An MMLV enzyme mutant, its preparation method and its application

Abstract

This invention provides an MMLV enzyme mutant, its preparation method, and its applications, relating to the field of biotechnology. The MMLV enzyme mutant is an amino acid sequence mutation based on the wild-type MMLV enzyme amino acid sequence shown in SEQ ID NO.1, and contains at least one amino acid mutation selected from S255K, P315W, and L486W. By introducing the S255K, P315W, or L486W mutation, the thermostability of this MMLV enzyme is significantly improved. This characteristic enables reverse transcription at high temperatures, thereby reducing erroneous products by increasing primer binding specificity and effectively unraveling the complex secondary structure of RNA, ultimately significantly improving the reverse transcription efficiency and full-length cDNA synthesis capability for difficult templates such as those with high GC content.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to an MMLV enzyme mutant, its preparation method, and its application. Background Technology

[0002] Reverse transcription, the process of synthesizing complementary deoxyribonucleic acid (cDNA) using ribonucleic acid (RNA) as a template, is a core technology in modern molecular biology and biotechnology. This process is mediated by a key catalytic enzyme—reverse transcriptase. Since its discovery, reverse transcription technology has become an indispensable tool in life science research and applications. In virology research, it is used to detect and quantitatively analyze the genomes of RNA viruses, providing a foundation for the diagnosis and monitoring of viruses such as influenza, human immunodeficiency virus (HIV), and various coronaviruses. In gene function and expression analysis, reverse transcription can convert intracellular messenger RNA (mRNA) populations into more stable cDNA libraries, allowing for precise measurement of gene expression levels using real-time quantitative PCR (RT-qPCR) or high-throughput sequencing technologies (such as RNA-Seq), thereby revealing complex biological processes and disease mechanisms. Therefore, the performance of reverse transcriptase directly determines the accuracy, sensitivity, and reliability of these downstream applications.

[0003] Currently, commercially available and widely used reverse transcriptases in laboratories are primarily derived from viruses, with the most representative being the Moloney Murine Leukemia Virus (MMLV) reverse transcriptase. These naturally occurring wild-type reverse transcriptases possess both RNA-dependent and DNA-dependent DNA polymerase activities, as well as RNA-degrading enzyme (RNase H) activity, enabling them to complete the entire process of synthesizing cDNA from an RNA template. Due to their mature application history and well-defined biochemical characteristics, MMLV reverse transcriptase has become a standard component in various cDNA synthesis, clinical diagnostic kits, and next-generation sequencing library construction processes. Under standard experimental conditions, these enzymes can efficiently reverse transcribe most simple RNA templates, meeting the needs of basic molecular biology experiments and forming the basis of current reverse transcription technology.

[0004] However, these wild-type reverse transcriptases derived from viruses have a fundamental inherent defect that limits their breadth and depth of application: their low thermal stability. Taking the most widely used MMLV reverse transcriptase as an example, its optimal catalytic reaction temperature range is only 37-42℃. This relatively low reaction temperature brings several problems. First, it directly affects the specificity of nucleic acid detection. At 37-42℃, the binding between primers and RNA templates is not tight enough, easily leading to non-specific annealing, i.e., primers incorrectly bind to similar but non-target sequences. This erroneous initiation results in the generation of a large amount of unexpected cDNA products, severely interfering with subsequent amplification and detection steps, and even causing false positive results, which is particularly prominent in clinical diagnostic applications requiring high precision. Second, low temperatures cannot effectively handle structurally complex RNA templates. Many RNA molecules, especially those sequences with high GC content, self-fold under physiological conditions, forming stable secondary structures such as hairpin loops and stem-loops. These structures can hinder the exposure of primer binding sites or create physical barriers as the reverse transcriptase extends along the template strand, causing the synthesis process to terminate prematurely and preventing the acquisition of full-length cDNA. At 37°C, the enzyme activity is insufficient to open these stable secondary structures, resulting in low efficiency or even complete failure of the reverse transcription reaction.

