Bst DNA polymerase mutant as well as preparation method and application thereof

Abstract

The invention discloses a Bst DNA polymerase mutant as well as a preparation method and application thereof. According to the invention, a wild type polymerase large fragment Bst-LF is subjected to single-site or double-site mutation to obtain a series of single-site or double-site mutants, and compared with a wild type Bst DNA polymerase, the enzyme activity of the obtained single-site or double-site mutants is remarkably improved; wherein the double-site mutant Bst-LF-P236S-M455L is applied to an LAMP (Loop-Mediated Isothermal Amplification) reaction, so that a plasmid template of 5 * 10 < 3 > copies / mL can be detected, and the sensitivity of the double-site mutant Bst-LF-P236S-M455L is higher than that of a wild type Bst DNA polymerase and a commercial enzyme; when the Bst DNA polymerase is applied to RT-LAMP amplification reaction, the sensitivity of the Bst DNA polymerase is also improved compared with that of wild type Bst DNA polymerase and commercial enzyme, and when the concentration of a used template is 1 * 10 <-5 > ng / mu L, bands can still be amplified.

Description

Technical Field

[0001] The invention relates to a DNA polymerase mutant, in particular to a Bst DNA polymerase mutant and a preparation method and application thereof, belonging to the field of DNA polymerase mutants and applications thereof. Background Art

[0002] Bst DNA polymerase is derived from Geobacillus stearothermophilus ( Geobacillus stearothermophilus , Bst), the large fragment (Bst large fragment, Bst-LF) after being hydrolyzed by Bacillus subtilisin has both 5′-3′ DNA polymerase activity and strong strand displacement activity, and has been widely used in various isothermal amplification reactions, especially in loop-mediated isothermal amplification (LAMP), where Bst-LF is the most mainstream polymerase.

[0003] LAMP technology was first proposed by Japanese scientist Notomi in 2000. This technology uses multiple pairs of specific primers under the action of DNA polymerase with chain displacement activity to achieve large-scale amplification of the target fragment at a single temperature. This method is simple to operate, low-cost, and has low equipment requirements (only constant temperature conditions are required). It can quickly produce results in less than 1 hour and is widely used in rapid detection and screening of etiologies in clinical scenarios, especially for animal disease prevention and control in farms. It not only saves manpower and testing costs, but also has flexible testing time, shortens the time period for disease diagnosis, and advances 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 assist in early detection of clinical epidemics and reduce missed detections. It is also a development trend and requirement formed in recent years with the promotion and use of LAMP technology. The development of BstDNA polymerase with high enzyme activity and high sensitivity is one of the keys to the promotion and use of LAMP technology. Summary of the invention

[0005] One of the objects of the present invention is to provide a mutant of Bst DNA polymerase.

[0006] The second purpose of the present invention is to provide a gene encoding a mutant of the Bst DNA polymerase.

[0007] The third object 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.

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

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] Another aspect of the present invention is to provide a gene encoding a mutant of the Bst DNA polymerase.

[0016] 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.

[0017] In a preferred embodiment of the present invention, the expression vector comprises a prokaryotic expression vector or a eukaryotic expression vector; wherein the eukaryotic expression vector is a Bombyx mori baculovirus expression vector.

[0018] In a preferred embodiment of the present invention, the host cell comprises a living insect or an insect cell line; wherein the insect cell line is a Bombyx mori BmN cell.

[0019] The present invention further provides a method for preparing the mutant, comprising: (1) constructing an expression vector containing a gene encoding 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 the mutant.

[0020] Another aspect 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.

[0021] The present invention performs single-site or double-site mutation on the wild-type polymerase Bst-LF to obtain a series of single-site or double-site mutants; compared with the wild-type Bst DNA polymerase, the enzyme activity of the single-site or double-site mutants is significantly improved. Among them, the double-site mutant Bst-LF-P236S-M455L can be used in the LAMP amplification reaction to detect 5×10 3 The sensitivity of the plasmid template was higher than that of wild-type Bst DNA polymerase and commercial enzyme when applied to RT-LAMP amplification reaction. The template concentration was 1×10 -5 ng / μL, the band can still be amplified.

