High-temperature-resistant reverse transcriptase for nucleic acid detection, and preparation method and application thereof

Abstract

The application discloses a high-temperature-resistant reverse transcriptase which can be used for nucleic acid detection, and a preparation method and application thereof. The application carries out random mutation on a reverse transcriptase gene, and carries out screening of high-temperature-resistant reverse transcriptase, and finally successfully screens a high-temperature-resistant M-MLV reverse transcriptase mutant M-MLV-M3, the amino acid sequence of which is shown in sequence 8. Compared with natural M-MLV and commercial high-temperature-resistant M-MLV reverse transcriptase, the M-MLV-M3 has better thermal stability, still has good reverse transcriptase activity at 65 DEG C, can be used for conventional reverse transcription reaction, and can also be used for fluorescence PCR detection, and is especially suitable for high-GC-content or complex-structure RNA templates. The application effectively improves the thermal stability of the reverse transcriptase and the performance of the reverse transcriptase product by optimizing and reforming the reverse transcriptase, and especially improves the research on complex-structure RNA and the nucleic acid detection level.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a high-temperature resistant reverse transcriptase that can be used for nucleic acid detection, and a preparation method and application thereof. Background Art

[0002] Reverse transcriptase, a subfamily of DNA polymerases, possesses both RNA-dependent DNA polymerase and RNase H activities, enabling DNA synthesis from RNA templates. Reverse transcriptase is currently the most widely used enzyme. The reverse transcription reaction it mediates, converting mRNA to cDNA, is an essential step in many gene expression studies and is indispensable for nearly all RNA-related research and clinical protocols. It is also an essential raw material for nucleic acid testing, and its performance directly determines its efficiency. The most commonly used reverse transcriptases are M-MLV reverse transcriptase, derived from Moloney murine leukemia virus, and AMV reverse transcriptase, derived from avian myeloblastoma virus. Compared to AMV reverse transcriptase, M-MLV reverse transcriptase has higher enzymatic activity and lower production costs, leading to its widespread use in nucleic acid testing.

[0003] The performance of natural M-MLV reverse transcriptase is not ideal. Since the optimal reaction temperature for natural M-MLV reverse transcriptase is 37°C, too low a reaction temperature can cause the primer to incorrectly bind to the RNA template, thereby reducing the specificity of nucleic acid detection and causing false-positive results. Moreover, for RNA templates with complex structures or high GC content, the RNA template will self-polymerize to form a hairpin loop secondary structure. This specific secondary structure can participate in the regulation of replication, transcription, and translation. 37°C makes it difficult to open the hairpin loop secondary structure, resulting in the inability of the primer to bind to the RNA template to initiate DNA synthesis, causing the reverse transcription reaction to fail, and ultimately leading to systematic deviations in the transcriptome analysis data or false-negative results in viral nucleic acid detection.

[0004] To address this issue, the reverse transcription reaction temperature needs to be increased. However, studies have shown that natural M-MLV reverse transcriptase rapidly and irreversibly loses its enzymatic activity at 49°C. To improve the thermal stability of M-MLV reverse transcriptase, several international research institutions and companies have attempted to modify M-MLV reverse transcriptase, primarily by modifying multiple key amino acid sites within the enzyme. These modifications have been shown to improve the thermal stability of the modified M-MLV reverse transcriptase. However, despite the impressive results achieved in modifying M-MLV reverse transcriptase, the maximum temperature tolerance of M-MLV reverse transcriptase has currently been increased to 60°C. Current RNA-related scientific research and clinical testing still require M-MLV reverse transcriptases with higher temperature tolerances. Summary of the Invention

[0005] The present application aims to provide a high-temperature resistant M-MLV reverse transcriptase for nucleic acid detection, and a preparation method and application thereof.

[0006] To achieve the above-mentioned object, the present application first provides a protein obtained by mutating glutamic acid at position 12, lysine at position 53, glutamine at position 84, glutamine at position 383, leucine at position 464, leucine at position 603, alanine at position 623 of the amino acid sequence of M-MLV protein to serine, glutamic acid, arginine, proline, glutamic acid, tryptophan, and arginine, respectively, and keeping other amino acid sequences of the M-MLV protein unchanged. The amino acid sequence of the M-MLV protein is shown in SEQ ID NO: 2 in the sequence listing.

[0007] The above-mentioned protein can be any one of the proteins described in (a1)-(a3) below:

[0008] (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing;

[0009] (a2) a protein having the same function obtained by substituting, deleting and / or adding one or several amino acid residues other than the amino acid residues at positions 12, 53, 84, 383, 464, 603 and 623 of the amino acid sequence shown in (a1);

[0010] (a3) a fusion protein obtained by connecting a tag to the N terminus or / and C terminus of the amino acid sequence shown in (a1) or (a2).

[0011] In the above-mentioned (a2), the substitution and / or deletion and / or addition of one or several amino acid residues is substitution and / or deletion and / or addition of not more than 10 amino acid residues, or not more than 9 amino acid residues, or not more than 8 amino acid residues, or not more than 7 amino acid residues, or not more than 6 amino acid residues, or not more than 5 amino acid residues, or not more than 4 amino acid residues, or not more than 3 amino acid residues, or not more than 2 amino acid residues, or not more than 1 amino acid residue.

[0012] In the above (a3), the tag refers to a polypeptide or protein fused with the target protein by DNA in vitro recombination technology, so as to facilitate the expression, detection, tracking and / or purification of the target protein. The tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0013] The protein in the above (a2) or (a3) can be artificially synthesized, or a gene encoding the protein can be synthesized first and then expressed biologically.

