M-MLV RT mutant, polynucleotide thereof, preparation method, enzyme activity detection method, RNA reverse transcription method, vector containing mutant, recombinant engineering cell strain and kit

Abstract

The invention provides a novel high-temperature-resistant and inhibitor-resistant M-MLV RT mutant, a polynucleotide molecule thereof, a preparation method, an enzyme activity detection method, an RNA reverse transcription method, a carrier containing the M-MLV RT mutant, a recombinant engineering cell strain and a kit. Specifically, the invention establishes a method for detecting the enzyme activity of M-MLV RT based on an RNA aptamer, and through site-specific mutagenesis and combinatorial mutation, a high-temperature-resistant M-MLV RT mutant (Max5.0) is finally screened out, and the amino acid sequence of the high-temperature-resistant M-MLV RT mutant is shown as SEQ ID NO.8. Compared with natural M-MLV RT and commercial SuperScript III reverse transcriptase, the M-MLV RT mutant can still be subjected to efficient reverse transcription reaction at the temperature of 65 DEG C, and meanwhile, the M-MLV RT mutant has good tolerance to KCl and formamide and is high in reaction sensitivity.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for detecting the activity of moloney murine leukemia virus reverse transcriptase (M-MLV RT) based on RNA aptamer, and provides an M-MLV RT mutant with high temperature resistance, inhibitor resistance and high reaction sensitivity. Background Art

[0002] In 1970, Renato Dulbecco, Howard Temin and David Baltimore discovered reverse transcriptase from RNA viruses and jointly won the Nobel Prize in Physiology or Medicine in 1975. This breakthrough discovery completely changed molecular biology and broke the traditional understanding. Different from the traditional DNA-to-RNA process of the core theory, it is the reverse transcription of RNA templates into cDNA molecules, indicating that genetic information can be transferred from RNA to DNA. It has promoted the research of molecular biology, biochemistry and virology and has become a powerful tool for studying these disciplines. Reverse transcription is a key step in many RNA experimental studies because it allows further analysis of cDNA through techniques such as molecular cloning and sequencing.

[0003] MLV and AMV reverse transcriptases are the two most commonly used reverse transcriptases in the field of molecular PCR detection. Moloney murine leukemia virus reverse transcriptase (M-MLV RT) consists of a single polypeptide chain and has multiple activities, including RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity, and RNase H activity. The RNase H activity of M-MLV reverse transcriptase is relatively low, and its reverse transcription ability is slightly higher. The structure of AMV reverse transcriptase is a heterodimer composed of two different subunits (α subunit: 65KDa and β subunit: 94KDa), and it has a higher reaction temperature (45°C - 50°C). However, compared with M-MLV reverse transcriptase, its RNase H activity is stronger, so the cDNA synthesis efficiency is lower and its application range is smaller.

[0004] Although the natural M-MLV reverse transcriptase has the disadvantages of low thermal stability and high RNase H activity, resulting in the inability to increase the temperature to open complex-structured RNA templates and truncated synthesized cDNA, it can be directionally modified through genetic engineering to improve the thermal stability of M-MLV reverse transcriptase, reduce RNase H activity, and increase the tolerance to common inhibitors. And there is a current demand in the field of RNA research for M-MLV reverse transcriptases with high temperature resistance, inhibitor tolerance, and high reaction sensitivity. Summary of the Invention

[0005] In the first aspect of the present invention, there is provided a mutant of Moloney murine leukemia virus reverse transcriptase (M-MLV RT) with high temperature resistance and inhibitor tolerance.

[0006] In the second aspect of the present invention, there is provided a polynucleotide molecule encoding the M-MLV RT mutant described in the first aspect of the present invention.

[0007] In the third aspect of the present invention, there is provided a recombinant vector containing the polynucleotide molecule described in the second aspect of the present invention.

[0008] In the fourth aspect of the present invention, there is provided a recombinant engineered cell line into which the recombinant vector described in the third aspect of the present invention is transferred.

[0009] In the fifth aspect of the present invention, there is provided a method for preparing an M-MLV RT mutant, comprising the steps of: culturing the recombinant engineered cell line described in the fourth aspect of the present invention under suitable conditions to induce the expression and isolate and purify the M-MLV RT mutant.

[0010] In another preferred example, the recombinant engineered cell line is Rosetta.

[0011] In another preferred example, the final concentration of isopropyl β-D-1-thiogalactopyranoside (IPTG) is 0.5 mM.

