Mycoplasma bovis protein RecD as well as coding gene, recombinant vector and application thereof

Abstract

The invention discloses a mycoplasma bovis protein RecD as well as a coding gene, a recombinant vector and application thereof, and aims to solve the technical problems that safe and effective commercial mycoplasma bovis vaccines are lacked in the current market and the function research on mycoplasma bovis genes is insufficient. A recombinant protein RecD is successfully expressed by optimizing a sequence of a mycoplasma bovis gene Mbov0848, constructing a recombinant vector and transforming the recombinant vector into escherichia coli, the recombinant protein RecD is confirmed to have ATP enzyme activity through purification and functional verification, the optimal reaction conditions of the recombinant protein RecD are further determined, meanwhile, an Mbov0848 gene deleted strain is constructed, and the recombinant protein RecD is transformed into escherichia coli. It is found that the viability of a strain without the gene in a host is reduced. The application has important significance for understanding the pathogenic mechanism of the diseased mycoplasma bovis and developing a new treatment strategy and a new mycoplasma bovis vaccine.

Description

Technical Field

[0001] The present invention relates to the technical field of prevention and treatment of animal infectious diseases, and specifically to a bovine mycoplasma protein RecD, its encoding gene, a recombinant vector and its application. Background Art

[0002] Mycoplasma ( Mycoplasma bovis,M.bovis ) belongs to the class Mollicutes ( Mollicutes ), Mycoplasmalales ( Mycoplasmatles ), Mycoplasma family, Mycoplasma genus, is a cell wall-less prokaryotic organism with a relatively small genome (0.58 to 1.4 Mb) and a low G+C content (23-40%). Mycoplasma bovis was first isolated from the milk of diseased cows in the United States in 1961, and then became widely prevalent throughout the world. Mycoplasma bovis pneumonia is closely related to transportation stress, with high morbidity and mortality rates. After calves are infected with Mycoplasma bovis, their immunity often decreases, and it cooperates with other pathogens to cause bovine respiratory disease syndrome (BRD), causing a series of serious diseases such as mastitis, pneumonia, arthritis, otitis media and reproductive tract infection. It is an important pathogen that endangers the health of calves.

[0003] At present, the resistance of bovine mycoplasma to antibiotics is increasing year by year. Due to the lack of cell walls in mycoplasma, the types of antibiotics that can be used to treat mycoplasma are greatly limited, which greatly increases the difficulty of effectively treating and controlling the disease. In addition, there is still a lack of safe and effective commercial bovine mycoplasma vaccines on the market, which further exacerbates the difficulty of disease prevention and control. In addition, the lack of research on the gene function of bovine mycoplasma has also become one of the important factors hindering the progress of research on the pathogenic mechanism of the pathogen.

[0004] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0005] Based on the screening of the Mbov_0848 gene from the genome of Mycoplasma bovis, the inventors of this application cloned, expressed and purified the protein encoded by the gene. Further in-depth research found that the RecD protein encoded by the gene has DNA-dependent ATPase activity and was verified by experiments. Combined with the phenomenon that the Mbov_0848 gene-deficient strain (deficient Mbov_0848 gene) showed growth lag under the co-culture conditions of EBL (embryonic bovine lung cells), it is shown that the Mbov_0848 gene is related to the growth metabolism of Mycoplasma bovis, and the gene and its encoded protein RecD are important potential targets for the study of the pathogenic mechanism of Mycoplasma bovis.

[0006] Based on this, the present application provides a bovine mycoplasma protein RecD, whose amino acid sequence is shown in SEQ ID NO.2.

[0007] The second aspect disclosed in the present application provides a gene encoding the above-mentioned bovine mycoplasma protein RecD, whose nucleotide sequence is shown in SEQ ID NO.1.

[0008] The third aspect disclosed in the present application provides a recombinant vector containing the above gene or a fragment thereof.

[0009] In a fourth aspect disclosed in the present application, the bovine Mycoplasma protein RecD, the gene or the recombinant vector is used in the preparation of an ATPase activity preparation.