[0014] In any of the above proteins, the protein has higher thermal stability than the M-MLV protein (sequence 2). Specifically, it still has good reverse transcriptase activity at 65°C.

[0015] To achieve the above-mentioned purpose, the present application further provides a nucleic acid molecule encoding the above-mentioned protein.

[0016] The nucleic acid molecule encoding the above-mentioned protein provided by the present application is any of the following (b1)-(b3) DNA molecules:

[0017] (b1) the nucleotide sequence is a DNA molecule represented by sequence 7 in the sequence listing;

[0018] (b2) a DNA molecule having 75% or more identity with the nucleotide sequence defined in (b1) and encoding the above-mentioned protein;

[0019] (b3) a DNA molecule hybridizing to the nucleotide sequence defined in (b1) or (b2) under stringent conditions and encoding the above-mentioned protein.

[0020] The nucleic acid molecule can be DNA, such as recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA. The nucleic acid molecule can be a nucleic acid molecule formed by a gene encoding the protein and its regulatory sequence.

[0021] Those skilled in the art can easily mutate the nucleotide sequence encoding the above-mentioned protein of the present application by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence encoding the above-mentioned protein, as long as they encode the above-mentioned protein and have the same function, are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application.

[0022] In the above (b2), the identity refers to sequence similarity with the natural nucleic acid sequence. The "identity" includes a nucleotide sequence having 75% or more, 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein consisting of the amino acid sequence shown in the coding sequence 8 of the present application. The identity can be evaluated by the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%) which can be used to evaluate the identity between related sequences.

[0023] The above 75% or more identity can be at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, or 100% identity.

[0024] In the above (b3), the stringent conditions can be hybridization in a solution of 2xSSC, 0.1% SDS at 68°C and washing the membrane twice for 5 min each time, or hybridization in a solution of 0.5xSSC, 0.1% SDS at 68°C and washing the membrane twice for 15 min each time.

[0025] To achieve the above object, the present application also provides a biological material according to any one of (c1) to (c3) described below:

[0026] (c1) an expression cassette containing the above nucleic acid molecule;

[0027] (c2) a recombinant vector containing the above nucleic acid molecule;

[0028] (c3) a recombinant bacterium containing the above nucleic acid molecule.

[0029] In the above (c1), the expression cassette refers to DNA capable of expressing the above protein in a host cell, which can include not only a promoter that initiates transcription of the above protein-encoding gene sequence, but also a terminator that terminates transcription of the above protein-encoding gene sequence. Further, the expression cassette can also include an enhancer sequence.

[0030] In the above (c2), the vector can be a plasmid, cosmid, bacteriophage, or viral vector. The recombinant vector is specifically a vector obtained by replacing the DNA molecule between the NcoI and XhoI enzyme sites of the pET26b vector with the nucleic acid molecule encoding the above protein.

[0031] In the above (c3), the recombinant bacterium can be a fungus or bacterium containing the above nucleic acid molecule.

[0032] Further, the bacterium can be Escherichia coli. The recombinant bacterium is a bacterium obtained by introducing the nucleic acid molecule encoding the above protein into Escherichia coli.

[0033] Further, the recombinant bacteria are obtained by introducing the recombinant vector into Escherichia coli.

[0034] The Escherichia coli is Escherichia coli BL21 (DE3).

[0035] To achieve the above object, the present application further provides a method for preparing the protein.

[0036] The method for preparing the protein provided by the present application comprises the following steps: expressing the nucleic acid molecule in a host bacteria to obtain the protein.

[0037] Further, the method comprises the following steps: fermenting and culturing the recombinant bacteria to obtain the protein.

[0038] Further, the method for fermenting and culturing can be performed according to the following steps: inoculating the recombinant bacteria into a culture medium to culture until OD 600nm = 0.7-0.8, then adding IPTG to induce culture to obtain a fermentation liquor containing the protein.

[0039] Specifically, the seed culture medium can be LB liquid culture medium containing ampicillin.

[0040] The final concentration of the IPTG in the fermentation culture system can be 0.5 mM.

[0041] The condition of the induction culture can be 37°C, 200 rpm (rotating radius of 13 mm) shaking induction for 5 hours.

[0042] To achieve the above object, the present application further provides a kit.

[0043] The kit provided by the present application comprises the protein.

[0044] Further, the kit further comprises other reagents for performing reverse transcription reaction except reverse transcriptase, such as one or more of RNA extraction reagent, reverse transcription reaction solution, dNTPs, water, RNase inhibitor and reverse transcription primer.

[0045] Further, the kit further comprises reagents for performing PCR reaction, such as one or more of reaction buffer, dNTPs, water, DNA polymerase and amplification primer.

[0046] To achieve the above object, the present application further provides the use of any one of the following (d1)-(d4):

[0047] (d1) the protein as reverse transcriptase;

[0048] (d2) use of the nucleic acid molecule or the biological material in the preparation of reverse transcriptase;

[0049] (d3) use of the protein or the nucleic acid molecule or the biological material or the method or the kit in performing reverse transcription reaction or fluorescent PCR reaction;

[0050] (d4) use of the protein or the nucleic acid molecule or the biological material or the method or the kit in nucleic acid detection.