[0005] In summary, the inherent low-temperature catalytic characteristic of wild-type reverse transcriptases, especially MMLV reverse transcriptases, which are widely used in existing technologies, is their core technological bottleneck. This deficiency not only reduces the specificity of detection and increases the risk of false positive results, but also severely limits their ability to process complex RNA templates. In practical applications, high temperatures are often required to unravel the secondary structure of RNA to improve the elongation efficiency and continuous synthesis capacity of reverse transcription. Therefore, the performance of current reverse transcriptases falls far short of meeting the urgent need for efficient and high-fidelity reverse transcription of long fragments of RNA with high GC content or complex secondary structures. Overcoming the series of problems caused by the thermal instability of enzymes and developing high-performance reverse transcriptases capable of continuously synthesizing long fragments is a key technical challenge that urgently needs to be solved in this field.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an MMLV enzyme mutant, its preparation method, and its application. The MMLV enzyme mutant improves thermal stability, thereby enhancing the specificity, efficiency, and ability to process complex templates in reverse transcription.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0009] In a first aspect, the present invention provides an MMLV enzyme mutant, wherein the MMLV enzyme mutant is a mutation of the amino acid sequence based on the wild-type MMLV enzyme amino acid sequence as shown in SEQ ID NO.1, and contains at least one selected from the amino acid mutations S255K, P315W and L486W.

[0010] In an optional embodiment, the amino acid sequence of the amino acid mutation S255K is as shown in SEQ ID NO.2; and / or, the amino acid sequence of the amino acid mutation P315W is as shown in SEQ ID NO.3; and / or, the amino acid sequence of the amino acid mutation L486W is as shown in SEQ ID NO.4.

[0011] Secondly, the present invention provides a polynucleotide molecule encoding the MMLV enzyme mutant as described in the foregoing embodiments.

[0012] Thirdly, the present invention provides a recombinant expression vector comprising the polynucleotide molecule as described in the foregoing embodiments.

[0013] Fourthly, the present invention provides a host cell containing the recombinant expression vector described in the foregoing embodiments; or, the chromosome is integrated with the polynucleotide molecule described in the foregoing embodiments.

[0014] Fifthly, the present invention provides a kit comprising the MMLV enzyme mutant as described in the foregoing embodiments.

[0015] Sixthly, the present invention provides a method for preparing the MMLV enzyme mutant as described in the foregoing embodiments, comprising:

[0016] Using a nucleic acid molecule encoding the wild-type MMLV enzyme as a template, a polynucleotide molecule encoding the MMLV enzyme mutant was obtained by site-directed mutagenesis, and then constructed into an expression vector to obtain a recombinant expression vector;

[0017] The recombinant expression vector was transformed into host cells and cultured and induced to express the MMLV enzyme mutant;

[0018] The host cells were lysed, and the expressed MMLV enzyme mutant was isolated and purified by affinity chromatography to obtain the MMLV enzyme mutant.

[0019] In optional embodiments, the site-directed mutagenesis treatment is rolling circle PCR; and / or, the host cell is Escherichia coli; and / or, the affinity chromatography is purification using a nickel column; and / or, the sequence of the nucleic acid molecule encoding the wild-type MMLV enzyme is shown in SEQ ID NO. 5.

[0020] In a seventh aspect, the present invention provides a method for reverse transcription of RNA, comprising:

[0021] The MMLV enzyme mutant described in the foregoing embodiments was used, and the reaction was carried out at a temperature of 50°C to 65°C.

[0022] Eighthly, the present invention provides the use of the MMLV enzyme mutant as described in the foregoing embodiments in the preparation of reagents or kits for RNA reverse transcription.

[0023] This invention provides an MMLV enzyme mutant, its preparation method, and its applications. This MMLV enzyme mutant, through the introduction of amino acid mutations S255K, P315W, or L486W at specific positions, significantly enhances the enzyme's thermostability. This means that compared to the unmodified wild-type MMLV enzyme, this mutant better maintains its three-dimensional structure and catalytic activity under high-temperature conditions. Experimental data show that after heating at 65°C, the mutant retains significantly higher activity than the wild-type enzyme, while the latter's activity decreases substantially. This improvement in fundamental properties directly brings a series of crucial advantages in practical applications.

[0024] First, the enhanced thermostability allows the reverse transcription reaction to proceed at higher temperatures. It is common knowledge that increasing the reaction temperature increases the rigor of nucleic acid hybridization. Wild-type MMLV enzymes, which operate only at low temperatures of 37-42°C, often experience non-specific binding between primers and RNA templates, leading to erroneous cDNA products, reduced detection specificity, and potentially false positives. This mutant enzyme, due to its thermostability, supports reactions at higher temperatures, thereby improving primer binding specificity, significantly reducing the generation of non-target products, and making subsequent quantitative analysis or detection results more accurate and reliable.