[0022] Definitions of terms used in this invention Unless defined otherwise, 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.

[0023] The terms "mutation" and "mutant" have their ordinary meanings herein and refer to genetic, naturally occurring or introduced changes in nucleic acid or polypeptide sequences, and their meanings are the same as those generally known to those skilled in the art.

[0024] The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector such as a plasmid or may be integrated into the host genome.

[0025] The term "transformation" refers to the genetic transfer of a polynucleotide or polypeptide into a host cell in such a way that the encoding gene is introduced into the interior of the host cell.

[0026] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a host cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure is the electrophoresis diagram of the purified 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.

[0028] Figure 2 The gel electrophoresis diagram of the LAMP amplification products of wild-type Bst-LF, double-site mutant Bst-LF-P236S-M455L, and commercial enzyme; M is DNA marker; the template amount of lanes 1 to 8 is 5×10 8 -5×10 1 copies / mL, lane 9 is a negative control with sterile enzyme-free water; Figure 2 -A is the gel electrophoresis of the LAMP amplification product of wild-type Bst-LF; Figure 2 -B is the gel electrophoresis of the LAMP amplification product of the double-site mutant Bst-LF-P236S-M455L; Figure 2 -C is the gel electrophoresis of the LAMP amplification product of the commercial enzyme.

[0029] Figure 3 Gel electrophoresis of RT-LAMP amplification products of wild-type Bst-LF, double-site mutant Bst-LF-P236S-M455L, and commercial enzyme; M is 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 enzyme-free water; Figure 3 -A is the gel electrophoresis of the RT-LAMP amplification product of wild-type Bst-LF; Figure 3 -B is the gel electrophoresis of the RT-LAMP amplification product of the double-site mutant Bst-LF-P236S-M455L; Figure 3 -C is the gel electrophoresis of the RT-LAMP amplification product of the commercial enzyme. DETAILED DESCRIPTION

[0030] 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 as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0031] Example 1 Expression, purification and detection of Bst-LF single-site mutants in silkworm bioreactors 1 Experimental methods 1.1 Gene acquisition of Bst-LF single-site mutant The corresponding sites were selected for mutation to improve the 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 resulting 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 pUC57 Vectors were used to form 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, respectively. The determined genes were sent to the company for synthesis, and a large number of mutants were quickly and preliminarily expressed and screened in the prokaryotic system.

[0032] The amino acid sequence of wild-type Bst DNA polymerase is shown below: MAKMAFTLADRVTEEMLADKAALVVEVVEENYHDAPIVGIAVVNEHGRFFLRPETALADPQFVAWLGDETKKKSMFDSKRAAVALKWKGIELCGVSFDLLLAAYLLDPAQGVDDVAAAAKMKQYEAVRPDEAVYGKGAKRAVPDEPVLAEHLVRKAAAIWELERPFLDELRRNEQDRLLVELEQPLSSILAEMEFAGVKVDTKRLEQMGKELAEQLGTVEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPYHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTKKVHTIFNQALTQTGRLSSTEPNLQNIPIRLEEGRKIRQAFVPSESDWLIFAADYSQIELRVLAHIAEDDNLMEAFRRDLDIHTKTAMDIFQVSEDEVTPNMRRQAKAVNFGIVYGISDYGLAQNLNISRKEAAEFIERYFESFPGVKRYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERMAMNTPIQGSAADIIKKAMIDLNARLKEERLQAHLLLQVHDELILEAPKEEMERLCRLVPEVMEQAVTLRVPLKVDYHYGSTWYDAK (SEQ ID NO.1).

[0033]

[0034] 1.2 Construction of recombinant baculovirus transfer vector 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 Bam HI and Eco RI double digestion, the digestion product was subjected to agarose gel electrophoresis, and the target fragment was recovered by gel recovery using the Tiangen agarose gel DNA recovery kit; the target fragment was connected to the baculovirus transfer vector pBR that had been double digested and inactivated using T4 DNA ligase. The ligation product was transformed into Escherichia coli competent cells TOP10, colonies were selected for culture, plasmids were extracted, and used respectively Bam HI and Eco After positive clones were identified by RI double digestion, sequencing verification was performed, and the correctly sequenced plasmids were 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.

