Bst DNA Polymerase Mutant and Its Preparation Method and Application

Abstract

The present invention discloses a Bst DNA polymerase mutant and its preparation method and application. In the present invention, a large fragment of wild-type polymerase Bst-LF is subjected to single-site or double-site mutation to obtain a series of single-site or double-site mutants. Compared with the wild-type Bst DNA polymerase, the enzyme activities of the obtained single-site or double-site mutants are significantly improved; among them, the double-site mutant Bst-LF-P236S-M455L can detect plasmid templates at a concentration of 5×10 3 copies / mL in the LAMP amplification reaction, and its sensitivity is higher than that of the wild-type Bst DNA polymerase and commercial enzymes; when applied to the RT-LAMP amplification reaction, its sensitivity is also improved compared with the wild-type Bst DNA polymerase and commercial enzymes, and when the template concentration is 1×10 ‑5 ng / μL, a band can still be amplified.

Description

Technical Field

[0001] The present invention relates to DNA polymerase mutants, in particular to Bst DNA polymerase mutants, their preparation methods and applications, and belongs to the field of DNA polymerase mutants and their applications. Background Art

[0002] Bst DNA polymerase is derived from Geobacillus stearothermophilus ( Geobacillus stearothermophilus , Bst). The large fragment (Bst large fragment, Bst-LF) obtained after hydrolysis by subtilisin has been widely used in various isothermal amplification reactions due to its simultaneous 5′-3′ DNA polymerase activity and powerful strand displacement activity. Especially in loop-mediated isothermal amplification (LAMP), Bst-LF is the most mainstream polymerase.

[0003] The LAMP technique was first proposed by Japanese scientist Notomi in 2000. This technique uses multiple pairs of specific primers to achieve a large amount of amplification of the target fragment at a single temperature under the action of a DNA polymerase with strand displacement activity. This method is simple to operate, low in cost, and low in equipment requirements (only requires a constant temperature condition), and can quickly obtain results in less than 1 hour. It is widely used in the rapid detection and screening of pathogens in clinical scenarios, especially suitable for the prevention and control of animal diseases in farms. It can not only save manpower and detection costs, but also has flexible detection time, shortening the time cycle for disease diagnosis and advancing the time window for farms to respond to the epidemic, laying a good foundation for disease prevention and control.

[0004] Faster and more sensitive LAMP detection will further contribute to the early discovery and less missed detection of clinical diseases. It is also a development trend and requirement formed in recent years during the popularization and use of the LAMP technique. Developing Bst DNA polymerase with high enzyme activity and high sensitivity is one of the keys to the popularization and use of the LAMP technique. Summary of the Invention

[0005] One object of the present invention is to provide mutants of Bst DNA polymerase.

[0006] Another object of the present invention is to provide the coding genes of the mutants of Bst DNA polymerase.

[0007] A third object of the present invention is to provide a recombinant expression vector containing the coding gene of the mutant or a recombinant host cell containing the recombinant expression vector.

[0008] The fourth purpose of the present invention is to use the mutant of the Bst DNA polymerase as a DNA polymerase in the LAMP amplification reaction of DNA or RNA.

[0009] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0010] One aspect of the present invention is to provide a mutant of Bst DNA polymerase.

[0011] The wild-type Bst DNA polymerase large fragment described in the present invention is from Geobacillus stearothermophilus ( Geobacillus stearothermophilus ) DNA polymerase; the amino acid sequence number of the wild-type Bst DNA polymerase large fragment on NCBI is PDB: 1L3S_A.

[0012] In a preferred embodiment of the present invention, the single-site mutant is a single-site mutant obtained by subjecting the amino acid sequence of the Bst DNA polymerase shown in SEQ ID NO.1 to any one of D176G, P236S, M455L or P500S single-site mutations.

[0013] The single-site mutant "Bst-LF-P500S" described in the present invention means that the 500th amino acid of the Bst DNA polymerase with the amino acid sequence shown in SEQ ID NO.1 is mutated from proline (P) to serine (S); the descriptions of the remaining single-site mutants of the present invention are similar.