[0051] To achieve the above-mentioned purposes, the present application finally provides (e1) or (e2) the method:

[0052] (e1) a method for performing reverse transcription reaction or fluorescent PCR reaction, comprising the step of performing reverse transcription reaction or fluorescent PCR reaction (such as one-step fluorescent RT-PCR) with the above-mentioned protein as reverse transcriptase;

[0053] (e2) a method for nucleic acid detection, comprising the step of performing reverse transcription reaction or fluorescent PCR reaction with the above-mentioned protein as reverse transcriptase.

[0054] In any of the above-mentioned applications or methods, the sample in the reverse transcription reaction or fluorescent PCR reaction or nucleic acid detection can be a viral sample, specifically an influenza virus sample (such as an influenza A virus sample or an influenza B virus sample), a new coronavirus sample and a respiratory syncytial virus sample.

[0055] The present application successfully screens a reverse transcriptase mutant strain with consistent enzyme activity with natural M-MLV reverse transcriptase and commercial M-MLV reverse transcriptase, but with greatly improved thermal stability, by randomly mutating reverse transcriptase genes and screening high-thermal-stability reverse transcriptase. Through sequencing of the reverse transcriptase mutant strain, M-MLV-M3, a mutant of M-MLV reverse transcriptase, is obtained. Compared with natural M-MLV and commercial high-temperature-resistant M-MLV, M-MLV-M3 has better thermal stability and still has good reverse transcriptase activity at 65℃. It can be used for conventional reverse transcription reaction, and also can be used for fluorescent PCR detection, especially for RNA templates with high GC content or complex structure. The present application effectively improves the thermal stability of reverse transcriptase and the performance of reverse transcriptase products by optimizing and modifying reverse transcriptase, especially improves the research on complex structure RNA and the level of nucleic acid detection. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 To detect the reverse transcription efficiency of natural M-MLV reverse transcriptase and different M-MLV reverse transcriptase mutants for 500bp fragments at different temperatures by agarose gel electrophoresis.

[0057] Figure 2 Agarose electrophoresis was used to detect the reverse transcription efficiency of natural reverse transcriptase and different M-MLV reverse transcriptase mutants at different temperatures on a 1700 bp fragment.

[0058] Figure 3 Agarose electrophoresis was used to detect the reverse transcription efficiency of commercial M-MLV reverse transcriptase and M-MLV reverse transcriptase mutant M-MLV-M3 at different temperatures on a 1700 bp fragment.

[0059] Figure 4 Agarose electrophoresis was used to detect the reverse transcription efficiency of commercial M-MLV reverse transcriptase and M-MLV reverse transcriptase mutant M-MLV-M3 at 65°C on a 2300 bp fragment.

[0060] Figure 5 Fluorescent PCR was used to detect the reverse transcription efficiency of commercial M-MLV reverse transcriptase and M-MLV reverse transcriptase mutant M-MLV-M3 at different temperatures on influenza virus RNA.

[0061] Figure 6 Fluorescent PCR was used to detect the reverse transcription efficiency of commercial M-MLV reverse transcriptase and M-MLV reverse transcriptase mutant M-MLV-M3 at different temperatures on coronavirus RNA.

[0062] Figure 7 Fluorescent PCR was used to detect the reverse transcription efficiency of commercial M-MLV reverse transcriptase and M-MLV reverse transcriptase mutant M-MLV-M3 at different temperatures on syncytial virus RNA. DETAILED DESCRIPTION

[0063] The present application will be further described in conjunction with the specific embodiments, and the examples given are only to illustrate the present application, not to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0064] The experimental methods in the following examples are all routine methods, unless otherwise specified, according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0065] Example 1, screening and obtaining of reverse transcriptase mutant genes

[0066] I. Preparation of M-MLV mutant expression strain

[0067] 1. The natural M-MLV reverse transcriptase gene was synthesized by Nanjing Kings River Company, and NcoI and XhoI restriction enzyme sites were added at both ends of the gene. The sequence of the synthesized natural M-MLV reverse transcriptase gene is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the protein encoded thereby is shown in SEQ ID NO: 2 in the sequence listing.

[0068] 2. Random mutations were introduced into the synthesized natural M-MLV reverse transcriptase gene using the mispriming PCR technique to obtain an M-MLV random mutation gene library. The operation steps were completed by using the GeneMorph II random mutation kit (Agilent, item number 200550) according to the instructions of the kit.

[0069] 3. The M-MLV random mutation gene library was cloned into the pET26b (Novagen, item number TB071) vector between the NcoI and XhoI enzyme digestion sites to obtain the pET26b recombinant plasmid into which the M-MLV random mutation gene was cloned.

[0070] 4. The pET26b recombinant plasmid into which the M-MLV random mutation gene was cloned was transformed into BL21 (DE3) E. coli competent cells (Quanta Biodesign, item number CD601-02), and LB plates containing 50 μg / mL ampicillin (Merck, item number 69-52-3) and 3.0 g / L lactose (Merck, item number 63-42-3) were coated. The LB plates were incubated at 30°C for 72 hours. The lactose in the LB plate can induce the pET26b recombinant plasmid to express the M-MLV mutant protein (recombinant protein). The recombinant protein is first secreted into the periplasmic space of the E. coli through the pelB signal peptide of the vector, and as the induction time is prolonged, the recombinant protein in the periplasmic space appears around the colonies.

