Application of mycoplasma bovis secretory protein MbovP570

A technology of Mycoplasma bovis and secreted protein, applied in the field of animal infectious disease prevention and control, can solve problems such as slow research progress

Active Publication Date: 2020-10-30
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 resear...

Method used

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  • Application of mycoplasma bovis secretory protein MbovP570
  • Application of mycoplasma bovis secretory protein MbovP570
  • Application of mycoplasma bovis secretory protein MbovP570

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: Expression of Mycoplasma bovis MbovP570 protein

[0019] 1. Cloning and expression of Mycoplasma bovis Mbov_0570 gene

[0020] The nucleotide sequence of the Mbov_0570 gene in Mycoplasma bovis HB0801 (genome GenBank accession number is CP002058) is shown in SEQ ID NO: 1, and the sequence size is 2265bp. 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 2265bp. 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 754 amino acids.

[0021] The amplified product of the synthesized Mbov_0570 gene was digested with Xho I and BamHI, while the pET-30a...

Embodiment 2

[0035] Embodiment 2: Mycoplasma bovis MbovP570 secretion verification

[0036] 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.

[0037] 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

[0039] Embodiment 3: Mycoplasma bovis MbovP570 natural antigenicity verification

[0040] The purified rMbovP570 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 discloses application of mycoplasma bovis secretory protein MbovP570 in preparation of mycoplasma bovis diagnostic reagents, drugs or vaccines, and belongs to the technical field of animal infectious disease prevention and treatment. The mycoplasma bovis secretory protein MbovP570 has a sequence as shown in SEQ ID NO: 1. 2 is an amino acid sequence shown in the specification; test proves that the mycoplasma bovis secretory protein MbovP570 has antigenicity and can be used for detecting mycoplasma bovis. The protein can react with mycoplasma bovis positive serum instead of mycoplasma bovis negative serum, the protein can induce bovine macrophages to highly express IL-8 and IL-12, IL-8 can activate neutrophils, and IL-12 can activate NK cells and T cells. Therefore, it is believed that MbovP570 can activate a host immune system and has important potential in research and development of new drugs and vaccines.

Description

technical field [0001] The invention belongs to the technical field of prevention and treatment of animal infectious diseases, and specifically relates to the application of a Mycoplasma bovis secretory protein MbovP570, which is identified as a secretory protein for the first time in the present invention, and protein function research proves that the protein can induce bovine macrophages to highly express IL-8 , IL-12. Background technique [0002] Mycoplasma bovis (M.bovis) belongs to the class Mollicutes, the order Mycoplasmatles, the family Mycoplasma, and the genus Mycoplasma. It is a minimal pathogenic microorganism that can replicate in vitro. Mycoplasma bovis can cause a variety of diseases in dairy cattle and beef cattle. The clinical symptoms after the onset of beef cattle are mainly pneumonia and arthritis, and the main symptoms of dairy cattle are mastitis. M. bovis can infect cattle of any age, but calves between 2 and 6 months of age are most susceptible (Sti...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/30C12N15/70G01N33/68G01N33/569A61K39/02A61P31/04
CPCC07K14/30C12N15/70G01N33/6854G01N33/56933A61K39/0241A61P31/04C12N2800/22G01N2469/20A61K2039/552
Inventor 郭爱珍赵刚伊赫萨努拉·希拉尼陈颖钰胡长敏陈曦陈建国
Owner HUAZHONG AGRI UNIV
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