[0012] In another preferred example, the temperature for protein induction is 18 °C.

[0013] In another preferred example, the low-temperature induction time for the protein is 16 h.

[0014] In the sixth aspect of the present invention, there is provided a method for detecting the enzyme activity of M-MLV RT based on RNA aptamers.

[0015] In another preferred example, the method for detecting enzyme activity includes: 1 μg of RNA aptamer.

[0016] In another preferred example, the method for detecting enzyme activity includes: taking the M-MLV RT mutant protein and incubating it at 55 °C, 60 °C, 65 °C or 70 °C for 15 min. Dilute the protein to 4 μM using 1×M-MLV buffer, take 2 μL of the protein and add it to the reverse transcription reaction system, and perform the detection of the remaining activity of M-MLV RT at 37 °C.

[0017] In the seventh aspect of the present invention, there is provided a kit for reverse transcription reaction, and the kit includes the M-MLV RT mutant described in the fifth aspect of the present invention.

[0018] In another preferred example, the kit further includes one or more of the following components: RNA extraction reagent, RNA template, RNA aptamer (Pepper), DEPC water, dNTPs, reverse transcription reaction buffer, reverse transcription reaction primer.

[0019] In another preferred example, the length of the nucleotides of the RNA template is between 1000 nt and 3000 nt.

[0020] In another preferred example, the length of the nucleotides of the RNA template is 1000 nt or 3000 nt.

[0021] In the eighth aspect of the present invention, there is provided a kit for fluorescence quantitative PCR reaction, and the kit for fluorescence quantitative PCR reaction includes the cDNA of the reverse transcription product described in the seventh aspect of the present invention.

[0022] In another preferred example, the kit for fluorescence quantitative PCR reaction further includes one or more of the following components: cDNA product, deionized water, EveGreen dye, dNTPs, reaction buffer, Easy Taq DNA polymerase, and DNA polymerization reaction primer.

[0023] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0024] (1) The present invention explores the induction expression conditions of M-MLV RT, enabling the high-level expression of M-MLV RT protein, improving the protein yield, and facilitating the subsequent purification and detection experiments of M-MLV RT mutant protein.

[0025] (2) The present invention provides a method for detecting the enzyme activity of M-MLV RT based on RNA aptamer.

[0026] (3) The present invention provides a heat-resistant M-MLV RT mutant. Compared with the commercial enzyme SuperScriptⅢ, the M-MLV RT mutant has significantly improved thermal stability and can react normally at 65°C.

[0027] (4) The present invention provides an inhibitor-resistant M-MLV RT mutant. Compared with the commercial enzyme SuperScriptⅢ, the M-MLV RT mutant has significantly improved tolerance to KCl and formamide and can react normally in a reaction system with a final concentration of 300 mM KCl or 20% formamide by volume.

[0028] (5) The present invention provides an M-MLV RT mutant with high reaction sensitivity. Compared with the commercial enzyme SuperScriptⅢ, the M-MLV RT mutant has improved reaction sensitivity and can detect an RNA template with a content as low as 50 ng at 70°C

[0029] under the condition. Description of the Drawings

[0030] Figure 1 It is the SDS-PAGE electrophoresis pattern of the supernatant after ultrasonic disruption and centrifugation of the Moloney murine leukemia virus reverse transcriptase (M-MLV RT) mutant in Example 1 of the present invention.

[0031] Figure 2 It is the SDS-PAGE electrophoresis pattern of the purified protein of the M-MLV RT mutant in Example 1 of the present invention.

[0032] Figure 3 It is the schematic diagram of the method for detecting the enzyme activity of M-MLV RT based on RNA aptamer in Example 2 of the present invention.

[0033] Figure 4 It is for detecting the thermal stability of the M-MLV RT mutant based on RNA aptamer in Example 2 of the present invention.

[0034] Figure 5 It is for detecting the reverse transcription efficiency of the M-MLV RT mutant and the commercial enzyme SuperScriptⅢ at different temperatures by fluorescence quantitative PCR in Example 3 of the present invention.

[0035] Figure 6 It is for detecting the reverse transcription efficiency of the M-MLV RT mutant and the commercial enzyme SuperScriptⅢ at different temperatures by agarose gel electrophoresis in Example 3 of the present invention.

[0036] Figure 7This is for detecting the reverse transcription efficiency of the M-MLV RT mutant and the commercial enzyme SuperScriptⅢ on a 3000nt RNA template at different temperatures in Example 4 of the present invention.