[0014] In a preferred specific embodiment of the present invention, the double-site mutant is a double-site mutant obtained by mutation of the amino acid sequence of the Bst DNA polymerase shown in SEQ ID NO.1 according to any one of the double-site mutations D176G-P500S, P236S-P500S, and M455L-P500S.

[0015] The double-site mutant "Bst-LF-D176G-P500S" described in the present invention means that the 176th amino acid of the Bst DNA polymerase with the amino acid sequence shown in SEQ ID NO.1 is mutated from aspartic acid (D) to glycine (G) and the 500th amino acid is mutated from proline (P) to serine (S); the description of the remaining double-site mutants of the present invention is similar.

[0016] Another aspect of the present invention is to provide a gene encoding a mutant of the Bst DNA polymerase.

[0017] Another aspect of the present invention is to provide a recombinant expression vector containing the gene encoding the mutant or a recombinant host cell containing the recombinant expression vector.

[0018] A preferred specific implementation of the present invention, the expression vector includes a prokaryotic expression vector or a eukaryotic expression vector; wherein, the eukaryotic expression vector is a baculovirus expression vector of silkworm.

[0019] A preferred specific implementation of the present invention, the host cell includes a living insect or an insect cell line; wherein, the insect cell line is BmN cell of silkworm.

[0020] The present invention further provides a preparation method for preparing the mutant, including: (1) constructing an expression vector containing the coding gene of the single-site mutant or double-site mutant; (2) transforming the constructed expression vector into a host cell, expressing the protein in the host cell, and purifying to obtain.

[0021] Another aspect of the present invention is to apply the mutant of the Bst DNA polymerase as a DNA polymerase to the LAMP amplification reaction of DNA or RNA.

[0022] The present invention mutated the wild-type polymerase Bst-LF at a single site or two sites to obtain a series of single-site or double-site mutants; compared with the wild-type Bst DNA polymerase, the enzyme activities of the single-site or double-site mutants were significantly improved. Among them, the double-site mutant Bst-LF-P236S-M455L can detect 5×10 3 copies / mL of plasmid template in the LAMP amplification reaction, and the sensitivity is higher than that of the wild-type Bst DNA polymerase and the commercial enzyme; when applied to the RT-LAMP amplification reaction, its sensitivity is improved compared with the wild-type Bst DNA polymerase and the commercial enzyme, and when the template concentration is 1×10 -5 ng / μL, a band can still be amplified.

[0023] Term definitions involved in the present invention

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0025] The terms "mutation" and "mutant" have their common meanings herein, referring to genetic, naturally occurring or introduced changes in a nucleic acid or polypeptide sequence, and their meanings are the same as those commonly known to those skilled in the art.

[0026] The term "host cell" or "recombinant host cell" means a cell that contains the polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f - mating or other methods known in the art. The exogenous polynucleotide can be maintained as a non - integrating vector such as a plasmid or can be integrated into the host genome.

[0027] The term "transformation" refers to the genetic transformation of a polynucleotide or polypeptide into a host cell by introducing an encoding gene into the interior of the host cell.

[0028] The term "expression": the transcription and / or translation of an endogenous gene or a transgene in a host cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Electrophoretogram for purifying the obtained wild - type Bst - LF and its mutants; wherein, M is the protein Marker; lane 1 is the purified wild - type Bst - LF; lane 2 is the purified single - site mutant Bst - LF - P500S; lane 3 is the purified double - site mutant Bst - LF - P236S - M455L; lane 4 is the purified double - site mutant Bst - LF - M455L - P500S.

[0030] Figure 2 Gel electrophoretogram of LAMP amplification products of wild - type Bst - LF, double - site mutant Bst - LF - P236S - M455L, and commercial enzyme; M is the DNA Marker; the template amounts in lanes 1 to 8 are 5×10 8 -5×10 1 copies / mL, lane 9 is the negative control with sterile and enzyme - free water added; wherein, Figure 2 -A is the gel electrophoretogram of the LAMP amplification product of wild - type Bst - LF; Figure 2 -B is the gel electrophoretogram of the LAMP amplification product of double - site mutant Bst - LF - P236S - M455L; Figure 2 -C is the gel electrophoretogram of the LAMP amplification product of commercial enzyme.

