-type MMLV reverse transcriptase (as shown in SEQ ID NO: 1): T246K, E286R, E302K, L435R, D524G. The thermostability of the enzyme is substantially equivalent to that of the thermostable MMLV reverse transcriptase in Example 2.

[0115] In other embodiments of the present invention, the heat-resistant MMLV reverse transcriptase can also be used in RT-PCR detection, RNA sequencing library construction, and RNA sequencing, and can be used to prepare RT-PCR detection reagents, RNA sequencing library construction reagents, and RNA sequencing universal reagents.

[0116] In other embodiments and comparative examples of the present invention, the MMLV reverse transcriptase mutants and other mutants shown in Table 4 were constructed according to the method basically the same as that in Example 1, and the expansion primers corresponding to each mutation site were shown in Table 5.

[0117] Experimental example

[0118] 1. Thermal stability test (fluorescent probe method)

[0119] The other MMLV reverse transcriptase mutants in Table 4 were constructed by referring to the method of Example 1. The SDS-PAGE results of the obtained crude enzyme solution are as follows: Figure 1As shown, the thermal stability of different mutants was tested. Experimental method: The crude enzyme solution of different MMLV reverse transcriptase mutants was prepared into a reverse transcription reaction system according to Table 6, first incubated at 25°C for 2min, then reacted at 42°C, 50°C, 55°C, and 60°C for 30min, and then reacted at 70°C for 15min to thermally inactivate the reverse transcriptase. Take 0.5μL of the reverse transcription product for PCR amplification. The amplification reaction system is shown in Table 7. The reaction procedure is: 95°C pre-reaction for 5min; 95°C for 15s, 60°C for 30s (collecting fluorescence), for a total of 40 cycles. The reverse transcription activity of the MMLV reverse transcriptase mutant was judged by the Ct value, and the results are shown in Table 8.

[0120] Table 6 Reverse transcription reaction system

[0121]

[0122] Note: 5×RT buffer: 250mM Tris-HCl, 375mM KCl, 15mM MgCl2, pH 8.3@25℃.

[0123] Table 7 PCR amplification reaction system

[0124] Components Dosage 2×Taq Mix (Yisheng) 10μL 10μM PCR Primer-F 0.3μL 10μM PCR Primer-R 0.3μL PCR probe-P 0.2μL Reverse transcription product 0.5μL <![CDATA[H2O]]> 8.7μL

[0125] Table 8 Thermal stability test results

[0126]

[0127] As can be seen from Table 8, 8 mutants have high reverse transcription activity at 50°C (Ct value <13), especially mutants T5, T7, and T11 still have high reverse transcription activity at 55°C (Ct value <13). In addition, the T246K single point mutation in mutant T7 can achieve the effect of the combined mutations of E286R, E302K, and L435R in T5, and mutant T11, as a combination of the two, has a further enhanced reverse transcription activity at 55°C than mutants T5 and T7. In particular, the reverse transcription activity of mutant T11 at 55°C is higher than that at 50°C, which shows that the optimal reaction temperature of the mutant is higher than 50°C, at around 55°C, indicating that the thermal stability of mutant T11 is significantly better than that of mutants T5 and T7.

[0128] 2. Enzyme activity test (fluorescent dye method)

[0129] Experimental method: Taking mutant T7 as an example, dilute its crude enzyme solution to 0.0071 mg / mL, add 0.5, 2, 3, 4, 5, 10 μL of input into the reverse transcription reaction system (as shown in Table 9), synthesize cDNA hybrid chain, the reaction procedure is: incubate at 25℃ for 2min; 55℃ for 30min (collect fluorescence), and inactivate the enzyme at 70℃ for 15min. After the reaction is completed, count and draw the standard curve of enzyme amount and fluorescence value, the results are shown in Table 10, Figure 2 shown.

[0130] Table 9 Reverse transcription reaction system

[0131]

[0132]

[0133] Table 10 Fluorescence values ​​measured at different enzyme amounts

[0134] Input Net fluorescence value Rn 10μL 1258.26 5μL 824.67 4μL 662.29 3μL 559.52 2μL 447.3 0.5μL 195.07

[0135] from Figure 2 It can be seen that when the crude enzyme solution was diluted to 0.0071 mg / mL, 0.5-10 μL of enzyme was added to the reverse transcription reaction system, and the fluorescence value showed an upward trend and did not reach saturation. Therefore, 5 μL of enzyme input was selected, and the mutants T5, T7, and T11 were reverse transcribed at 55°C for 30 min, respectively. The increased fluorescence values ​​of the three mutants were compared to reflect the reverse transcription efficiency. The results are shown in Table 11.