[0071] II. Primary screening of heat-resistant M-MLV

[0072] The target mutant M-MLV expression strain was screened by detecting the M-MLV activity around the colonies. The specific steps are as follows:

[0073] 1. The transformed plate incubated for 72 hours was placed at 65°C for 10 minutes, and the mutant M-MLV with poor heat stability lost its enzyme activity, and only the mutant M-MLV with good heat stability retained its enzyme activity, and its reverse transcriptase activity was detected, so that the heat-resistant mutant M-MLV was screened.

[0074] 2. Reverse transcriptase activity determination: Reverse transcriptase synthesizes DNA using RNA as a template. In this determination scheme, polyribonucleic acid cytosine {poly(rC)} (Biosynthesis) was used as a template, and α-[ 32P]Labeled dGTP (Shanghai Hengyuan Biotech, PN109), during DNA synthesis, alpha-[ 32 P]Labeled dGTP will be incorporated into newly synthesized DNA, and by detecting the radioactivity of the DNA, the activity of the reverse transcriptase can be determined.

[0075] 3. Prepare the reaction system: 50 mM Tris pH 8.3, 75 mM KCl, 8 mM MgCl2, 20 μg / mL poly(rC), 50 μg / mL oligo(dG), 10 mM DTT, 50 mM dGTP and 0.5 μCi alpha-[ 32 P]dGTP.

[0076] 4. Cut a PVDF membrane (Merck, 32031613) of the same size as the bacterial culture plate and soak it in the reaction solution prepared in step 3 for 1 hour.

[0077] 5. Take out the soaked PVDF membrane and place it on a culture dish containing E. coli transformed with recombinant plasmid, mark the corresponding position of the PVDF membrane and the culture dish, and react at 65°C for 30 minutes.

[0078] 6. Gently remove the PVDF membrane and place it in a 55°C oven until completely dry.

[0079] 7. Rinse the dried PVDF membrane with 2xSSC buffer (Merck, S6630) twice and air dry.

[0080] 8. Overlap the air-dried PVDV membrane with a Kodak X-OMAT BT film and press it with a film presser overnight, and process the X-ray film with a developing and fixing reagent kit (Yubo Biotech, YB0020).

[0081] 9. Find the corresponding colonies according to the light points on the X-ray film, pick the colonies and inoculate them into a 96-well culture plate containing liquid LB medium containing 50 μg / mL ampicillin (Merck, 69-52-3), and incubate at 37°C until the bacterial solution concentration OD 600nm = 0.7-0.8.

[0082] 10. Add isopropyl-beta-D-thiogalactopyranoside (IPTG, Merck, 16758) to a final concentration of 0.5 mM, induce at 30°C, 200 rpm (rotational radius 13 mm) for 24 hours.

[0083] 11. Centrifuge the 96-well culture plate at 5000g for 10 minutes, collect the bacterial solution supernatant, and further screen and identify.

[0084] Results showed that: after the recombinant plasmid transformed BL21 (DE3) E. coli, each plate grew about 500 colonies on average, a total of 2000 plates were transformed, and about 10 million single colonies were generated. Through this round of screening, a total of 52 high-temperature-resistant mutant strains were obtained.

[0085] III. Secondary screening of high-temperature-resistant M-MLV

[0086] One-step fluorescence PCR was used to identify the enzyme activity and thermal stability of M-MLV reverse transcriptase mutants expressed by the 52 high-temperature-resistant mutant strains obtained by screening. RNA extracted from the national reference of influenza A virus nucleic acid (China Food and Drug Inspection Research Institute, batch number 370051-201801) was used as the template, and one-step qRT-PCR kit (Takara, product number RR064A) was used for reverse transcription and PCR reaction. The specific primer and probe for influenza A virus were published by the US Centers for Disease Control and Prevention (CDC), and the specific reaction system and reaction conditions are shown in Table 1 and Table 2. The specific primer sequences are as follows:

[0087] IFNAF: 5'-GACCRATCCTGTCACCTCTGAC-3';

[0088] IFNAR: 5'-AGGGCATTYTGGACA AAKCGTCTA-3';

[0089] IFNAP: FAM-5'-TGCAGTCCTCGCTCACTGGGCACG-3'-BHQ1.

[0090] Table 1, fluorescence PCR reaction system

[0091] Component Volume (μL) 2x RT-PCR buffer 12.5 Taq enzyme 0.5 Mutant M-MLV 5 Upstream primer IFNAF (40 μΜ) 0.5 Downstream primer IFNAR (40 μΜ) 0.5 Probe IFNAP (10 μΜ) 0.5 RNA template 5.5 Total volume 25

[0092] Table 2, fluorescence PCR amplification conditions

[0093]

[0094] Note: *FAM fluorescence is collected at 55°C extension stage.

[0095] Select the mutant M-MLV that can form an amplification S curve in the fluorescence PCR result, and has a low PCR Ct value and a high peak, as a candidate variant, and sequence the candidate variant to confirm the mutation site.

[0096] Results showed that: 52 M-MLV reverse transcriptase mutants were identified by fluorescence PCR, of which 3 M-MLV reverse transcriptase mutants could form good RT-PCR reactions, and were named M-MLV-M1, M-MLV-M2, and M-MLV-M3, respectively.

[0097] Further gene sequencing analysis of the three M-MLV reverse transcriptase mutants showed that there were multiple point mutations in the three M-MLV reverse transcriptase mutants by comparing with the gene sequence of the natural M-MLV reverse transcriptase. After removing the nonsense mutations, the amino acid mutation sites of the M-MLV reverse transcriptase mutant M-MLV-M1 were S67L, E302K, G386V, P485Q, and L624I, respectively; the amino acid mutation sites of the M-MLV reverse transcriptase mutant M-MLV-M2 were R241P, G290R, A623G, and K668R, respectively; and the amino acid mutation sites of the M-MLV reverse transcriptase mutant M-MLV-M3 were E12S, K53E, Q84R, Q383P, L464P, L603W, and A623G, respectively.