[0025] Secondly, for RNA templates with complex secondary structures or high GC content, wild-type enzymes struggle to perform effective reverse transcription at a low temperature of 37°C. This is because RNA molecules fold themselves into stable spatial structures such as hairpins and stem-loops, which hinder primer binding or impede enzyme progression, leading to interruption or complete failure of cDNA synthesis. This mutant enzyme, however, is heat-tolerant, allowing the reaction system to proceed at temperatures sufficient to open these RNA secondary structures (e.g., reaching 65°C). Under high temperatures, the RNA template is unstretched into a more accessible single-stranded state, significantly improving the ability to process complex templates and the success rate of reverse transcription.

[0026] Finally, because the high temperature effectively eliminates the physical barriers of RNA secondary structure to the enzyme elongation process, this mutant enzyme can synthesize DNA strands more smoothly and continuously along the RNA template. This directly improves the enzyme's elongation efficiency and continuous synthesis capability. The result is the production of longer cDNA products that more completely cover the entire length of the RNA transcript. This is invaluable for subsequent applications requiring full-length cDNA, such as long-read sequencing, gene cloning, or research on long non-coding RNAs. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 The above are the electrophoresis results of the purified crude enzymes of the MMLV variant and wild type in the embodiments of this application;

[0029] Figure 2 The results show the difference in reverse transcription performance between the MMLV variant and the wild type before and after heating in the embodiments of this application. Detailed Implementation

[0030] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0031] In this application embodiment, an MMLV enzyme mutant is provided, wherein the MMLV enzyme mutant is a mutation of the amino acid sequence based on the wild-type MMLV enzyme amino acid sequence shown in SEQ ID NO.1, and contains at least one selected from the amino acid mutations S255K, P315W and L486W.

[0032] The aforementioned MMLV enzyme mutant is a biological macromolecule, essentially a catalytically active enzyme derived from the reverse transcriptase of Moloney Murine Leukemia Virus. The term "mutant" signifies that the enzyme's molecular structure (i.e., amino acid sequence) has undergone specific, artificial alterations compared to its naturally occurring "wild-type" form.

[0033] The basic structure of this mutant is the amino acid sequence of the wild-type MMLV enzyme. This basic sequence is explicitly designated as SEQ ID NO.1. This constitutes the vast majority of the protein molecule. The mutated portion, defining the product as a "mutant," is the key component and is at least one amino acid substitution that occurs in its amino acid sequence.

[0034] Regarding the specific substitution sites and types, the specific parts that are allowed to undergo substitution are clearly defined, namely, at least one of the following three substitutions must be selected:

[0035] (1) S255K: At position 255 of the sequence, the serine (S) is replaced with lysine (K).

[0036] (2) P315W: At position 315 of the sequence, proline (P) is replaced with tryptophan (W).

[0037] (3) L486W: At position 486 of the sequence, leucine (L) is replaced with tryptophan (W).

[0038] This substance is constructed based on the principles of protein engineering and site-directed mutagenesis. Its core principle is that a protein's specific function (such as catalytic activity and stability) is determined by its specific three-dimensional spatial structure, which in turn is determined by its primary structure (i.e., the amino acid sequence). By precisely altering one or several key sites in the amino acid sequence, it is possible to change the local or overall structure of the protein, thereby optimizing a particular performance. In this example, by replacing the amino acids at sites S255, P315, or L486 with specific other amino acids, the internal interactions within the enzyme molecule are altered, making its overall structure more stable under thermal stress and less prone to misfolding or disintegration.

[0039] The enzyme's thermostability enables it to perform reverse transcription at higher temperatures, offering several advantages. First, the higher temperature increases the binding tightness between the primer and the RNA template, reducing non-specific binding and thus lowering the risk of false positives. Second, the higher temperature effectively breaks down stable secondary structures (such as hairpin loops) in the RNA template caused by high GC content or its own sequence characteristics, solving the problem that wild-type enzymes cannot process such templates at low temperatures. By eliminating these physical barriers to secondary structures, the enzyme can synthesize more smoothly and continuously. This not only successfully reverse transcribes "difficult" templates but also helps improve the ability to synthesize long-chain cDNA and the proportion of full-length products obtained.

[0040] In some embodiments, the amino acid sequence of the amino acid mutation S255K is shown in SEQ ID NO.2.

[0041] In some embodiments, the amino acid sequence of the amino acid mutant P315W is shown in SEQ ID NO.3.

[0042] In some embodiments, the amino acid sequence of the amino acid mutation L486W is shown in SEQ ID NO.4.