[0136] Table 11 Fluorescence values ​​measured for different mutants

[0137] RT crude enzyme solution Net fluorescence value Rn Yin ginseng (water) 0 T5 810 T7 815 T11 1020

[0138] As can be seen from Table 11, the MMLV reverse transcriptase mutants were reacted at 55°C for 30 minutes, and the changes in fluorescence values ​​were monitored every 30 seconds. The results showed that the reverse transcription activity of mutant T7 was similar to that of mutant T5, further proving that the T246K single point mutation can achieve the effect of the combined mutation of E286R, E302K and L435R. The reverse transcription activity of mutant T11 was significantly higher than that of mutants T5 and T7, indicating that the combined mutation of T246K, E286R, E302K and L435R is more conducive to improving the reverse transcription activity of MMLV reverse transcriptase at 55°C.

[0139] Based on the above experimental results, it can be seen that the present invention significantly improves the thermal stability and reverse transcription activity of the enzyme by introducing the T246K mutation in the DNA polymerase active domain of the wild-type MMLV reverse transcriptase. Further introduction of E286R, E302K, and L435R mutations in the thumb domain and the linker domain can synergize with the T246K mutation to improve the heat resistance of the enzyme. Compared with other mutants or commercial enzymes, the optimal reaction temperature of the heat-resistant MMLV reverse transcriptase is higher than 50°C, which is suitable for high-temperature reverse transcription reactions and has broad application prospects and high economic value in the fields of molecular diagnosis and medical testing.

[0140] 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 thermostable MMLV reverse transcriptase, characterized in that: Compared with the amino acid sequence of wild-type MMLV reverse transcriptase, it contains the following mutation sites: T246K, mutating the threonine at position 246 to lysine; The amino acid sequence of the wild-type MMLV reverse transcriptase is shown in SEQ ID NO:

1.

2. The thermostable MMLV reverse transcriptase according to claim 1, characterized in that: The amino acid sequence of the heat-resistant MMLV reverse transcriptase has more than 90% sequence homology with the wild-type MMLV reverse transcriptase.

3. The thermostable MMLV reverse transcriptase according to claim 1, characterized in that: The thermostable MMLV reverse transcriptase further comprises one or more of the following mutation sites: E286R, glutamic acid at position 286 mutated to arginine; E302K, i.e., glutamic acid at position 302 is mutated to lysine; L435R, i.e. the leucine at position 435 is mutated to arginine.

4. The thermostable MMLV reverse transcriptase according to any one of claims 1 to 3, characterized in that: The thermostable MMLV reverse transcriptase further comprises one or more of the following mutation sites: D524G, i.e., the aspartic acid at position 524 was mutated to glycine; E562Q, i.e., glutamic acid at position 562 mutated to glutamine; D583N, i.e., the aspartic acid at position 583 is mutated to asparagine.

5. The thermostable MMLV reverse transcriptase according to claim 4, characterized in that: The thermostable MMLV reverse transcriptase comprises the following mutations: T246K, D524G, E562Q and D583N; Alternatively, the thermostable MMLV reverse transcriptase comprises the following mutation: T246K, E286R, E302K, L435R, D524G, E562Q, and D583N.

6. A gene encoding the thermostable MMLV reverse transcriptase according to any one of claims 1 to 5.

7. A biological material comprising the heat-resistant MMLV reverse transcriptase according to any one of claims 1 to 5, or the encoding gene according to claim 6.

8. A use of the thermostable MMLV reverse transcriptase according to any one of claims 1 to 5, or the encoding gene according to claim 6, or the biomaterial according to claim 7, characterized in that: Include one or more of the following: (1) Application in reverse transcription and cDNA synthesis; (2) Application in RT-PCR detection; (3) Application in RNA sequencing library construction; (4) Application in RNA sequencing.

9. A use of the thermostable MMLV reverse transcriptase according to any one of claims 1 to 5, or the encoding gene according to claim 6, or the biomaterial according to claim 7, characterized in that: Include one or more of the following: (1) Application in the preparation of reverse transcription reagents; (2) Application in the preparation of RT-PCR detection reagents; (3) Application in the preparation of RNA sequencing library construction reagents; (4) Application in the preparation of universal reagents for RNA sequencing.

10. A reverse transcription reagent, RT-PCR detection reagent, RNA sequencing library construction reagent or RNA sequencing universal reagent, characterized in that: The method comprises at least one container, wherein the container contains the heat-resistant MMLV reverse transcriptase according to any one of claims 1 to 5.

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