[0098] The gene sequence of the M-MLV-M1 mutant is shown in SEQ ID NO: 3 in the sequence listing, and the amino acid sequence of the M-MLV-M1 mutant encoded thereby is shown in SEQ ID NO: 4 in the sequence listing; the gene sequence of the M-MLV-M2 mutant is shown in SEQ ID NO: 5 in the sequence listing, and the amino acid sequence of the M-MLV-M2 mutant encoded thereby is shown in SEQ ID NO: 6 in the sequence listing; and the gene sequence of the M-MLV-M3 mutant is shown in SEQ ID NO: 7 in the sequence listing, and the amino acid sequence of the M-MLV-M3 mutant encoded thereby is shown in SEQ ID NO: 8 in the sequence listing.

[0099] Example 2, Preparation and purification of M-MLV reverse transcriptase mutants

[0100] I. Protein expression

[0101] 1. The recombinant plasmids pET26b-M-MLV, pET26b-M-MLV-M1, pET26b-M-MLV-M2, and pET26b-M-MLV-M3 containing the natural M-MLV reverse transcriptase and the three M-MLV reverse transcriptase mutants, respectively, were transformed into BL21(DE3) competent cells, and LB plates containing 50 μg / mL ampicillin were coated. The culture was incubated at 37°C for 15 hours.

[0102] The recombinant plasmid pET26b-M-MLV was obtained by replacing the DNA molecule between the NcoI and XhoI enzyme cutting sites of the pET26b vector with the DNA molecule shown in SEQ ID NO: 1 in the sequence listing, while keeping other sequences of the pET26b vector unchanged.

[0103] The recombinant plasmid pET26b-M-MLV-M1 was obtained by replacing the DNA molecule between the NcoI and XhoI enzyme cutting sites of the pET26b vector with the DNA molecule shown in SEQ ID NO: 3 in the sequence listing, while keeping other sequences of the pET26b vector unchanged.

[0104] The recombinant plasmid pET26b-M-MLV-M2 is obtained by replacing the DNA molecule between the Ncol and Xhol enzyme cutting sites of the pET26b vector with the DNA molecule shown in SEQ ID NO: 5 in the sequence listing, while keeping other sequences of the pET26b vector unchanged.

[0105] The recombinant plasmid pET26b-M-MLV-M3 is obtained by replacing the DNA molecule between the Ncol and Xhol enzyme cutting sites of the pET26b vector with the DNA molecule shown in SEQ ID NO: 7 in the sequence listing, while keeping other sequences of the pET26b vector unchanged.

[0106] 2. A single colony was picked and inoculated into LB liquid medium containing 50 μg / mL ampicillin, and incubated at 37°C with shaking at 100 rpm (rotating radius of 13 mm) until the OD 600nm = 0.7-0.8, and 0.5 mM IPTG was added to induce expression at 37°C with shaking at 200 rpm (rotating radius of 13 mm) for 5 hours.

[0107] 3. The culture was centrifuged at 5000 g for 10 min, and the bacterial pellet (induced bacteria) was collected. The pellet was resuspended in 30 mM Tris-HCl pH 8.0, 20% sucrose, 1 mM EDTA (80 mL per gram of bacteria), and then placed in an ice bath and gently shaken for 10 min.

[0108] 4. The bacteria were centrifuged at 8000 g for 10 min, and the supernatant was removed. The bacterial pellet was resuspended in 5 mM MgSO4, placed in an ice bath, and gently shaken for 10 min.

[0109] 5. The bacteria were centrifuged at 12000 g for 15 min, and the supernatant was collected as the solution of the expressed protein in the periplasmic space (recombinant reverse transcriptase protein solution).

[0110] II. Protein purification

[0111] The protein solution prepared in step one was purified respectively, and finally four kinds of M-MLV recombinant proteins were obtained, which were natural M-MLV reverse transcriptase, three M-MLV reverse transcriptase mutants M-MLV-M1, M-MLV-M2, and M-MLV-M3. The specific purification steps are as follows: the protein solution in the periplasmic space prepared in step one was diluted 5 times with binding buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 8.0), and then filtered with a 0.22 μm filter needle (Corning, item number CLS431229). The affinity chromatography column HisTrap FF (GELife, item number 17-5286-01) was selected for purification, and the sample was added to the chromatography column at a speed of 0.3 mL / min, and the impurities were washed with washing buffer (20 mM Tris, 500 mM NaCl, 60 mM imidazole, pH 8.0); the target protein was eluted with elution buffer (20 mM Tris, 500 mM NaCl, 200 mM imidazole, pH 8.0); the target elution peak was collected, and the collected elution peak liquid was further passed through the HiLoad Superdex 200 size exclusion chromatography column (GELife, item number 17-1071-01), and the target protein was separated at a speed of 0.4 mL / min with glycerol-free storage buffer (40 mM Tris, 200 mM NaCl, 0.25 mM EDTA, 0.01% NP-40, 2.5 mM DTT, pH 7.5), and the target elution peak was collected, and the purified recombinant reverse transcriptase solution was filtered with a 0.22 μm filter needle (Corning, item number CLS431229). The purified reverse transcriptase protein solution was quantified by BCA method (ThermoFisher, item number A53225), and an equal volume of sterilized glycerol was added, and stored at -20°C.