[0043] In this application embodiment, a polynucleotide molecule is provided that encodes the MMLV enzyme mutant as described in the foregoing embodiments.

[0044] In this embodiment, a recombinant expression vector is provided, which comprises the polynucleotide molecule as described in the foregoing embodiments.

[0045] In this application embodiment, a host cell is provided, the host cell containing the recombinant expression vector described in the foregoing embodiments; or, the chromosome is integrated with the polynucleotide molecule described in the foregoing embodiments.

[0046] In this application embodiment, a kit is provided, the kit comprising the MMLV enzyme mutant as described in the foregoing embodiments.

[0047] In some embodiments, the kit further includes at least one of reverse transcription buffer, primers, and RNase inhibitors.

[0048] The above content provides a kit. In the field of biotechnology, a kit is a commercial product that pre-packages the core or all components required for a specific experiment for convenient use by the end user. The above content provides a kit that can be used to construct a reverse transcription reaction system, or a kit for RNA reverse transcription. In the field of biotechnology, a kit is a commercial product that pre-packages the core or all components required for a specific experiment for convenient use by the end user.

[0049] Furthermore, in addition to containing the MMLV enzyme mutant, the kit must also contain at least one of the following additional components: Reverse transcription buffer: a solution that provides the enzyme with an optimal chemical environment, including pH and ion concentration. Primer: a short nucleic acid fragment that provides the starting point for the reverse transcription reaction, such as the oligo(dT)18 primer mentioned in the disclosure. RNase inhibitor: a reagent used to protect the RNA template from degradation by trace amounts of RNase in the environment.

[0050] In this application embodiment, a method for preparing the MMLV enzyme mutant as described in the foregoing embodiments is provided, comprising:

[0051] Step S1: Using the nucleic acid molecule encoding the wild-type MMLV enzyme as a template, a polynucleotide molecule encoding the MMLV enzyme mutant is obtained by site-directed mutagenesis, and then constructed into an expression vector to obtain a recombinant expression vector.

[0052] The above steps are the starting point of the entire preparation process, namely, creating the "blueprint" encoding the target mutant enzyme at the gene level. Starting template: This method begins with a plasmid containing the wild-type MMLV gene. Specifically, this template can be a pET30a plasmid containing the wild-type MMLV sequence.

[0053] The above-described site-directed mutagenesis method employs a technique called "site-directed mutagenesis" to introduce mutations (such as S255K) at specific sites in wild-type genes. After rolling circle PCR amplification, the plasmid containing the original wild-type template is digested with DpnI, while the newly synthesized plasmid carrying the mutated gene (i.e., the recombinant expression vector) is retained for subsequent steps.

[0054] Step S2: The recombinant expression vector is transformed into host cells for culture and induction to express the MMLV enzyme mutant.

[0055] The above steps involve sending the genetic "blueprint" into a biological "factory" (host cell) and instructing the factory to begin producing the target protein.

[0056] The recombinant expression vector obtained in the previous step is introduced into a host cell capable of producing the protein. Suitable host cells include *E. coli* competent cells BL21. The transformation process may include standard steps such as ice bath, 42°C heat shock, and recovery. The transformed cells need to be cultured to allow for mass proliferation. When the cells reach a certain density (OD600 approximately 0.6), the inducer IPTG (final concentration 1 mM) is added to the culture medium. IPTG activates the switch on the expression vector, instructing *E. coli* to begin synthesizing the target MMLV enzyme mutant protein in large quantities. The induction process is carried out overnight at 20°C.

[0057] Step S3: Lyse the host cells and separate and purify the expressed MMLV enzyme mutant by affinity chromatography to obtain the MMLV enzyme mutant.

[0058] The above steps involve precisely extracting and purifying the single desired product—the MMLV enzyme mutant—from a large number of cellular components in a biological "factory."

[0059] Specifically, for example, after protein expression, the bacterial cells are first collected by centrifugation, then a lysis buffer containing lysozyme and Triton X-100 is added, and the cell wall and cell membrane are disrupted through repeated freeze-thaw cycles, releasing all intracellular proteins, including the target enzyme. Affinity chromatography is a highly efficient purification technique. For example, a specific method using a nickel column can be employed. This is because the expressed protein is pre-designed to carry a special "his-tag" that specifically binds to the nickel column. When the cell lysate supernatant passes through the nickel column, the tagged target enzyme is "captured" and adsorbed onto the column, while other contaminating proteins flow through.