[0037] Figure 8 This is for detecting the tolerance of the M-MLV RT mutant and SuperScriptⅢ to KCl by agarose gel electrophoresis in Example 5 of the present invention.

[0038] Figure 9 This is for detecting the tolerance of the M-MLV RT mutant and SuperScriptⅢ to formamide by agarose gel electrophoresis in Example 6 of the present invention.

[0039] Figure 10 This is for detecting the reaction sensitivity of the M-MLV RT mutant and SuperScriptⅢ to different RNA template concentrations at different temperatures by agarose gel electrophoresis in Example 7 of the present invention. Detailed implementation manners

[0040] The following examples are included herein to more clearly and explicitly illustrate the technical solutions of the present invention by way of exemplification. Those skilled in the art should understand according to the disclosure herein that many changes can be made in the specific embodiments disclosed, but still similar or analogous results can be obtained without departing from the spirit and scope of the present invention. The specific implementation manners of the present invention are only used to explain the present invention and are not intended to limit the present invention in any way.

[0041] Although any methods and materials similar or equivalent to those described in the present invention can be used in the implementation or testing of the present invention, preferred methods and materials are listed herein for experiments.

[0042] The experimental methods in the embodiments of the present invention are all conventional experimental methods unless otherwise specified.

[0043] The experimental materials and reagents used in the embodiments of the present invention can be obtained through commercial channels unless otherwise specified.

[0044] Example:

[0045] I. Experimental materials:

[0046] The Moloney murine leukemia virus reverse transcriptase (M-MLV RT) gene in the examples was synthesized by Nanjing GenScript Biotech Co., Ltd. All PCR primers were synthesized by Shanghai BGI Genomics Co., Ltd. The M-MLV RT mutants constructed in the examples were determined by Shanghai Jie Li Sequencing Co., Ltd. to confirm that the mutants were correct. PrimeStar Max DNA polymerase used in each example was purchased from Takara, and Easy Taq DNA polymerase was purchased from Shanghai Yisheng Biotechnology Co., Ltd. The above two DNA polymerases were purchased with corresponding polymerase buffer and dNTPs. EveGreen dye was purchased from Shanghai Yisheng Biotechnology Co., Ltd.

[0047] The DNA purification kit used in the examples was purchased from BBI, the RNA extraction solution was purchased from Kingclone Biotechnology Co., Ltd., the plasmid extraction kit was purchased from Tiangen Biotechnology Co., Ltd., the Trans5a engineering plasmid strain was purchased from Beijing Quanshijin Biotechnology Co., Ltd., the Rosetta engineering protein expression strain was homemade in the laboratory, the nickel column affinity chromatography column was purchased from GE healthcare, the ultrafiltration column was purchased from Merck Millipore, and the protein Marker was purchased from Shanghai Yishen Biotechnology Co., Ltd.

[0048] II. Experimental reagents:

[0049] (1) 5×BufferA (PH=8.0): 100mM Tris Base, 1000mM NaCl, 50%

[0050] (v / v) glycerol.

[0051] (2) BufferB (PH=8.0): 20mM Tris Base, 200mM NaCl, 10% (v / v)

[0052] Glycerol, 250 mM imidazole.

[0053] (3) Stock Buffer (PH=7.4): 20mM Tris Base, 200mM KCl, 0.2mMEDTA.

[0054] III. Experimental instruments:

[0055] Main instrument and equipment used in the examples: Biotek Synergy 2 multi-functional microplate reader (BioTek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge22R tabletop high-speed refrigerated centrifuge (Beckman, USA), PCR amplifier (Biometra, Germany), ultrasonic disruptor (Ningbo Xinzhi Company), nucleic acid electrophoresis apparatus (Shenneng Bojin Company), fluorescence spectrophotometer (Varian, USA), CO 2 constant temperature cell incubator (Sanyo Electric Co., Ltd., SANYO).