[0112] Example 3, identification of M-MLV reverse transcriptase mutants

[0113] I. Enzyme activity identification at different temperatures

[0114] 1. For influenza A virus genome, primers of different lengths were designed to evaluate the reverse transcription reaction efficiency of each M-MLV reverse transcriptase mutant (M-MLV-M1, M-MLV-M2, M-MLV-M3), and the primer sequences are shown in Table 3.

[0115] Table 3, PCR primer sequences

[0116]

[0117] 2. Reverse transcription was performed using RNA extracted from the National Reference Material for Influenza A Virus Nucleic Acid (China Food and Drug Inspection Institute, batch number: 370051-201801) as a template. The reverse transcription reaction system was prepared according to the formula shown in Table 4, and then the reaction was carried out at 42°C or 65°C for 30 minutes.

[0118] Table 4. Reverse transcription reaction system

[0119] Component Volume (μL) 5x RT Buffer (Gold, Cat. No. AT101) 4 10 mM dNTP 1 Native or mutant M-MLV protein (30 μg / mL) 1 RNase inhibitor (50 units / μL) (Gold, Cat. No. AI101) 0.5 Upstream primer (40 μΜ) 1 RNA template 5 Pure water 7.5 Total volume 20

[0120] 3. The DNA obtained by reverse transcription was used as a template for further PCR reaction using a PCR kit (Full Formula Gold, catalog number AS111-11). The reaction system and reaction conditions are shown in Tables 5 and 6.

[0121] Table 5. PCR reaction system

[0122] Component Volume (μL) 2x PCR mix 12.5 Upstream primer (10 μΜ) 0.5 Downstream primer (10 μΜ) 0.5 DNA template 2 Pure water 9.5 Total volume 25

[0123] Table 6. PCR amplification conditions

[0124]

[0125] 4. Identify the PCR amplification product by agarose electrophoresis. Figure 1 and Figure 2 As shown in the figure, N represents natural M-MLV reverse transcriptase, M1 represents M-MLV-M1, M2 represents M-MLV-M2, M3 represents M-MLV-M3, and M represents molecular weight standard.

[0126] The results show that there is no significant difference between the native M-MLV reverse transcriptase and the three M-MLV reverse transcriptase mutants at 42°C for small fragments (500bp), but when the reaction temperature is raised to 65°C, the PCR bands of the native M-MLV reverse transcriptase and the M-MLV reverse transcriptase mutant M-MLV-M1 become significantly weaker, indicating that the enzyme activity is significantly reduced, while the PCR bands of the M-MLV reverse transcriptase mutants M-MLV-M2 and M-MLV-M3 remain obvious, indicating that they can still maintain good enzyme activity at 65°C. For large fragments (1700bp), at 42°C, the PCR bands of the native M-MLV reverse transcriptase, M-MLV reverse transcriptase mutant M-MLV-M1 and M-MLV-M2 are significantly weaker than M-MLV reverse transcriptase mutant M-MLV-M3, but when the reaction temperature is raised to 65°C, only M-MLV reverse transcriptase mutant M-MLV-M3 can achieve DNA synthesis, and the other three M-MLV (native M-MLV reverse transcriptase, M-MLV reverse transcriptase mutant M-MLV-M1 and M-MLV-M2) cannot achieve effective enzymatic reaction, indicating that the DNA synthesis efficiency of M-MLV reverse transcriptase mutant M-MLV-M3 is superior to that of the native M-MLV reverse transcriptase and M-MLV reverse transcriptase mutants M-MLV-M1 and M-MLV-M2. Therefore, it can be concluded that the high temperature resistance and DNA polymerization ability of M-MLV reverse transcriptase mutant M-MLV-M3 are superior to those of the native M-MLV reverse transcriptase and M-MLV reverse transcriptase mutants M-MLV-M1 and M-MLV-M2

[0127] II. Comparison with commercial high-temperature-resistant M-MLV activity

[0128] In order to further confirm the high temperature resistance and DNA polymerization ability of the M-MLV reverse transcriptase mutant M-MLV-M3 obtained by screening, two kinds of commercial high-temperature-resistant M-MLV reverse transcriptases were selected for comparison with M-MLV reverse transcriptase mutant M-MLV-M3. The two commercial high-temperature-resistant M-MLV reverse transcriptases are SuperScript III M-MLV reverse transcriptase (ThermoFisher, item number 18080044) and Induro M-MLV reverse transcriptase (NEB, item number M0681L). The technical data of SuperScript III (SSIII) M-MLV reverse transcriptase shows that its optimum reaction temperature is 50°C, and the technical data of Induro M-MLV reverse transcriptase shows that its optimum reaction temperature is 45°C-60°C. The three M-MLV reverse transcriptases were compared by using conventional reverse transcription PCR method and one-step fluorescence PCR method, respectively.

[0129] 1. Conventional reverse transcription PCR

[0130] The reverse transcription PCR was respectively selected for the fragments with the size of 1700bp and 2300bp. The RNA extracted from the national reference of influenza A virus nucleic acid (China Institute for Food and Drug Control, batch number 370051-201801) was used as the template for the reverse transcription reaction, the primers were respectively selected as For2 or For3 in Table 3, the reaction system was prepared according to the formula shown in Table 7, and for the fragments with the size of 1700bp, the reaction was carried out at 50℃, 60℃ and 65℃, and for the fragments with the size of 2300bp, only the reaction was carried out at 65℃.