[0035] 1.3 Preparation, purification and amplification of recombinant silkworm baculovirus The mutants predicted and initially proved to have good mutation effects in the prokaryotic expression system experiment were further optimized and expressed in the eukaryotic system. The steps are as follows: The well-growing BmN cells were plated in a six-well plate at a density of about 60% for overnight culture; according to the amount of each sample, 2 μL of liposomes and 50 μL of sterile ultrapure water were taken, mixed at room temperature, and placed for 5 min for use; 1 μg of Bombyx mori baculovirus parent strain BmBac DNA preserved in the laboratory, 5 μg of pBR-Bst-LF-F62S and other transfer plasmids, and 2 μL of liposomes were added to a sterile centrifuge tube in sequence, and sterile ultrapure water was added to make up to 60 μL, gently mixed, and placed at room temperature for 15 min, and then added dropwise to the overnight cultured BmN cells for co-transfection, sealed with a sealing film, and placed in a 27°C cell culture incubator for 4 h, and then 1.5 mL of serum-free culture medium and 300 μL of FBS and 3 μL of double antibody, seal with sealing film and culture at 27°C for 4-5 days until the cells fall off and float up, collect the cell culture fluid, and obtain recombinant viruses containing the target genes 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, respectively.

[0036] The purification and amplification methods of the recombinant silkworm baculovirus are as follows: the mutants with good mutation effects are purified and amplified according to the following method.

[0037] The cells were inoculated into 35 mm small dishes at a density of about 70%-80%. After they adhered to the wall, the culture medium was aspirated and the cell culture medium containing the recombinant virus obtained above with different dilutions was added (1 mL / dish). After being gently distributed evenly, the cells were infecting at 27°C for 1 h. The non-infected virus liquid was aspirated and 4 mL of gel solution (preparation method: 2% agarose gel was melted in a 60°C water bath, cooled to 40°C and mixed evenly with isothermal 2×TC-100 culture medium containing 20% ​​FBS) was added to each dish. After the gel solution solidified, it was sealed with a sealing film and inverted cultured at 27°C for 3-5 days and observed under a microscope. Pick out the plaques and repeat the above steps. After 2-3 rounds of purification, pure recombinant silkworm baculovirus 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 were obtained.

[0038] The recombinant silkworm baculovirus was used to infect normally growing BmN cells, and the supernatant was collected after culturing for 3-5 days. The supernatant 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.

[0039] 1.4 Expression of Bst-LF single-site mutants The recombinant virus culture medium was heated to 10 5 5-day-old silkworms were injected with a dose of 1.0 PFU / head and cultured under the conditions of humidity 70%-80% and temperature 27°C. After about 3.5 d-4.5 d, symptoms such as swollen body segments, abnormal behavior, and decreased appetite were observed in the larvae. When they stopped eating, it indicated that the 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 efficiently expressed under the action of the polyhedron gene promoter. At this time, the hemolymph was collected and stored at -20°C for later use.

[0040] 1.5 Purification of expression products of Bst-LF single-site mutants The collected hemolymph was ultrasonically disrupted in an ice bath, the supernatant obtained by centrifugation was denatured at 60°C for 20 min, and impurities were removed by centrifugation. Then, the results of the above studies were comprehensively applied (Huang Weihua, Zhai Feng, Hong Guofan. Cloning, expression, properties and application of 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. 1981;145(1):21-26) in the method of heat treatment denaturation of impurity proteins, ammonium persulfate precipitation, ion exchange chromatography, and heparin affinity chromatography, etc., after elution, it is concentrated using an ultra-Amicon® Ultra centrifugal ultrafiltration tube and replaced into an enzyme storage buffer solution, the enzyme storage buffer solution formula is: 100 mM KCl, 10 mM Tris-HCl, 0.1mM EDTA, 1 mM DTT, 0.1% Triton ® X-100, 50% glycerol, pH 7.4. Take an appropriate amount of the harvested protein solution for SDS-PAGE analysis of the purification effect; the harvested protein solution is filtered and sterilized, quantified by the BCA method and diluted to 1 mg / mL with enzyme storage buffer, take an appropriate amount of sample for SDS-PAGE analysis of the purification effect, package under sterile conditions, and freeze at -80℃.