[0056] Example 1: Construction and purification of M-MLV RT mutants

[0057] I. Construction of M-MLV RT mutants

[0058] Download the original sequence of M-MLV RT gene (Entry: 4MH8) from the PDB database, send the gene sequence to Nanjing Genscript for codon optimization and synthesis, clone the gene sequence onto the vector pET28a, and finally synthesize the plasmid pET28a-M-MLVWT plasmid. The amino acid sequence is shown in SEQ ID NO: 1. The synthesized sequence contains a 6×His tag at the N-terminus for protein purification. An NheI restriction site is added at the 5' end, a TAA stop codon and an EcoRI restriction site are added at the 3' end, and a kanamycin gene is included for screening of the constructed plasmid. After the synthesis arrives, transformation is carried out, and it is spread on a solid culture dish containing kanamycin and placed in an incubator at 37°C for overnight culture. Trans5a (TransGen Biotech) is used as the engineering strain for plasmid construction, and Rosetta is used as the engineering strain for protein expression. Single colonies are picked, cultured in a shaker, plasmids are extracted and sent for sequencing to ensure the correct gene synthesis. On the basis of confirming the correct sequence, site-directed mutagenesis primers are synthesized, and pET28a-M-MLV-WT is used as the initial mutant plasmid template to construct M-MLV RT mutants.

[0059] II. Induced expression and purification of M-MLV RT mutant proteins

[0060] 1. Exploration of M-MLV RT expression conditions

[0061] Explore the IPTG concentration and induction time for protein induction. IPTG concentration: 0.1 mM - 1 mM. Time: 16 h, 20 h. The experimental results are as Figure 1 shown.

[0062] The experimental results show that when the final concentration of IPTG is 0.6 mM and the induction time is 16 h, the protein can be highly expressed.

[0063] 2. Induced expression of M-MLV RT mutant:

[0064] Take the solid medium cultured overnight, pick a single colony and add it to a 10 mL shaking tube containing 100 mg / mL kanamycin, and place it in a shaking incubator at 37 °C and 220 rpm for 6 h. According to a ratio of 1:100, take 1 mL of the primary bacterial solution and add it to a 100 mL flask containing 100 mg / mL kanamycin for further scale-up culture. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the optical density (OD) of the bacterial solution 600 When OD 600 is between 0.4 and 0.6, add 60 μL of 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) for induction, so that the final concentration of IPTG is 0.6 mM; place it in a shaking incubator at 18 °C and 180 rpm for 16 h for low-temperature induction for protein expression.

[0065] 3. Purification of M-MLV RT mutant:

[0066] The purification steps include:

[0067] (1) Transfer 100 mL of the bacterial solution to a 50 mL centrifuge tube, centrifuge at 4 °C and 4000 rpm for 15 min to harvest the bacteria. Discard the culture medium and retain the bacterial cell pellet, and resuspend the cell pellet with 30 mL of 1×Buffer A.

[0068] (2) Use a Laneige ultrasonic cell disruptor to perform ultrasonic disruption of the bacteria, and set the power to: 350 W, 25 min.

[0069] (3) After the bacteria are disrupted, place it at 4 °C and 4000 rpm, centrifuge for 30 min, collect the supernatant, and discard the pellet.

[0070] (4) Loading: Load the supernatant, and after loading, equilibrate with 1×Buffer A for 5 column volumes to wash away some miscellaneous proteins.

[0071] (5) Washing away impurities: Wash away the miscellaneous proteins with a 60 mM imidazole solution, and use Bradford solution to detect whether the miscellaneous proteins are washed clean.

[0072] (6) Elution: Add 6 mL of 1×Buffer B for elution, and the final concentration of imidazole is 250 mM.

[0073] (7) The target protein eluted is desalted and concentrated using a 10 kDa ultrafiltration column: centrifuge at 4 °C and 4000 rpm for 1 h. When the volume of the protein retained on the ultrafiltration membrane is 200 μL - 500 μL, Stock Buffer can be added for buffer exchange.

[0074] (8) Add 15 mL of StockBuffer to the ultrafiltration column, centrifuge at 4000 rpm for 1 h at 4 °C, so that the liquid remaining in the filter membrane of the ultrafiltration tube is 200 μL - 500 μL.

[0075] (9) Repeat step (8).

[0076] (10) Pipette the protein solution from the ultrafiltration column into a 1.5 mL centrifuge tube, add an equal volume of glycerol and mix well for protein preservation.

[0077] (11) Protein quantification: Measure A280, and dilute all the protein to 1 mg / mL using the protein preservation buffer. Store at -80 °C in the refrigerator.

[0078] 4. SDS-PAGE electrophoresis:

[0079] The electrophoresis procedure includes:

[0080] (1) The total mass of protein loaded is 10 μg. Denature the protein using SDS Loading Buffer, and perform denaturation treatment at 95 °C for 3 min in a thermal cycler. After the incubation ends, quickly insert the sample into ice for cooling.

[0081] (2) Preparation of electrophoresis buffer: Dilute 10×SDS-Mops Running Buffer with deionized water to 1×SDS-Mops Running Buffer.