[0131] Table 7, reverse transcription reaction system

[0132] Component Volume (μL) 5x RT Buffer (ThermoFisher, Cat. No. 18080044) 4 10 mM dNTP 1 0.1 M DTT 1 Commercial M-MLV or M-MLV-M3 1 RNase inhibitor (50 units / μL) (Gold, Cat. No. AI101) 0.5 Upstream primer (40 μΜ) 1 RNA template 5 Pure water 6.5 Total volume 20

[0133] The DNA obtained by reverse transcription was used as the template for the PCR reaction, the PCR reagent kit (Quangen, item number AS111-11) was used for the reaction, and the reaction system and reaction conditions are shown in Table 8 and Table 9.

[0134] Table 8, PCR reaction system

[0135]

[0136]

[0137] Table 9, PCR amplification conditions

[0138]

[0139] The PCR products were identified by agarose electrophoresis. The electrophoresis diagram is shown in Figure 3 and Figure 4 , wherein SSIII represents SSIIIM-MLV, Induro represents Induro M-MLV, M3 represents M-MLV-M3, and M represents the molecular weight standard.

[0140] The results show that for a 1700 bp fragment, the band brightness of the PCR products is consistent for the two commercial M-MLV reverse transcriptases and the M-MLV reverse transcriptase mutant M-MLV-M3 when the reverse transcription reaction is performed at 50°C, indicating that the reverse transcription reaction efficiencies are similar. When the reaction temperature is increased to 60°C, the band brightness of the PCR products of the SSIII M-MLV reverse transcriptase is weaker than that of the M-MLV reverse transcriptase mutant M-MLV-M3 and the Induro M-MLV, indicating that the reverse transcription reaction efficiency of the SSIII M-MLV reverse transcriptase is lower than that of the other two reverse transcriptases at 60°C. When the reaction temperature is further increased to 65°C, the band brightness of the PCR products of the SSIII M-MLV reverse transcriptase and the Induro M-MLV reverse transcriptase is weaker than that of the M-MLV reverse transcriptase mutant M-MLV-M3, while the M-MLV reverse transcriptase mutant M-MLV-M3 can still achieve good enzymatic reaction, indicating that the high-temperature resistance of the M-MLV reverse transcriptase mutant M-MLV-M3 is better than that of the other two high-temperature resistant commercial reverse transcriptases. In order to confirm the results, a longer fragment (2300 bp) is further tested, and the results show that at 65°C, the band brightness of the PCR products of the SSIII M-MLV reverse transcriptase and the Induro M-MLV reverse transcriptase is weaker than that of the M-MLV reverse transcriptase mutant M-MLV-M3, thereby confirming that the M-MLV reverse transcriptase mutant M-MLV-M3 has better high-temperature resistance than the two high-temperature resistant commercial reverse transcriptases.

[0141] 2. One-step fluorescent RT-PCR

[0142] In order to verify the application potential of the M-MLV reverse transcriptase mutant M-MLV-M3 in the fluorescent RT-PCR kit, three common viruses (influenza virus, new coronavirus and syncytial virus) are selected as detection targets, and one-step fluorescent RT-PCR methods are established using the commercial reverse transcriptases SSIII M-MLV and Induro M-MLV and the M-MLV reverse transcriptase mutant M-MLV-M3. By comparing the PCR Ct values and peak shapes at different reverse transcription temperatures, the reverse transcriptase performance of the M-MLV reverse transcriptase mutant M-MLV-M3 is determined. The specific steps are as follows:

[0143] The influenza B virus nucleic acid standard material (Institute of Metrology Science and Technology of China, standard material number NIM-RM 4056), the new coronavirus nucleic acid standard material (Institute of Metrology Science and Technology of China, standard material number GBW(E)091089), and the respiratory syncytial virus nucleic acid standard material (Institute of Metrology Science and Technology of China, standard material number NIM-RM 4058) were used as templates, and one-step qRT-PCR kit (Takara, product number RR064A) was used for reverse transcription and PCR reaction, the sequences of the three virus-specific primers and probes are shown in Table 10, and the specific reaction system and reaction conditions are shown in Tables 11 and 12.

[0144] Table 10, sequences of fluorescence PCR primers and probes

[0145]

[0146] Table 11, fluorescence PCR reaction system

[0147]

[0148]

[0149] Table 12, fluorescence PCR amplification conditions

[0150]

[0151] Note: *FAM fluorescence is collected at the extension stage of 55℃.