[0031] Figure 3 Gel electrophoretogram of RT - LAMP amplification products of wild - type Bst - LF, double - site mutant Bst - LF - P236S - M455L, and commercial enzyme; M is the DNA Marker; the template amounts in lanes 1 to 8 are 1×10 1 -1×10 -6 ng / μL, lane 9 is the negative control with sterile and enzyme - free water added; wherein, Figure 3 -A is the gel electrophoretogram of the RT - LAMP amplification product of wild - type Bst - LF;Figure 3 -B is the gel electrophoresis pattern of the RT-LAMP amplification product of the double-site mutant Bst-LF-P236S-M455L; Figure 3 -C is the gel electrophoresis pattern of the RT-LAMP amplification product of the commercial enzyme. Specific implementation manners

[0032] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the described embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0033] Example 1 Expression, purification and detection of Bst-LF single-site mutant in silkworm bioreactor

[0034] 1 Experimental methods

[0035] 1.1 Gene acquisition of Bst-LF single-site mutant

[0036] Corresponding sites were selected for mutation to improve the enzyme activity of Bst DNA polymerase. The amino acid sequence of the wild-type Bst DNA polymerase shown in SEQ ID NO.1 was subjected to the following amino acid single-site mutations: F62S, E131K, D176G, P236S, M455L, P500S. The obtained mutants were named Bst-LF-F62S, Bst-LF-E131K, Bst-LF-D176G, Bst-LF-P236S, Bst-LF-M455L, Bst-LF-P500S. The determined mutant genes were inserted into the pUC57 vector to form plasmids pUC57-Bst-LF-F62S, pUC57-Bst-LF-E131K, pUC57-Bst-LF-D176G, pUC57-Bst-LF-P236S, pUC57-Bst-LF-M455L, pUC57-Bst-LF-P500S respectively. The determined genes were sent to the company for synthesis, and a large number of mutants were rapidly and preliminarily expressed and screened in the prokaryotic system.

[0037] The amino acid sequence of wild-type Bst DNA polymerase is shown below: MAKMAFTLADRVTEEMLADKAALVVEVVEENYHDAPIVGIAVVNEHGRFFLRPETALADPQFVAWLGDETKKKSMFDSKRAAVALKWKGIELCGVSFDLLLAAYLLDPAQGVDDVAAAAKMKQYEAVRPDEAVYGKGAKRAVPDEPVLAEHLVRKAAAIWELERPFLDELRRNEQDRLLVELEQPLSSILAEMEFAGVKVDTKRLEQMGKELAEQLGTVEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPYHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTKKVHTIFNQALTQTGRLSSTEPNLQNIPIRLEEGRKIRQAFVPSESDWLIFAADYSQIELRVLAHIAEDDNLMEAFRRDLDIHTKTAMDIFQVSEDEVTPNMRRQAKAVNFGIVYGISDYGLAQNLNISRKEAAEFIERYFESFPGVKRYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERMAMNTPIQGSAADIIKKAMIDLNARLKEERLQAHLLLQVHDELILEAPKEEMERLCRLVPEVMEQAVTLRVPLKVDYHYGSTWYDAK (SEQ ID NO.1).

[0038]

[0039] 1.2 Construction of recombinant baculovirus transfer vector

[0040] Plasmids pUC57 - Bst - LF - F62S, pUC57 - Bst - LF - E131K, pUC57 - Bst - LF - D176G, pUC57 - Bst - LF - P236S, pUC57 - Bst - LF - M455L, and pUC57 - Bst - LF - P500S were respectively subjected to Bam HI and Eco RI double digestion. The digested products were subjected to agarose gel electrophoresis, and the target fragments were recovered from the gel using the Tiangen agarose gel DNA recovery kit. The target fragments were ligated to the baculovirus transfer vector pBR that had been double digested and inactivated using T4 DNA ligase. The ligation products were transformed into Escherichia coli competent cells TOP10, colonies were selected for culture, plasmids were extracted, and after double digestion with Bam HI and Eco RI to identify positive clones, sequencing verification was performed. The plasmids with correct sequencing were respectively named pBR - Bst - LF - F62S, pBR - Bst - LF - E131K, pBR - Bst - LF - D176G, pBR - Bst - LF - P236S, pBR - Bst - LF - M455L, and pBR - Bst - LF - P500S.