[0010] In a fifth aspect disclosed in the present application, the Mycoplasma bovis protein RecD, the gene or the recombinant vector is used in the preparation of Mycoplasma bovis antibodies or vaccines.

[0011] In a sixth aspect disclosed in the present application, the bovine Mycoplasma protein RecD, the gene or the recombinant vector is used in the preparation of a reagent for detecting bovine Mycoplasma.

[0012] In a seventh aspect disclosed in the present application, the Mycoplasma bovis protein RecD, the gene or the recombinant vector is used in the preparation of an anti-Mycoplasma bovis drug.

[0013] In an eighth aspect disclosed in the present application, the Mycoplasma bovis protein RecD, the gene or the recombinant vector is used in regulating the growth and metabolism of Mycoplasma bovis.

[0014] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: 1. For the first time, it was determined that the bovine Mycoplasma protein RecD has ATPase activity, and that the Mbov_0848 gene is related to the growth and metabolism of bovine Mycoplasma. This is of great significance for understanding the pathogenic mechanism of bovine Mycoplasma and developing new treatment strategies, and it is expected to be developed and utilized as a molecular target.

[0015] 2. The optimal reaction conditions for the ATPase activity of bovine Mycoplasma protein RecD were determined, namely, the ATPase activity was highest when the reaction temperature was 40℃, the concentration of bovine Mycoplasma protein RecD was 0.8nM, the substrate was ssDNA, and the substrate concentration and length were 40μM and 40mer respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the plasmid map of the empty plasmid pET-32a in one embodiment of the present application.

[0017] Figure 2This is a map of the recombinant plasmid pET-32a-Mbov_0848 prepared in one example of the present application.

[0018] Figure 3 This is an SDS-PAGE diagram of the purified bovine Mycoplasma RecD and empty protein in an example of the present application; wherein, A: line M, PageRuler Prestained Protein Ladder; line 1: purified RecD protein; B: line M, PageRuler Prestained Protein Ladder; line 2, purified empty protein.

[0019] Figure 4 This is the ATPase activity determination of the purified bovine Mycoplasma RecD in an example of the present application; wherein, empty protein: ATPase activity of empty protein; empty protein + ssDNA: ATPase activity of empty protein in the presence of ssDNA; RecD: ATPase activity of RecD protein; RecD + ssDNA: ATPase activity of RecD protein in the presence of ssDNA.

[0020] Figure 5 The optimal reaction conditions for the ATPase activity of the purified Mycoplasma bovis RecD in an example of the present application; wherein A: ATPase activity of RecD protein at different temperatures; B: Effects of different types of nucleotides on the ATPase activity of RecD protein; C: Effects of different ssDNA concentrations on the ATPase activity of RecD protein; D: Effects of different RecD concentrations on ATPase activity.

[0021] Figure 6 Figure 2 shows the growth of Mycoplasma bovis HB0801 and Mbov_0848 gene-deficient strain T8.365 in culture medium in one embodiment of the present application; wherein, A: the growth of Mycoplasma bovis HB0801 and Mbov_0848 gene-deficient strain T8.365 cultured in PPLO medium for 48 h; B: the growth of Mycoplasma bovis HB0801 and Mbov_0848 gene-deficient strain T8.365 co-cultured with EBL cells at 37°C, 5% CO2 for 72 h. DETAILED DESCRIPTION

[0022] The specific implementation modes of the present invention are described below in conjunction with examples. However, the following examples are only used to illustrate the present invention in detail and are not intended to limit the scope of the present invention in any way.

[0023] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents and materials involved are all conventional commercial products unless otherwise specified; the tests and detection methods involved are all conventional methods unless otherwise specified.

[0024] Example 1. Expression of Mycoplasma bovis RecD protein 1. Cloning and expression of the gene of Mycoplasma bovis Mbov_0848 First, the amino acid sequence of the RecD protein of Mycoplasma bovis HB0801 strain was retrieved from the GenBank database (protein_id=AFM52192.1). In order to gain a deeper understanding of the characteristics of the protein, its physicochemical properties, signal peptide, transmembrane region, and secondary structure were predicted using tools such as SignalP 6.0, TMHMM 2.0, Expasy, and Protean. In addition, the KEGG database was used to analyze the metabolic pathways that it may be involved in.