[0041] 1.6 Determination of enzyme activity of Bst-LF and its single-site mutants The mutants with good expression were subjected to enzyme activity assay, and according to the principle and method disclosed in patent publication number CN 117946997 A, the DNA polymerase activity and strand displacement activity of the obtained mutants were detected with wild-type Bst-LF as a control.

[0042] 2 Experimental Results 2.1 SDS-PAGE identification results The purified wild-type Bst-LF and its single-site mutants were analyzed by SDS-PAGE electrophoresis. Figure 1 As 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.

[0043] 2.2 Enzyme activity assay results The results of enzyme activity determination 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 improved to varying degrees compared with the wild-type Bst-LF, and they were effective mutations.

[0044] Table 1 Detection results of enzyme activity of single-site mutants of Bst-LF

[0045] Note: Some mutants with poor expression and activity are not shown in the article.

[0046] Example 2 Expression, purification and detection of Bst-LF double-site mutant in silkworm bioreactor 1. Implementation Methods 1.1 Obtaining the gene of Bst-LF double-site mutant The four preferred single-site mutations Bst-LF-D176G, Bst-LF-P236S, Bst-LF-M455L, and Bst-LF-P500S were combined in pairs for double-site mutation. There are a total of six combinations of double-site mutations: 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, respectively. 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, pUC57-Bst-LF-M455L-P500S, the confirmed genes were handed over to a DNA synthesis company for synthesis.

[0047] 1.2 Construction of recombinant baculovirus transfer vector The experimental method was the same as 1.2 of the experimental method in Example 1. The correctly identified recombinant plasmids were sequenced. The correctly sequenced plasmids 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.

[0048] 1.3 Preparation, purification and amplification of recombinant silkworm baculovirus 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.

[0049] 1.4 Expression of Bst-LF double-site mutant The experimental method is the same as that in 1.4 of Example 1.

[0050] 1.5 Purification of the expression product of the Bst-LF double-site mutant The experimental method was the same as that in 1.5 of Example 1, and the mutant was purified and analyzed by SDS-PAGE.

[0051] 1.6 Enzyme activity assay of Bst-LF and its double-site mutants The experimental method is the same as 1.6 of the experimental method in Example 1.

[0052] 2 Experimental Results 2.1 SDS-PAGE identification results The purified Bst-LF and its double-site mutants were analyzed by SDS-PAGE electrophoresis. Figure 1 As shown, lane 1 is the purified wild-type Bst-LF, lane 3 is the purified Bst-LF-P236S-M455L, and lane 4 is the purified Bst-LF-M455L-P500S. An obvious and relatively single target band appeared near the expected size of 67 kDa, indicating that the Bst-LF double-site mutant was successfully purified.

[0053] 2.2 Enzyme activity assay results The results of enzyme activity determination are shown in Table 2. The enzyme activity of Bst-LF (Bst DNA polymerase) double-site mutants Bst-LF-P236S-M455L, Bst-LF-D176G-P500S or Bst-LF-M455L-P500S is higher than that of the wild type and single-site mutants. The activities of the other groups of sites remain unchanged or decrease after mutation, indicating that the combined mutation of these three double sites is an effective mutation, which can achieve the purpose of further improving the enzyme activity of Bst-LF single-site mutants.