[0041] 1.3 Preparation, purification and amplification of recombinant silkworm baculovirus

[0042] The mutants that were predicted and initially demonstrated to have good mutation effects in the prokaryotic expression system experiments were further optimized for expression in the eukaryotic system. The steps are as follows:

[0043] Seed well-grown BmN cells into a six-well plate at a density of about 60% and culture overnight. According to the amount of each sample, take 2 μL of liposome and 50 μL of sterile ultrapure water, mix at room temperature and let stand for 5 min for later use. Sequentially add 1 μg of the silkworm baculovirus parental strain BmBac DNA preserved in the laboratory, 5 μg of transfer plasmid such as pBR-Bst-LF-F62S, and 2 μL of liposome into a sterile centrifuge tube, make up to 60 μL with sterile ultrapure water, gently mix, let stand at room temperature for 15 min, then add dropwise to the BmN cells cultured overnight for co-transfection. Seal with parafilm and place in a 27°C cell culture incubator for 4 h. Then add 1.5 mL of serum-free medium, 300 μL of FBS and 3 μL of double antibody, seal with parafilm and incubate at 27°C for 4 - 5 days until the cells detach and float. Collect the cell culture supernatant to obtain recombinant viruses rBmBac-Bst-LF-F62S, rBmBac-Bst-LF-E131K, rBmBac-Bst-LF-D176G, rBmBac-Bst-LF-P236S, rBmBac-Bst-LF-M455L, rBmBac-Bst-LF-P500S containing the target genes respectively.

[0044] The purification and amplification method of the recombinant silkworm baculovirus is as follows: The mutants with good mutation effects are purified and amplified according to the following method.

[0045] Inoculate cells into a 35 mm small petri dish at a density of about 70% - 80%. After they adhere, aspirate the culture medium, add the cell culture supernatant containing the recombinant virus obtained above diluted with different concentrations (1 mL / dish), gently distribute evenly and incubate at 27°C for 1 h. Aspirate the virus solution that has not infected. Add 4 mL of gel solution to each petri dish (preparation method: melt 2% agarose gel in a 60°C water bath, cool to 40°C and mix evenly with an equal-temperature 2×TC-100 medium containing 20% FBS). After the gel solution solidifies, seal with parafilm and culture upside down at 27°C for 3 - 5 days, and observe under a microscope. Pick out the plaques and repeat the above steps. After 2 - 3 rounds of separate purification, obtain pure recombinant silkworm baculoviruses rBmBac-Bst-LF-F62S, rBmBac-Bst-LF-E131K, rBmBac-Bst-LF-D176G, rBmBac-Bst-LF-P236S, rBmBac-Bst-LF-M455L, rBmBac-Bst-LF-P500S.

[0046] The recombinant Bombyx mori baculovirus was used to infect normally growing BmN cells. After culturing for 3 - 5 days, the supernatant was collected, which contained a large amount of recombinant viruses, namely rBmBac - Bst - LF - F62S, rBmBac - Bst - LF - E131K, rBmBac - Bst - LF - D176G, rBmBac - Bst - LF - P236S, rBmBac - Bst - LF - M455L, and rBmBac - Bst - LF - P500S.

[0047] 1.4 Expression of single - site mutants of Bst - LF

[0048] The recombinant virus culture solution was injected into the 5 - day - old newly molted silkworms at a dose of 10 5 PFU / head and cultured under the conditions of 70% - 80% humidity and 27°C temperature. After about 3.5 d - 4.5 d, symptoms such as swelling of body segments, abnormal behavior, and decreased appetite of silkworm larvae could be observed. When the silkworms stopped eating, it indicated that the silkworm larvae had reached the late growth stage. The mutants Bst - LF - F62S, Bst - LF - E131K, Bst - LF - D176G, Bst - LF - P236S, Bst - LF - M455L, and Bst - LF - P500S were highly expressed under the action of the polyhedrin gene promoter; at this time, the hemolymph was collected and stored at - 20°C for future use.