[0025] Using the Mbov_0848 gene in Mycoplasma bovis HB0801 (GenBank accession number CP002058) as a template, the nucleotide sequence of the gene was comprehensively optimized according to the codon preference of Escherichia coli; for example, in order to prevent Escherichia coli from prematurely terminating during protein expression (the codon UGA encoding tryptophan in Mycoplasma bovis is used as a terminator in Escherichia coli), the codon UGA encoding tryptophan in the Mycoplasma bovis gene was mutated to its synonymous codon UGG to conform to the codon preference of Escherichia coli, and the optimized sequence was then sent to a biological company for synthesis.

[0026] The nucleotide sequence of the bovine Mycoplasma gene Mbov_0848 after a series of strategy optimization is shown in SEQ ID NO: 1, and its length is 2283 bp; the amino acid sequence of the RecD protein encoded by the gene is shown in SEQ ID NO: 2, which encodes 756 amino acids.

[0027] The nucleotide sequence of the bovine Mycoplasma gene Mbov_0848 was transformed into Escherichia coli DH5α by constructing a recombinant plasmid vector pET-32a-Mbov_0848 to obtain a recombinant Escherichia coli, and the recombinant Escherichia coli was named Escherichia coli pET-32a-Mbov_0848. The specific steps are as follows: The Mbov_0848 gene was amplified by PCR and the amplified product was combined with the pET-32a plasmid ( Figure 1 ) (purchased from Novagen) XOt I and BamThe Mbov_0848 gene and pET-32a plasmid were ligated by T4 DNA ligase (purchased from Takara) to successfully obtain the recombinant plasmid pET-32a-Mbov_0848 ( Figure 2 ). After the recombinant plasmid pET-32a-Mbov_0848 was transformed into Escherichia coli DH 5α, it was placed in a 37°C shaker at 180 r / min for 12 hours, the bacterial solution plasmid was extracted, and the correctly sequenced plasmid was transformed into Escherichia coli Rosetta2 (DE3) pLysS (purchased from Shanghai Weidi Biological Company), and placed in a 37°C shaker at 180 r / min for 12 hours. The Escherichia coli was inoculated into 1L LB liquid culture medium and cultured in a 37°C shaker at 180 r / min until OD = 0.6. 1mL of bacterial solution was taken as a pre-induction control, and isopropylthiogalactoside (IPTG) was added to the remaining bacterial solution to a final concentration of 0.2mM, and the expression was induced at 16°C shaker for 20 hours. Take 1mL of bacterial solution before and after induction and do the following treatments: centrifuge at 12000r / min for 1min, discard the supernatant, add 1mL PBS (formula: KCl 0.2g, NaCl 8g, Na2HPO41.44g, KH2PO40.24g, distilled water 1000mL, pH=7.48), resuspend and centrifuge again (12000r / min, 1min), discard the supernatant, add 30uL PBS to resuspend. Add 7.5μL loading buffer (1M Tris-HCl (pH=6.8) 1mL, 200mM DDT 0.31g, 4% SDS 0.4g, 0.2% bromophenol blue 0.02g, 20% glycerol 2mL, 7mL ultrapure water) to the resuspended solution. Boil in 100℃ boiling water for 10min. Use SDS-PAGE gel electrophoresis to identify whether it is expressed.

[0028] 2. Purification of Recombinant RecD Protein of Mycoplasma bovis The E. coli culture liquid that has been successfully induced to express was centrifuged at 8000r / min for 10min, and the supernatant was discarded. It was resuspended with 500mL of PBS and centrifuged again at 8000r / min for 10min, and repeated once. After two centrifugations, the supernatant was discarded, and then 30mL of PBS was added for resuspending. Protease inhibitors (purchased from Shanghai Roche Pharmaceuticals Co., Ltd.) were added, and ultrasonic disruption was performed. After disruption, it was centrifuged at 12000r / min for 30min, and 80μL of supernatant was added to 20μL of loading buffer and boiled in boiling water for 10min as the supernatant group. 30mL of PBS was added to the precipitate for resuspending, and 80μL of the precipitate sample was added to 20μL of loading buffer and boiled for 10min as the precipitate group. After SDS-PAGE gel electrophoresis, it was determined that most of the RecD protein was expressed in the supernatant ( Figure 3 ).