[0082] (3) Loading: Perform electrophoresis using YoungPAGE protein precast gel (GenScript). Before loading, pipette and rinse each loading well to wash away residues such as glycerol in the precast gel wells, to avoid affecting electrophoresis.

[0083] (4) Electrophoresis: First, perform electrophoresis at a voltage of 100 V for 20 min, and then perform electrophoresis at a voltage of 200 V for 40 min.

[0084] (5) Staining: After electrophoresis, take out the protein gel, rinse it briefly, and place it in a container with staining solution for staining at room temperature for 20 min.

[0085] (6) Decolorization: After staining, add decolorizing solution, place it on a small shaker, and perform overnight decolorization treatment.

[0086] (7) Imaging: Use a gel imaging device to take fluorescence photos to determine the purification status of M-MLV RT. The purification results are as Figure 2 shown.

[0087] The experimental results show that the M-MLV RT protein can be purified normally and there are basically no impurity bands. From 100 mL of bacterial solution, 2 mg of the native M-MLV RT protein can be purified, while in the present invention, 10 mg of the M-MLV RT mutant (Max5.0) can be obtained, indicating an increase in protein yield.

[0088] Example 2. Detection method for the enzymatic activity of the RNA aptamer M-MLV RT

[0089] The RNA aptamer (Pepper) is used as the template for detecting enzymatic activity. When the RNA aptamer (Pepper) binds to a specific dye, a fluorescence signal is generated. In the absence of the RNA aptamer, the free dye molecules cannot produce fluorescence. Once the reverse transcription reaction occurs, the RNA aptamer forms an RNA-DNA hybrid double strand through primer complementarity. At this time, there is no single-stranded RNA aptamer, which cannot bind to the dye, resulting in a decrease in the fluorescence signal. The activity of the reverse transcriptase can be analyzed based on the fluorescence decrease. The nucleotide sequence of Pepper is shown in SEQ ID NO: 2, and the nucleotide sequence of the Pepper primer (Pepper-Primer) is shown in SEQ ID NO: 3. The detection principle is as Figure 3 shown, and the enzymatic activity detection system is shown in Table 1 below.

[0090] Table 1

[0091]

[0092] The M-MLV (D524G) protein sequence is grouped by domain and input into the Rosetta software: the first group (41aa - 124aa, 160aa - 192aa), the second group (1aa - 40aa, 125aa - 159aa, 193aa - 275aa), the third group (360aa - 468aa), the fourth group (469aa - 671aa), and the fifth group (276aa - 359aa). Calculate ΔΔG, select the sites with ΔΔG < -3 for site-directed mutagenesis, and conduct the detection of thermal stability. The specific operation is as follows: Take 6 μg of the protein and incubate it in a thermal cycler at 60 °C for 15 min. After incubation, dilute the protein to 4 μM using 1×M-MLV buffer. Take 2 μL of the protein and add it to the reverse transcription reaction system in Table 1, and conduct the remaining activity detection at 37 °C for 1 h. The larger the △RFU, the greater the remaining activity and the higher the thermal stability.

[0093] Select the better mutant sites for site stacking. Then, combined with charge engineering, a series of Max series mutants with significantly improved thermal stability are finally screened out and named Max1.0, Max2.0, Max3.0, Max4.0, and Max5.0. The detection results of the thermal stability mutants are as Figure 4 shown.

[0094] The experimental results show that Max4.0 and Max5.0 have the best thermal stability. The amino acid sequences of natural M-MLV RT and Max series mutants are as shown in SEQ ID NO: 4 to SEQ ID NO: 8, and the mutation sites are shown in Table 2 below.

[0095] Table 2 Mutation Sites

[0096]

[0097] Example 3. Detection of Thermal Stability of M-MLV RT Mutants by Fluorescent Quantitative PCR

[0098] The thermal stability of M-MLV RT mutants was verified by two-step fluorescent quantitative PCR. Max1.0, Max2.0, Max3.0, Max4.0, and Max5.0 were incubated at 60 °C, 65 °C, and 70 °C for 10 min respectively for reverse transcription reaction, and the reaction system is shown in Table 3 below. Fluorescent quantitative PCR was performed using the reverse transcription product cDNA, and the reaction system is shown in Table 4 below. Then, agarose gel electrophoresis was performed using the fluorescent quantitative PCR product.