[0049] 1.5 Purification of the expression products of single - site mutants of Bst - LF

[0050] The collected hemolymph was ultrasonically disrupted in an ice bath. The supernatant obtained by centrifugation was denatured at 60°C for 20 min, and the impurities were removed by centrifugation. Then, referring to the references comprehensively (Huang Weihua, Zhai Feng, Hong Guofan. Cloning, expression, properties and application of the large fragment of Bst DNA polymerase [J]. Acta Biochimica et Biophysica Sinica, 1999, (04): 379 - 385; Kaboev OK, Luchkina LA, Akhmedov AT, Bekker ML. Purification and properties of deoxyribonucleic acid polymerase from Bacillus stearothermophilus. J Bacteriol. The steps of heat treatment denaturation of impurity proteins, ammonium persulfate precipitation, ion exchange chromatography, and heparin affinity chromatography were carried out in sequence according to the method in (1981;145(1):21-26). After elution, it was concentrated using an ultra Amicon® Ultra centrifugal ultrafiltration tube and replaced into the enzyme storage buffer solution. The formula of the enzyme storage buffer solution is: 100 mM KCl, 10 mM Tris-HCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton® X-100, 50% glycerol, and the pH value is 7.4. An appropriate amount of the harvested protein solution was taken for SDS-PAGE analysis of the purification effect; the harvested protein solution was filtered and sterilized, quantified by the BCA method, diluted to 1 mg / mL with the enzyme storage buffer, an appropriate amount of the sample was taken for SDS-PAGE analysis of the purification effect, aliquoted under sterile conditions, and stored at -80 °C.

[0051] 1.6 Enzyme activity assay of Bst-LF and its single-site mutants

[0052] The mutants with good expression were subjected to enzyme activity assay. According to the principle and method disclosed in the patent publication number CN 117946997 A, using wild-type Bst-LF as a control, the DNA polymerase activity and strand displacement activity of the obtained mutants were detected.

[0053] 2 Experimental results

[0054] 2.1 SDS-PAGE identification results

[0055] The purified wild-type Bst-LF and its single-site mutants were analyzed by SDS-PAGE electrophoresis. The experimental results are as Figure 1 shown. Lane 1 is the purified wild-type Bst-LF, and lane 2 is the purified mutant Bst-LF-P500S, which is consistent with the expected size of 67 kDa, proving that a Bst-LF single-site mutant with higher purity was successfully obtained.

[0056] 2.2 Enzyme activity assay results

[0057] The enzyme activity assay results are shown in Table 1. The enzyme activities of the mutants Bst-LF-D176G, Bst-LF-P236S, Bst-LF-M455L, and Bst-LF-P500S were all improved to varying degrees compared with wild-type Bst-LF, which are effective mutations.

[0058] Table 1 Detection results of the enzyme activities of single-site mutants of Bst-LF

[0059]

[0060] Note: Some mutants with poor expression and activity were not presented in this paper.

[0061] Example 2 Expression, Purification and Detection of Bst-LF Double-site Mutants in Silkworm Bioreactor

[0062] 1 Implementation Method

[0063] 1.1 Obtaining Genes of Bst-LF Double-site Mutants

[0064] Four preferred single-site mutants, Bst-LF-D176G, Bst-LF-P236S, Bst-LF-M455L, and Bst-LF-P500S, were combined pairwise for double-site mutation. There were a total of six combinations of double-site mutations, named Bst-LF-D176G-P500S, Bst-LF-D176G-P236S, Bst-LF-D176G-M455L, Bst-LF-P236S-P500S, Bst-LF-P236S-M455L, and Bst-LF-M455L-P500S. The determined mutant genes were inserted into the pUC57 vector to obtain plasmids pUC57-Bst-LF-D176G-P500S, pUC57-Bst-LF-D176G-P236S, pUC57-Bst-LF-D176G-M455L, pUC57-Bst-LF-P236S-P500S, pUC57-Bst-LF-P236S-M455L, and pUC57-Bst-LF-M455L-P500S respectively. The determined genes were submitted to a DNA synthesis company for synthesis.