[0060] Finally, the target enzyme bound to the nickel column can be eluted with an imidazole-containing elution buffer, followed by dialysis to remove impurities and buffer replacement, ultimately yielding a high-purity crude MMLV enzyme mutant. The obtained product can be verified by SDS-PAGE electrophoresis.

[0061] In some embodiments, the site-directed mutagenesis treatment is rolling circle PCR.

[0062] The aforementioned rolling circle PCR, also known as rolling circle amplification (RCA), is a nucleic acid amplification technique that rapidly and efficiently amplifies circular DNA or RNA molecules at a constant temperature (isothermal). It differs from traditional PCR techniques, which require repeated temperature cycling.

[0063] In some embodiments, the host cell is Escherichia coli.

[0064] In some embodiments, the affinity chromatography is a purification process using a nickel column.

[0065] In some embodiments, the sequence of the nucleic acid molecule encoding the wild-type MMLV enzyme is shown in SEQ ID NO.5.

[0066] In this application embodiment, a method for reverse transcription of RNA is provided, comprising:

[0067] The MMLV enzyme mutant described in the foregoing embodiments was used, and the reaction was carried out at a temperature of 50°C to 65°C. For example, the temperature could be 50°C, 55°C, 60°C, 65°C, etc.

[0068] In this application embodiment, the use of the MMLV enzyme mutant as described in the foregoing embodiments in the preparation of reagents or kits for RNA reverse transcription is provided.

[0069] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0070] Example 1: Construction of MMLV variants

[0071] In this embodiment, the pET30a plasmid containing the MMLV wild-type sequence was used as a template for rolling circle PCR construction.

[0072] Experimental methods:

[0073] (1) Based on the wild-type MMLV nucleic acid sequence (SEQ ID NO.1), rollover amplification primers for point mutations S255K, P315W and L486W were designed. See the primer synthesis summary table 1. The rolling circle PCR amplification system was prepared according to Table 2 (rolling circle amplification procedure is shown in Table 3). After digesting the template with DpnI, the cells were transformed into Escherichia coli competent cells BL21.

[0074] Table 1. Summary of primers used for MMLV variant construction

[0075]

[0076] Table 2. Rolling Loop PCR Amplification System for MMLV Variant Construction

[0077]

[0078] Table 3. Rolling Loop PCR Amplification Procedure for MMLV Variant Construction

[0079]

[0080] (2) Take out 100 µL of BL21 competent cells from the -80 ℃ freezer and thaw them on ice. Add 10 μL of the digested product to each competent cell, gently pipette to mix, and let stand on ice for 30 min. Then heat shock in a 42 ℃ water bath for 90 s, immediately cool in an ice water bath, add 700 μL of LB liquid medium after 3 min, and resuscitate in a shaker at 37 ℃ for 60 min.

[0081] (3) After resuscitation, centrifuge at 5000 rpm for 2 min. Remove the supernatant, resuspend the bacteria in the remaining 100 μL of liquid, and spread it evenly on LB Kan solid plates. Incubate upside down at 37 ℃ for about 12 h. Sequencing of single colonies on the plates confirms the correct sequence and obtains the MMLV variant.

[0082] Example 2: Preparation of crude enzymes from MMLV variants and wild-type enzymes

[0083] In this embodiment, crude enzymes of MMLV variant and wild type were prepared.

[0084] Experimental methods:

[0085] 1. Culture induction of MMLV variants and wild-type:

[0086] (1) Primary seed culture: The variant and wild type were inoculated into 15 ml centrifuge tubes (2 mL LB medium, 50 μg / mL Kan antibiotic) and cultured overnight at 37 ℃ and 220 rpm.

[0087] (2) Secondary induction: 1 mL of culture medium was transferred from the primary seed culture to a secondary 250 mL shake flask (50 mL LB medium, 50 μg / mL Kan), and cultured at 37 °C and 250 rpm until OD. 600 Approximately 0.6, add a certain amount of IPTG (final concentration 1mM) to induce enzyme expression, and induce overnight at 20℃ and 220 rpm.

[0088] 2. Cell lysis:

[0089] Centrifuge the cultured secondary bacterial culture to collect the bacterial cells, add 5 mL of cell lysis buffer (1% Triton X-100, 20 mM Tris-HCl, 500 mM NaCl, 0.3% lysozyme), mix well, incubate at -80 ℃ for 30 min, and then treat at 37 ℃ for 30 min. Centrifuge at 12000 rpm for 20 min and collect the supernatant.