[0099] Table 3 Reverse Transcription Reaction System

[0100]

[0101] Table 4 qPCR Reaction System

[0102]

[0103] Thermal stability was detected by fluorescent quantitative PCR, and the fluorescence amplification pattern is as Figure 5 shown. The Ct values are shown in Table 5 below. The experimental results show that:

[0104] (1) When the reverse transcription reaction temperature was 60 °C, the Ct of Max5.0 was 15.17 ± 0.16, while the Ct of the commercial enzyme SuperScriptⅢ was 17.94 ± 0.18, indicating that the thermal stability of Max5.0 is higher than that of the commercial enzyme SuperScriptⅢ.

[0105] (2) When the reverse transcription reaction temperature was 65 °C, the Ct of Max5.0 was 15.51 ± 0.18, while the Ct of the commercial enzyme SuperScriptⅢ was 19.94 ± 0.14, indicating that the thermal stability of Max5.0 is higher than that of the commercial enzyme SuperScriptⅢ.

[0106] (3) When the reverse transcription reaction temperature was 70 °C, the Ct value of Max5.0 was 21.14 ± 0.15, while the Ct value of the commercial enzyme SuperScriptⅢ was 30.16 ± 0.06, indicating that the thermal stability of Max5.0 was higher than that of the commercial enzyme SuperScriptⅢ.

[0107] Table 5 Ct values

[0108] Name 60 °C, 10 min 65 °C, 10 min 70 °C, 10 min Max 1.0 18.87±0.23 20.55±0.14 29.76±0.2 Max 2.0 17.55±0.23 18.38±1.13 18.38±1.13 Max 3.0 15.79±0.28 16.33±0.22 26.55±2.59 Max 4.0 15.03±0.32 15.44±0.13 22.45±0.13 Max 5.0 15.17±0.16 15.51±0.18 21.14±0.15 SuperScriptⅢ 17.94±0.18 19.94±0.14 30.16±0.06 WT 21.48±0.16 28.34±0.12 29.12±0.7

[0109] Agarose gel electrophoresis was performed using the qPCR products, and the experimental results are as Figure 6 shown. The experimental results showed that:

[0110] (1) When the reverse transcription reaction temperatures were 37 °C and 60 °C, the fluorescence band intensities of native M-MLV RT, the commercial enzyme SuperScriptⅢ, and the M-MLV mutant were basically the same, suggesting that their enzyme activities were basically the same.

[0111] (2) When the reaction temperatures were increased to 65 and 70 °C, there were basically no fluorescence bands for the Max1.0 mutant, native M-MLV RT, and the commercial enzyme SuperScriptⅢ. The Max2.0 mutant had weak fluorescence bands, and the Max3.0, Max4.0, and Max5.0 mutants had relatively bright fluorescence bands, suggesting that they still had relatively good enzyme activities.

[0112] Example 4. Detection of the synthesis ability of M-MLV RT mutants for 3000nt RNA templates

[0113] To further determine the reverse transcription ability of M-MLV RT mutants for longer RNA templates at different temperatures. At 37 °C, 60 °C, 65 °C, and 70 °C respectively, a 20-minute reverse transcription reaction was carried out on a 3000nt RNA template, and the reaction system was as shown in Table 3 above. The reverse transcription product cDNA was diluted 10 times and subjected to ordinary PCR, and the reaction system was as shown in Table 6 below. The PCR products were taken for agarose gel electrophoresis, and the agarose gel was photographed using a gel imaging system. The synthesis ability of the reverse transcriptase mutants was judged according to the band size and the fluorescence intensity of the bands.

[0114] Table 6 Ordinary PCR reaction system

[0115]

[0116]

[0117] The agarose gel electrophoresis results of the detection of the reverse transcription ability for 3000nt RNA templates are as Figure 7 shown. The experimental results showed that:

[0118] (1) Reverse transcription of a 3000 nt RNA template was carried out by incubating at 37 °C for 20 min. Natural M-MLV RT and the commercial enzyme SuperScriptⅢ had relatively weak fluorescent bands, while Max4.0 and Max5.0 had stronger fluorescent bands. This indicates that the synthesis ability of Max4.0 and Max5.0 is higher than that of natural M-MLV RT and the commercial enzyme SuperScriptⅢ.

[0119] (2) Reverse transcription of a 3000 nt RNA template was carried out by incubating at 60 °C for 20 min. Natural M-MLV RT, the commercial enzyme SuperScriptⅢ, and Max1.0 had no fluorescent bands at all, while Max2.0, Max3.0, Max4.0, and Max5.0 had relatively bright fluorescent bands. This indicates that the synthesis ability of Max4.0 and Max5.0 mutants is higher than that of natural M-MLV RT and the commercial enzyme SuperScriptⅢ.