[0029] The specific purification steps of RecD protein are as follows: (1) Filling with nickel: Add 1 mL of Ni-NTA metal chelated His protein purification medium (purchased from GE) to the affinity chromatography column; (2) Washing the column: Add 12 mL of ddH2O to the affinity chromatography column for washing; (3) Balancing the column: Add 12 mL of binding buffer (20 mM Na3PO4, 0.5 M NaCl, 20 mM imidazole, pH = 7.48) to balance the column; (4) Protein hanging column: Add the protein expression supernatant filtered through a 0.22 μm pore size filter to the affinity chromatography column; Collect the first few drops of filtered liquid, numbered 1; (5) Balancing the column again: Add 50 mL of binding buffer to balance the column, collect the first few drops of liquid, numbered 2; (6) Washing impurities: Add 50 mL of washing buffer (20 mM Na3PO4, 0.5 M NaCl, 60mM imidazole, pH=7.48) to wash away impurities, collect the first few drops, numbered 3; (7) Collect the target protein: add 12mL elute buffer (20mM Na3PO4, 0.5M NaCl, 1M imidazole, pH=7.48) to elute the target protein, collect the first few drops, numbered 4; (8) Sample preparation: take 80μL from each tube numbered 1 to 4 and put it into a 1.5mL centrifuge tube, and add 20μL loading buffer to boil for 10min; (9) SDS-PAGE gel electrophoresis: prepare SDS-PAGE polyacrylamide gel, add the treated sample to the well (20μL / well), and then perform electrophoresis (the electrophoresis condition of the concentrated gel is a DC voltage of 80 volts, and the electrophoresis condition of the separation gel is a DC voltage of 120 volts). After the electrophoresis is completed, remove the gel and stain it with Coomassie Brilliant Blue overnight. After staining, decolorize it to confirm that the purified target protein has been obtained.

[0030] Example 2: Functional verification of bovine mycoplasma protein RecD This example uses Na + K + -ATPase detection kit (purchased from Beijing Solaibao Company) was used to verify the function of bovine mycoplasma protein RecD. According to the instructions of the kit, the ATPase activity of four test samples, RecD, 32a empty protein, RecD + ssDNA, and 32a empty protein + ssDNA, was verified.

[0031] The verification process is as follows: 1. Enzyme activity reaction: Take two 1.5mL centrifuge tubes, mark them as control tube and assay tube respectively. Add 65μL of reagent 1, 40μL of reagent 2 and 20μL of reagent 3 to the control tube; add 45μL of reagent 1, 40μL of reagent 2, 20μL of reagent 3, 20μL of reagent 4 and 100μL of the sample to be tested to the assay tube, mix them respectively, and accurately water bath them at 25℃ for 10min. Then add 25μL of reagent 5 and 100μL of sample to the control tube; add 25μL of reagent 5 to the assay tube, mix them respectively, centrifuge them at 4000g for 10min at room temperature, and take the supernatant.

[0032] 2. Phosphorus determination: Four 1.5mL centrifuge tubes are marked as blank tube, standard tube, control tube and assay tube. Add 20μL of distilled water to the blank tube, add 20μL of 0.5μmol / mL standard phosphorus application solution to the standard tube, and add 20μL of the corresponding enzyme activity reaction supernatant to the control tube and the assay tube. Add 200μL of phosphorus determination reagent to all centrifuge tubes. Vortex to mix, place in a 40℃ water bath for 10 minutes, and finally record the absorbance of each tube at 660nm, and record them as A blank, A standard, A control, and A assay respectively.