[0090] 3. Crude enzyme preparation:

[0091] The supernatant was passed through a nickel column to adsorb the target protein. After washing three times with 3 ml of washing buffer (20 mM Tris-HCl, 500 mM NaCl, 30 mM imidazole), elution was performed with 0.3 ml of elution buffer (20 mM Tris, 20 mM NaCl, 300 mM imidazole). The resulting eluent was transferred to a 30 kD dialysis bag and placed in dialysis fluid. The dialysis fluid was changed every 2-3 hours, and dialysis was performed overnight. The crude enzyme was collected. The expression of the crude enzyme was verified by SDS-PAGE, and the protein concentration was quantified according to BSA standards. See [link to relevant documentation]. Figure 1 .

[0092] Example 3: Thermal stability testing of MMLV variants and wild types

[0093] In this embodiment, thermal stability tests were performed on MMLV variants and wild-type.

[0094] Experimental methods:

[0095] 1. Thermal stability testing of MMLV variants and wild types.

[0096] (1) Preparation of reverse transcription and amplification systems:

[0097] Table 4. MMLV variants and wild-type reverse transcription systems

[0098]

[0099] Prepare the reverse transcription system in the PCR tube according to the proportions in the table above. After preparation, vortex the mixture for 10 seconds and centrifuge for a few seconds.

[0100] Table 5. Amplification systems for MMLV variants and wild-type

[0101]

[0102] Prepare the reverse transcription system in the PCR tube according to the proportions in the table above. After preparation, vortex the mixture for 10 seconds and centrifuge for a few seconds.

[0103] (2) Amplification reaction:

[0104] Table 6. MMLV crude enzyme reverse transcription program

[0105]

[0106] Table 7. MMLV crude enzyme amplification program

[0107]

[0108] Place the PCR tubes in the PCR instrument and perform the above procedure;

[0109] 2. Gel migration experiment of amplification reaction products

[0110] (1) After completion, take 5µL of reaction product, add 1µL of 6× loading buffer, mix well, and spot the sample into the well of the agarose gel electrophoresis.

[0111] (2) Use 1×TAE electrophoresis solution and run the gel for 25 minutes at 120 V.

[0112] (3) The product was photographed using ultraviolet conditions on the ChampGel 5000 imaging system (Cycler), and the brightness of the target bands of the MMLV variant and wild type was analyzed by ImageJ (grayscale analysis).

[0113] Example 4: Reverse transcription and amplification of crude MMLV variant enzyme before and after heat treatment

[0114] Experimental methods:

[0115] In this embodiment, the wild-type and variant MMLV were not treated and were treated at 65°C for 30 min, respectively. The crude enzymes of the MMLV variant and wild-type were prepared according to Tables 4 and 5, and reverse transcription and amplification were performed according to Tables 6 and 7, respectively.

[0116] Experimental results are as follows Figure 2 As shown, and for Figure 2 The results of the grayscale analysis are shown in Table 8. Figure 2 M is a 2000bp marker. Samples 1, 2, 3, and 4 are bands amplified after reverse transcription without heat treatment, while samples 5, 6, 7, and 8 are bands after reverse transcription with heat treatment.

[0117] Table 8. Statistics Figure 2 Differences in reverse transcription performance between MMLV variants and wild-type after heating

[0118]

[0119] The results are shown in Table 8. The brightness of the target bands of wild-type MMLV, MMLV:S255K, MMLV:P315W and MMLV:L486W were 1.81, 1.79 and 1.92 times that of wild-type MMLV, respectively, indicating that the MMLV variants have stronger stability than wild-type MMLV.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An MMLV enzyme mutant, characterized in that, The MMLV enzyme mutant is a mutation of the amino acid sequence based on the wild-type MMLV enzyme amino acid sequence shown in SEQ ID NO.1, and is any one of the amino acid mutations S255K, P315W and L486W.

2. A polynucleotide molecule, characterized in that, Encodes the MMLV enzyme mutant as described in claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide molecule as described in claim 2.

4. A host cell, characterized in that, The host cell contains the recombinant expression vector of claim 3; or, the chromosome is integrated with the polynucleotide molecule of claim 2.

5. A reagent kit, characterized in that, The kit includes the MMLV enzyme mutant as described in claim 1.

6. A method for reverse transcription of RNA, characterized in that, include: The MMLV enzyme mutant as described in claim 1 was used, and the reaction was carried out at a temperature of 50°C to 65°C.

7. Use of the MMLV enzyme mutant as described in claim 1 in the preparation of reagents or kits for RNA reverse transcription.

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