[0120] (3) Reverse transcription of a 3000 nt RNA template was carried out by incubating at 65 °C for 20 min. Only Max4.0 and Max5.0 had relatively bright fluorescent bands. This indicates that the synthesis ability of Max4.0 and Max5.0 mutants is higher than that of natural M-MLV RT and the commercial enzyme SuperScriptⅢ.

[0121] (4) Reverse transcription of a 3000 nt RNA template was carried out by incubating at 70 °C for 20 min. Natural M-MLV RT, the commercial enzyme SuperScriptⅢ, and all mutants had no fluorescent bands. This indicates that all enzymes lost their activity.

[0122] Example 5. Comparison of the KCl tolerance of M-MLV RT mutants

[0123] The KCl tolerance of natural M-MLV RT, the commercial enzyme SuperScriptⅢ, and M-MLV RT mutants was compared. Different concentrations of KCl (75 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM) were prepared in the reverse transcription reaction system, and reverse transcription was carried out under the condition of incubating at 37 °C for 10 min to synthesize the first-strand cDNA. The reaction system is shown in Table 3. After the reaction ended, the product was diluted 10 times for PCR, and the reaction system is shown in Table 6. The PCR product was subjected to agarose gel electrophoresis.

[0124] The results of agarose gel electrophoresis for the detection of KCl tolerance are as Figure 8 shown. The experimental results show that:

[0125] (1) Incubate for 10 min in a reaction system with a KCl concentration of 75 mM to 150 mM at 37°C for reverse transcription of a 1000-nt RNA template. The fluorescence band intensities of native M-MLV RT, commercial enzyme SuperScriptⅢ, and the M-MLV RT mutant are basically the same, indicating that their tolerances to 75 mM to 150 mM KCl are basically the same.

[0126] (2) Incubate for 10 min in a reaction system with a KCl concentration of 200 mM at 37°C for reverse transcription of a 1000-nt RNA template. The fluorescence band intensities of native M-MLV RT and commercial enzyme SuperScriptⅢ become dim, while the Max5.0 mutant still has a very bright fluorescence band. This indicates that the Max5.0 mutant has the highest tolerance to KCl.

[0127] (3) Incubate for 10 min in a reaction system with a KCl concentration of 250 mM to 300 mM at 37°C for reverse transcription of a 1000-nt RNA template. The fluorescence band intensities of native M-MLV RT and commercial enzyme SuperScriptⅢ become significantly dim, while the Max5.0 mutant still has a very bright fluorescence band. This indicates that the Max5.0 mutant has the strongest tolerance to KCl.

[0128] Example 6. Comparison of the formamide tolerance of M-MLV RT mutants

[0129] Compare the formamide tolerances of native M-MLV RT, commercial enzyme SuperScriptⅢ, and M-MLV RT mutants. Prepare formamide with different volume ratios in the reaction system (1%, 2%, 5%, 10%, 15%, 20%), and carry out reverse transcription reaction at 37°C for 10 min to synthesize the first-strand cDNA. The reaction system is shown in Table 3. After the reaction, the product is diluted 10 times for PCR, and the reaction system is shown in Table 6. The PCR products are subjected to agarose gel electrophoresis.

[0130] The results of agarose gel electrophoresis for formamide tolerance detection are as Figure 9 shown. The experimental results show that:

[0131] (1) Incubate for 10 min in a reaction system with a formamide ratio of 1% to 10% at 37°C for reverse transcription of a 1000-nt RNA template. The fluorescence band intensities of native M-MLV RT, commercial enzyme SuperScriptⅢ, and the M-MLV RT mutant are basically the same.

[0132] (2) Incubate for 10 min in a reaction system with 15% - 20% formamide at 37°C for reverse transcription of a 1000 nt RNA template. The fluorescence band brightness of native M-MLV RT and the commercial enzyme SuperScriptⅢ significantly darkened, while the Max5.0 mutant still had a relatively bright fluorescence band. This indicates that the Max5.0 mutant has the strongest tolerance to formamide.

