Secretory adhesion protein MbovP58 of mycoplasma bovis

A technology of mycoplasma bovis and protein secretion, applied in biochemical equipment and methods, microbes, peptides, etc., can solve problems such as slow research progress

Inactive Publication Date: 2020-07-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, because most of the genes in the M. bovis genome are unknown genes, and the research

Method used

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  • Secretory adhesion protein MbovP58 of mycoplasma bovis
  • Secretory adhesion protein MbovP58 of mycoplasma bovis
  • Secretory adhesion protein MbovP58 of mycoplasma bovis

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Embodiment 1: Mycoplasma bovis Mbov_0581 gene cloning and expression purification

[0025] 1. Cloning and expression of Mycoplasma bovis Mbov_0581 gene and purification of MbovP0581

[0026] See SEQ ID NO: 1 for the nucleotide sequence of the Mbov_0581 gene in Mycoplasma bovis HB0801 (genome GenBank accession number is CP002058), and the sequence size is 2103bp. Using the original sequence as a template, according to the codon preference of Escherichia coli, the codon UGA of the gene was mutated into the codon UGG capable of expressing tryptophan in Escherichia coli, the mutated sequence is shown in SEQ ID NO: 3, The sequence size is 2103bp. The sequence shown in SEQ ID NO:3 was sent to a commercial gene cloning company for synthesis. The protein sequence encoded by the original gene sequence and the mutated gene sequence is identical (see SEQ ID NO: 2 for the sequence), encoding 700 amino acids.

[0027] The amplified product of the synthesized Mbov_0581 gene was di...

Embodiment 2

[0041] Embodiment 2: Mycoplasma bovis MbovP581 secretory verification

[0042] The Mycoplasma bovis HB0801 strain was cultivated to the logarithmic phase in PPPLO medium, centrifuged at 140000 g for 20 minutes, and the supernatant was filtered with a 0.22 μm filter. Add TCA reagent to the supernatant to a final concentration of 10%, and incubate overnight at 4°C. Centrifuge at 65000g for 20 minutes, remove the supernatant and wash the precipitate with pre-cooled acetone. The washed precipitate was dissolved with lysis buffer (8Murea, 4% CHAPS, 2M thiourea, 60mM DTT, 2% Amidosulfobetaine-14 (ASB-14), 40mM Tris-HCl pH 8.8), which was the secretome extract, using The 2D Quant Kit measures the protein concentration, adds PMSF to the proteome to prevent protein degradation, and stores at -80°C.

[0043] At the same time, extract whole bacterial protein according to the following method: Take 10ml of Mycoplasma bovis grown to the logarithmic phase, centrifuge at 12000r / min for 10m...

Embodiment 3

[0045] Embodiment 3: Mycoplasma bovis MbovP581 natural antigenicity verification

[0046] The purified rMbovP581 protein was treated with 5×SDS-PAGE protein loading buffer and boiled for 5 minutes. After cooling to room temperature, protein samples were directly loaded onto the wells of the SDS-PAGE gel. The upper gel electrophoresis voltage was 80V, and the lower gel electrophoresis voltage was 120V. After the electrophoresis is completed, the protein is transferred using a nitrocellulose membrane. In this embodiment, the wet transfer method is adopted, the voltage is 100V, and the transfer time is 1 hour. After transfer, the membrane was blocked with 5% skim milk and placed at 4°C overnight. Wash the membrane three times with TBST, with an interval of 5 minutes each time, and then incubate at room temperature for 3 hours with the mixed serum (reserved in the laboratory) of 5 cows in the artificial infection experiment of Mycoplasma bovis HB0801 diluted with TBST (volume ra...

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Abstract

The invention belongs to the technical field of animal infectious disease prevention and control, and in particular relates to a secretory adhesion protein MbovP58 of mycoplasma bovis. The nucleotidesequence of the gene of the protein is shown as SEQ ID NO:1, and the amino acid sequence encoded by the protein is shown as SEQ ID NO:2. According to codon preference of escherichia coli, an Mbov_0581gene is mutated, and the sequence of the mutated Mbov_0581 gene is shown as SEQ ID NO:3. The protein is proved to be a secretory protein by an immune transfer printing method and can react with infected bovine serum. Adhesion of a recombinant protein rMbovP581 expressed in escherichia coli to embryonic bovine lung cells (EBL) is detected by immunofluorescence assay and flow cytometry, and it is found that the MbovP581 has good adhesion to the EBL cells. Through integration of the secretion characteristics, immunogenicity and adhesion of the protein, the protein is expected to be applied to preparation of mycoplasma bovis diagnostic reagents, medicaments and vaccines.

Description

technical field [0001] The invention belongs to the technical field of prevention and treatment of animal infectious diseases, and in particular relates to a mycoplasma bovis secretory mucin MbovP581. Background technique [0002] Mycoplasma bovis (Mycoplasma bovis, M.bovis) belongs to the ancient mollicutes class (Mollicutes), Mycoplasma order (Mycoplasmatles), Mycoplasma family, Mycoplasma genus, can cause important diseases such as beef cattle and dairy cattle pneumonia, mastitis, arthritis, etc. Cause reproductive tract inflammation, conjunctivitis, otitis media and other symptoms, the average incidence rate is 40%, and the case fatality rate is 10%. Mycoplasma bovis was first isolated from the milk of cows suffering from mastitis in the United States in 1961, and it was confirmed to be an important pathogen causing pneumonia in 1976. In 2008, respiratory diseases were prevalent in beef cattle imported from other places in Hubei Province. The cattle became ill about 2 w...

Claims

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Application Information

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IPC IPC(8): C07K14/30C12N1/20C12R1/35
CPCC07K14/30C12N1/205C12R2001/35
Inventor 郭爱珍穆罕默德·祖贝尔赵刚张慧陈颖钰胡长敏陈曦陈建国
Owner HUAZHONG AGRI UNIV
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