[0054] Table 2 Detection results of enzyme activity of double-site mutants of Bst-LF

[0055] Test Example 1: LAMP Detection Test for Bovine Infectious Rhinotracheitis Virus Conserved genes of bovine infectious rhinotracheitis virus (IBRV) B(GenBank Accession No. DQ006857.1) was used as the target, and the whole gene was synthesized and cloned into the pUC18 plasmid (pUC18-gB). The primers used for LAMP were designed and synthesized for the target gene. The nucleotide sequences of the primers are 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]. Journal of Biotechnology, 2018, 34(10): 1587-1595). In combination with Examples 1 and 2, the Bst DNA polymerase mutant Bst-LF-P236S-M455L with the highest enzyme activity was selected for LAMP reaction, and the wild-type Bst-LF and commercial NEB polymerase (Bst 3.0 DNA polymerase, catalog number: M0374S) were used 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. The template plasmid was diluted 10 times with sterile enzyme-free water, and the concentration range was set to 5×10 8 -5×10 1 copies / mL.

[0056] Table 3 Primer sequences

[0057] Table 4 LAMP reaction system

[0058] After the LAMP amplification reaction, the amplified product was detected by 2% agarose electrophoresis. The electrophoresis results were as follows: Figure 2 As shown, the negative control group with sterile enzyme-free water showed no amplification, while the natural wild-type Bst-LF, double-site mutant Bst-LF-P236S-M455L and commercial enzyme were able to form ladder-like DNA bands ( Figure 2 -A, Figure 2 -B, Figure 2 -C), and the amplification effect can meet the requirements of LAMP detection; at the same time, the LAMP amplification reaction using the double-site mutant Bst-LF-P236S-M455L can detect 5×10 3 copies / mL of plasmid template, and the sensitivity was higher than that of wild-type Bst-LF and commercial enzymes ( Figure 2 -B, Figure 2 -C).

[0059] Test Example 2 RT-LAMP Detection Test of Bovine Viral Diarrhea Virus The 5′UTR sequence of bovine viral diarrhea virus (BVDV) was used as the target, and the primers used in the reference (Zhang Yuming, Li Shufan, Chen Zhiyuan, et al. Establishment of a visualized one-step RT-LAMP detection method for bovine viral diarrhea virus [J]. Chinese Journal of Veterinary Science, 2022, 52(08): 985-991) were used. The nucleotide sequences of the primers are shown in Table 5. The RT-LAMP amplification reaction was performed according to the reaction conditions and reaction system of Experimental Example 1, except that the gradient plasmid DNA template was modified to a BVDV RNA (5.0 ng / μL) template, which was diluted 10 times with sterile enzyme-free water, and the concentration range was set to 1×10 1 -1×10 -6 ng / μL.

[0060] Table 5 Primer sequences

[0061] After the RT-LAMP reaction, the amplified product was detected by 2% agarose electrophoresis. Figure 3 As shown, the negative control group with sterile enzyme-free water did not amplify, and the wild-type Bst-LF did not amplify, which is not suitable for RT-LAMP detection ( Figure 3 -A), while the double-site mutant Bst-LF-P236S-M455L and the commercial enzyme have good reverse transcription activity and can form ladder-like DNA bands. The amplification effect can meet the requirements of RT-LAMP detection ( Figure 3 -B, Figure 3 -C); when the double-site mutant Bst-LF-P236S-M455L was used for RT-LAMP amplification, the sensitivity was improved compared with the wild-type Bst-LF and the commercial enzyme. The template concentration used was 1×10 -5 ng / μL, the BVDV band 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 subjecting the amino acid sequence of the Bst DNA polymerase shown in SEQ ID NO.1 to any single-site mutation of D176G, P236S, M455L or P500S.

2. A double-site mutant of Bst DNA polymerase, characterized in that The double-site mutant is a double-site mutant obtained by mutating 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 or M455L-P500S.

3. A gene encoding 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 Bombyx mori baculovirus expression vector. A recombinant host cell comprising the expression vector according to claim 4.

7. The recombinant host cell according to claim 6, characterized in that The recombinant host cell is a recombinant insect cell.

8. The method for preparing the single-site mutant according to claim 1 or the double-site mutant according to claim 2, characterized in that: include: (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 the protein.

9. The preparation method according to claim 8, characterized in that: The expression vector is a Bombyx mori baculovirus expression vector; The host cell includes a living insect 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 as a DNA polymerase in a LAMP amplification reaction of DNA or RNA.

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