[0133] Example 7. Reaction sensitivity of M-MLV RT mutants

[0134] To further detect the detection sensitivity of the Max5.0 mutant, the commercial enzyme SuperScriptⅢ was used as a control group. The RNA detection template was serially diluted 10-fold (0 ng / μL, 0.005 ng / μL, 0.05 ng / μL, 0.5 ng / μL, 5 ng / μL, 50 ng / μL, 500 ng / μL). At 60°C, 65°C, and 70°C respectively, incubate for 10 min for reverse transcription of a 1000 nt RNA template. After the reaction, take 2 μL of the reverse transcription product for PCR, and perform agarose gel electrophoresis on the PCR product to detect the reaction sensitivity.

[0135] The results of agarose gel electrophoresis sensitivity detection are as Figure 10 shown:

[0136] (1) At 60°C, incubate for 10 min for reverse transcription of a 1000 nt RNA template. The commercial enzyme SuperScriptⅢ can detect an RNA template amount of 50 pg, while the Max5.0 mutant can detect an RNA template amount as low as 5 pg under the same conditions, which is 10 times the reaction sensitivity of the commercial enzyme SuperScriptⅢ.

[0137] (2) At 65°C, incubate for 10 min for reverse transcription of a 1000 nt RNA template. The commercial enzyme SuperScriptⅢ can detect an RNA template amount of 0.5 ng, while the Max5.0 mutant can detect an RNA template amount as low as 50 pg under the same conditions, which is 10 times the reaction sensitivity of the commercial enzyme SuperScriptⅢ.

[0138] (3) At 70°C, incubate for 10 min for reverse transcription of a 1000 nt RNA template. The commercial enzyme SuperScriptⅢ cannot carry out the reverse transcription reaction, while the Max5.0 mutant can detect an RNA template amount as low as 50 ng under the same conditions.

[0139] Finally, it should be noted that the preferred embodiments of the present invention have been disclosed above, but they are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above six embodiments, for anyone familiar with this technology, without departing from the spirit and scope of the present invention, appropriate modifications and decorations can still be made to the technical solutions described in the above embodiments. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. An M-MLV RT mutant, characterized in that: In the M-MLV enzyme mutant, the alanine at position 54 is mutated to proline, the glutamic acid at position 69 is mutated to arginine, the proline at position 162 is mutated to histidine, the threonine at position 246 is mutated to histidine, the histidine at position 204 is mutated to arginine, the glutamic acid at position 302 is mutated to arginine, the threonine at position 306 is mutated to arginine, and the aspartic acid at position 524 is mutated to glycine; the amino acid sequence of the M-MLV enzyme mutant is shown in SEQ ID NO:

8.

2. A polynucleotide molecule, characterized in that: The polynucleotide molecule encodes the M-MLVRT mutant according to claim 1.

3. A carrier, characterized in that: The vector contains the polynucleotide molecule according to claim 2.

4. A recombinant engineered cell strain, characterized in that: The recombinant engineered cell line contains the vector as claimed in claim 3.

5. A method for preparing the M-MLV RT mutant according to claim 1, characterized in that: The polynucleotide molecule according to claim 2 is transformed with the vector according to claim 3, expressed and induced in the host cell according to claim 4, and purified to obtain the M-MLV RT mutant according to claim 1.

6. A kit, characterized in that: The kit comprises the M-MLV RT mutant according to claim 1.

7. The kit according to claim 6, characterized in that: The kit further comprises one or more of an RNA extraction reagent, an RNA template, an RNA adaptor Pepper, a reverse transcription reaction buffer, dNTPs, DEPC water, and a reverse transcription reaction primer.

8. The kit according to claim 6, characterized in that: The nucleotide length of the RNA template is between 1000 nt and 3000 nt. 9 . A method for detecting M-MLV RT enzyme activity based on RNA aptamers, characterized in that: using RNA aptamers to detect the enzyme activity of the M-MLV RT mutant according to claim 1 .

10. The method for detecting M-MLV RT enzyme activity according to claim 9, characterized in that: The concentration of the RNA aptamer was 500 ng / μL.

11. A method for reverse transcription of RNA, characterized in that: The RNA template is reverse transcribed to generate cDNA using the M-MLV RT mutant described in claim 1 and the kit described in claim 6.

12. The method according to claim 11, characterized in that: Reverse transcription was performed at 37°C to 70°C for 10 to 20 min to generate the cDNA.

13. The method according to claim 11, characterized in that: In the reverse transcription reaction system, the concentration of KCl is 75 mM to 300 mM, and the proportion of formamide in the reverse transcription reaction system is 1% to 20%.

14. The method according to claim 11, characterized in that: In the reverse transcription reaction system, the concentration of the RNA template is 1 ng / μL to 500 ng / μL.

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