[0033] According to the formula: Na + K + -ATPase activity (U / mL) = 7.5* (A assay - A control) / (A standard - A blank) to calculate the ATPase activity of the protein. The blank tube and the standard tube were repeated 2 times, and the four samples were repeated 3 times each. The results showed that the RecD protein has DNA-dependent ATPase activity ( Figure 4 ).

[0034] Example 3: Verification of the optimal reaction conditions for RecD protein ATPase activity In order to further determine the optimal reaction conditions for the ATPase activity of the RecD protein, based on the operating steps in Example 2, the reaction temperature of the system (4-60°C), the concentration of the RecD protein (0.05-0.8 nm), the type of nucleic acid substrate (ssDNA, dsDNA, circular dsDNA, 5'-OV, 3'-OV, forked end, blunt end, RNA), the length of ssDNA (5-80 mer), and the concentration of the substrate 40mer ssDNA (2.5-120 μM) were changed to determine the enzyme activity.

[0035] The results showed that the optimal reaction conditions for RecD protein ATPase activity were: temperature 40°C, RecD concentration 0.8 nM, nucleotide chain type ssDNA, ssDNA concentration 40 μM, ssDNA length 40mer ( Figure 5 ). Under this condition, the ATPase activity of RecD protein is the highest.

[0036] Example 4. Growth detection of Mbov_0848 gene deletion strain The strain T8.365 lacking the Mbov_0848 gene was constructed and the wild-type Mycoplasma bovis strain HB0801 and the gene-deficient strain T8.365 were isolated at 10 4 CFU / mL were inoculated into PPLO medium and cultured for 48h; at the same time, EBL (embryonic bovine lung cells) were cultured at 2×10 4 cell / cm 2 The wild-type strain HB0801 of Mycoplasma bovis and the Mbov_0848 gene-deficient strain T8.365 were plated onto a 24-well cell culture plate, and inoculated into EBL cells at an infection ratio of 0.5, and co-cultured at 37°C and 5% CO2 for 72 hours. After the culture, the number of Mycoplasma bovis colonies was quantitatively determined by colony counting. The results showed that there was no significant difference in the growth of HB0801 and T8.365 in PPLO culture, but the growth of T8.365 was inhibited in the co-culture of EBL cells ( Figure 6 ).

[0037] The above examples illustrate that the Mbov_0848 gene and its encoded protein RecD are potential targets for antibacterial drugs, which are of great significance for the development and research of new anti-mycoplasma drugs; at the same time, it also further confirms the core role of this gene in the pathogenicity of bovine Mycoplasma, that is, as a potential virulence factor, it is of great significance for in-depth understanding of its pathogenesis and design of targeted prevention and control strategies.

[0038] Although some preferred embodiments of the present application have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present application.

[0039] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the inventive concept. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A bovine mycoplasma protein RecD, whose amino acid sequence is shown in SEQ ID NO.

2.

2. A gene encoding the bovine Mycoplasma protein RecD according to claim 1, wherein the nucleotide sequence thereof is shown in SEQ ID NO.

1.

3. A recombinant vector containing the gene or a fragment thereof according to claim 3.

4. Use of the bovine Mycoplasma protein RecD according to claim 1, the gene according to claim 2 or the recombinant vector according to claim 3 in the preparation of an ATPase activity preparation.

5. Use of the bovine Mycoplasma protein RecD according to claim 1, the gene according to claim 2 or the recombinant vector according to claim 3 in the preparation of anti-bovine Mycoplasma antibodies or vaccines.

6. Use of the bovine Mycoplasma protein RecD according to claim 1, the gene according to claim 2 or the recombinant vector according to claim 3 in the preparation of a bovine Mycoplasma detection reagent.

7. Use of the bovine Mycoplasma protein RecD according to claim 1, the gene according to claim 2 or the recombinant vector according to claim 3 in the preparation of an anti-bovine Mycoplasma drug.

8. Use of the Mycoplasma bovis protein RecD according to claim 1, the gene according to claim 2 or the recombinant vector according to claim 3 in regulating the growth and metabolism of Mycoplasma bovis.

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