[0065] 1.2 Construction of Recombinant Baculovirus Transfer Vectors

[0066] The experimental method was the same as 1.2 in Example 1. The correctly identified recombinant plasmids were sequenced, and the plasmids with correct sequencing were named pBR-Bst-LF-D176G-P500S, pBR-Bst-LF-D176G-P236S, pBR-Bst-LF-D176G-M455L, pBR-Bst-LF-P236S-P500S, pBR-Bst-LF-P236S-M455L, and pBR-Bst-LF-M455L-P500S respectively.

[0067] 1.3 Preparation, Purification and Amplification of Recombinant Silkworm Baculovirus

[0068] The experimental method was the same as 1.3 of the experimental method in Example 1. The recombinant viruses obtained after recombination, purification, and amplification were named rBmBac-Bst-LF-D176G-P500S, rBmBac-Bst-LF-D176G-P236S, rBmBac-Bst-LF-D176G-M455L, rBmBac-Bst-LF-P236S-P500S, rBmBac-Bst-LF-P236S-M455L, and rBmBac-Bst-LF-M455L-P500S, respectively.

[0069] 1.4 Expression of Bst-LF double-site mutants

[0070] The experimental method was the same as 1.4 of the experimental method in Example 1.

[0071] 1.5 Purification of the expression products of Bst-LF double-site mutants

[0072] The experimental method was the same as 1.5 of the experimental method in Example 1. The purified mutants were analyzed by SDS-PAGE.

[0073] 1.6 Enzyme activity assay of Bst-LF and its double-site mutants

[0074] The experimental method was the same as 1.6 of the experimental method in Example 1.

[0075] 2 Experimental results

[0076] 2.1 SDS-PAGE identification results

[0077] The purified Bst-LF and its double-site mutants were analyzed by SDS-PAGE electrophoresis. The experimental results are as Figure 1 shown. Lane 1 was the purified wild-type Bst-LF, lane 3 was the purified Bst-LF-P236S-M455L, and lane 4 was the purified Bst-LF-M455L-P500S. An obvious and relatively single target band appeared at a position near the expected size of 67 kDa, indicating the successful purification of the Bst-LF double-site mutants.

[0078] 2.2 Enzyme activity assay results

[0079] The results of enzyme activity assays are shown in Table 2. The enzyme activities of the Bst-LF (Bst DNA polymerase) double-site mutants Bst-LF-P236S-M455L, Bst-LF-D176G-P500S, or Bst-LF-M455L-P500S are higher than those of the wild type and single-site mutants. After mutation at the remaining several sites, the activities remain unchanged or decrease, indicating that the combined mutations at these three double sites are effective mutations and can achieve the purpose of further improving the enzyme activity of the Bst-LF single-site mutants.

[0080] Table 2 Detection results of the enzyme activities of the double-site mutants of Bst-LF

[0081]

[0082] Experimental Example 1 LAMP detection assay for bovine infectious rhinotracheitis virus

[0083] Using the conserved gene of bovine infectious rhinotracheitis virus (IBRV) gB (GenBank Accession No. DQ006857.1) as the target, the full gene was synthesized and cloned into the pUC18 plasmid (pUC18-gB). Primers for LAMP were designed and synthesized for this target gene. The nucleotide sequences of the primers are specifically shown in Table 3 (Dong Shijuan, Feng Meng, Yu Ruisong, et al. Establishment and application of a visual LAMP detection method for bovine infectious rhinotracheitis [J]. Chinese Journal of Biotechnology, 2018, 34(10): 1587-1595). Combining Examples 1 and 2, the Bst DNA polymerase mutant Bst-LF-P236S-M455L with the highest enzyme activity was selected for the LAMP reaction, with the wild-type Bst-LF and the commercial NEB polymerase (Bst 3.0 DNA polymerase, catalog number: M0374S) as controls. The LAMP reaction was carried out according to the reaction system shown in Table 4. The LAMP amplification conditions were 65°C and the reaction time was 30 minutes. Among them, the template plasmid was serially diluted 10-fold with sterile and enzyme-free water, and the concentration range was set to 5×10 8 -5×10 1 copies / mL.