[0152] The fluorescence PCR amplification peak map is as Figure 5 、 Figure 6 and Figure 7The results show that for influenza B virus, the PCR Ct values of the two commercial reverse transcriptases (SSII M-MLV and Induro M-MLV) and the M-MLV reverse transcriptase mutant M-MLV-M3 are consistent at 50°C, only the peak height is slightly different, suggesting that the enzyme activities of the three reverse transcriptases are close at 50°C; when the reverse transcription temperature is increased to 65°C, the peak type of the M-MLV reverse transcriptase mutant M-MLV-M3 is normal, suggesting that it can still perform reverse transcription reaction normally at 65°C, while the amplification peak types of the reverse transcriptases SSII M-MLV and Induro M-MLV are significantly lower, and the Ct values are larger, suggesting that their enzyme activities are greatly affected, proving that the high temperature resistance performance of the M-MLV reverse transcriptase mutant M-MLV-M3 is better than that of the other two high temperature resistant commercial reverse transcriptases. For the new coronavirus, the PCR Ct values of the two commercial reverse transcriptases (SSII M-MLV and Induro M-MLV) and the M-MLV reverse transcriptase mutant M-MLV-M3 are similar at 50°C, only the peak height is slightly different, suggesting that the enzyme activities of the three reverse transcriptases are close at 50°C; when the reverse transcription temperature is increased to 65°C, the peak type of the M-MLV reverse transcriptase mutant M-MLV-M3 is normal, suggesting that it can still perform reverse transcription reaction normally at 65°C, while the amplification peak types of the reverse transcriptases SSII M-MLV and Induro M-MLV are significantly lower, and the Ct values are larger, suggesting that their enzyme activities are greatly affected, proving that the high temperature resistance performance of the M-MLV reverse transcriptase mutant M-MLV-M3 is better than that of the other two high temperature resistant commercial reverse transcriptases. For syncytial virus, the PCR Ct values of the two commercial reverse transcriptases (SSII M-MLV and Induro M-MLV) and the M-MLV reverse transcriptase mutant M-MLV-M3 are consistent at 50°C, only the peak height is slightly different, suggesting that the enzyme activities of the three reverse transcriptases are close at 50°C; when the reverse transcription temperature is increased to 65°C, the peak type of the M-MLV reverse transcriptase mutant M-MLV-M3 is normal, suggesting that it can still perform reverse transcription reaction normally at 65°C, while the amplification peak types of the reverse transcriptases SSII M-MLV and Induro M-MLV are significantly lower, and the Ct values are larger, suggesting that their enzyme activities are greatly affected, proving that the high temperature resistance performance of the M-MLV reverse transcriptase mutant M-MLV-M3 is better than that of the other two high temperature resistant commercial reverse transcriptases.

[0153] In summary, the three reverse transcriptases gave consistent fluorescent PCR results for the three viral RNA templates, which corresponded to the conventional reverse transcription PCR results. Therefore, it can be concluded that the thermostability of the M-MLV reverse transcriptase mutant M-MLV-M3 is superior to the other two thermostable commercial reverse transcriptases.

[0154] The above results show that the M-MLV reverse transcriptase mutant M-MLV-M3 screened in the present application has better thermostability than the natural M-MLV reverse transcriptase and the commercial thermostable M-MLV reverse transcriptase, and still has good reverse transcriptase activity at 65℃, which can be used for conventional reverse transcription reactions, and also can be used for fluorescent PCR detection, especially for RNA templates with high GC content or complex structure.

[0155] The present application has been described in detail. For those skilled in the art, without departing from the spirit and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that the present application can be further improved. In summary, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application. Some basic features can be applied according to the scope of the following attached claims.

Claims

1. A protein, wherein the glutamic acid at position 12 of the amino acid sequence of the M-MLV protein is mutated to serine, the lysine at position 53 is mutated to glutamic acid, the glutamine at position 84 is mutated to arginine, the glutamine at position 383 is mutated to proline, the leucine at position 464 is mutated to glutamic acid, the leucine at position 603 is mutated to tryptophan, and the alanine at position 623 is mutated to arginine, while the rest of the amino acid sequence of the M-MLV protein remains unchanged; The protein is as follows (a1) or (a2): (a1) a protein consisting of the amino acid sequence shown in Sequence 8 in the Sequence Listing; (a2) A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in (a1).

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleic acid molecule is a DNA molecule whose nucleotide sequence is shown as Sequence 7 in the sequence table.

4. The biomaterial described in any one of the following (c1) to (c3): (c1) an expression cassette containing the nucleic acid molecule according to claim 2 or 3; (c2) a recombinant vector containing the nucleic acid molecule according to claim 2 or 3; (c3) A recombinant bacterium containing the nucleic acid molecule according to claim 2 or 3.

5. The biomaterial according to claim 4, characterized in that: The recombinant vector is a vector obtained by replacing the DNA molecule between the NcoI and XhoI restriction sites of the pET26b vector with a nucleic acid molecule encoding the protein according to claim 1.

6. A method for preparing the protein of claim 1, comprising the steps of expressing the nucleic acid molecule of claim 2 or 3 in a host bacterium to obtain the protein of claim 1.

7. A kit comprising the protein of claim 1.

8. Applications for purposes other than disease diagnosis and treatment as described in any of the following (d1) to (d4): (d1) Use of the protein according to claim 1 as a reverse transcriptase; (d2) Use of the nucleic acid molecule according to claim 2 or 3 or the biological material according to claim 4 or 5 in the preparation of reverse transcriptase; (d3) Use of the protein according to claim 1, the nucleic acid molecule according to claim 2 or 3, the biological material according to claim 4 or 5, the method according to claim 6, or the kit according to claim 7 in a reverse transcription reaction or a fluorescent PCR reaction; (d4) Use of the protein according to claim 1, the nucleic acid molecule according to claim 2 or 3, the biological material according to claim 4 or 5, the method according to claim 6, or the kit according to claim 7 in nucleic acid detection.

9. Methods for non-disease diagnosis and treatment purposes as described in (e1) or (e2) below: (e1) A method for performing a reverse transcription reaction or a fluorescent PCR reaction, comprising the step of performing a reverse transcription reaction or a fluorescent PCR reaction using the protein of claim 1 as a reverse transcriptase; (e2) A method for nucleic acid detection, comprising the step of performing a reverse transcription reaction or a fluorescent PCR reaction using the protein according to claim 1 as a reverse transcriptase.

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