[0084] Table 3 Primer sequences

[0085]

[0086] Table 4 LAMP reaction system

[0087]

[0088] After the LAMP amplification reaction, 2% agarose gel electrophoresis was used to detect the amplification products. The electrophoresis results were as follows: Figure 2 As shown, there was no amplification in the negative control group with sterile and enzyme-free water added. The native wild-type Bst-LF, the double-site mutant Bst-LF-P236S-M455L, and the commercial enzyme could all form ladder-like DNA bands ( Figure 2 -A, Figure 2 -B, Figure 2 -C), and the amplification effects could all meet the requirements of LAMP detection. At the same time, the LAMP amplification reaction using the double-site mutant Bst-LF-P236S-M455L could detect plasmid templates at 5×10 3 copies / mL, and the sensitivity was higher than that of the wild-type Bst-LF and the commercial enzyme ( Figure 2 -B, Figure 2 -C).

[0089] Experimental Example 2 RT-LAMP Detection Test for Bovine Viral Diarrhea Virus

[0090] Using the 5′UTR sequence of bovine viral diarrhea virus (BVDV) as the target, referring to the primers used in the reference (Zhang Yuming, Li Shufan, Chen Zhiyuan, et al. Establishment of a Visual One-Step RT-LAMP Detection Method for Bovine Viral Diarrhea Virus [J]. Chinese Veterinary Science, 2022, 52(08): 985-991), the nucleotide sequences of the primers are shown in Table 5. Referring to the reaction conditions and reaction system in Experimental Example 1, RT-LAMP amplification reaction was carried out, and only the gradient plasmid DNA template was modified to BVDV RNA (5.0 ng / μL) template, which was serially diluted 10-fold with sterile and enzyme-free water, and the concentration range was set to 1×10 1 -1×10 -6 ng / μL.

[0091] Table 5 Primer Sequences

[0092]

[0093] After the RT-LAMP reaction, 2% agarose gel electrophoresis was used to detect the amplification products. The electrophoresis results were as follows: Figure 3 As shown, there was no amplification in the negative control group with sterile and enzyme-free water added, and there was no amplification with the wild-type Bst-LF, which was not applicable to RT-LAMP detection ( Figure 3 -A), while the double-site mutant Bst-LF-P236S-M455L and the commercial enzyme had good reverse transcription activity and could form ladder-like DNA bands, and the amplification effects could all meet the requirements of RT-LAMP detection ( Figure 3 -B, Figure 3-C); Meanwhile, when the double-site mutant Bst-LF-P236S-M455L is applied to RT-LAMP amplification, the sensitivity is improved compared with that of the wild-type Bst-LF and commercial enzymes. When the template concentration is 1×10 -5 ng / μL, the band of BVDV can still be amplified ( Figure 3 -B, Figure 3 -C).

Claims

1. A single-site mutant of Bst DNA polymerase, characterized in that, The single-site mutant is a single-site mutant obtained by performing any one of the single-site mutations of D176G, P236S, M455L, or P500S on the amino acid sequence of Bst DNA polymerase shown in SEQ ID NO.

1.

2. Double-site mutant of Bst DNA polymerase, characterized in that, The double-site mutant is a double-site mutant obtained by performing any one of the double-site mutations of D176G-P500S, P236S-M455L, or M455L-P500S on the amino acid sequence of Bst DNA polymerase shown in SEQ ID NO.

1.

3. The coding gene of the single-site mutant according to claim 1 or the double-site mutant according to claim 2.

4. An expression vector containing the coding gene according to claim 3.

5. The expression vector according to claim 4, characterized in that, The expression vector is a baculovirus expression vector of Bombyx mori.

6. A recombinant host cell containing the expression vector according to claim 4.

7. The recombinant host cell according to claim 6, wherein The recombinant host cell is a recombinant insect cell.

8. The preparation method of the single-site mutant according to claim 1 or the double-site mutant according to claim 2, characterized in that, Comprising: (1) Constructing an expression vector containing the coding gene of the single-site mutant or double-site mutant; (2) Transforming the constructed expression vector into a host cell, expressing the protein in the host cell, and purifying to obtain the product.

9. The preparation method according to claim 8, characterized in that, The expression vector is a baculovirus expression vector of Bombyx mori; The host cell includes an insect in vivo or an insect cell line.

10. Use of the single-site mutant according to claim 1 or the double-site mutant according to claim 2 in the preparation of reagents for LAMP amplification reaction of DNA.

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