Application of mycoplasma bovis secretory protein MbovP274

A technology of Mycoplasma bovis and secreted protein is applied in the application field of secreted protein MbovP274, which can solve the problems of slow research progress and the like

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 research on the secretory proteins of M. bovis has just started, the related research progress is slow.

Method used

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  • Application of mycoplasma bovis secretory protein MbovP274
  • Application of mycoplasma bovis secretory protein MbovP274
  • Application of mycoplasma bovis secretory protein MbovP274

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: Expression of Mycoplasma bovis MbovP274 protein

[0019] 1. Cloning and expression of Mycoplasma bovis Mbov_0274 gene

[0020] The nucleotide sequence of the Mbov_0274 gene in Mycoplasma bovis HB0801 (genome GenBank accession number is CP002058) is shown in SEQ ID NO: 1, and the sequence size is 1770bp. 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 1770bp. 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 589 amino acids.

[0021] The amplified product of the synthesized Mbov_0274 gene was digested with Xho I and BamHI, while the pET-30a...

Embodiment 2

[0035] Embodiment 2: Mycoplasma bovis MbovP274 secretory 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 MbovP274 natural antigenicity verification

[0040] The purified rMbovP274 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 MbovP274 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 MbovP274 has an amino acid sequence as shown in SEQ ID NO: 2; gene annotation information shows that the amino acid sequence is a functionally-unknown protein; and experiments show that the MbovP274 protein has antigenicity and can react with mycoplasma bovis positive serum instead of mycoplasma bovis negative serum; the MbovP274 protein is identified as a secretory protein through western blot; it is further verified that the protein can induce bovine macrophages to highly express IL-8 and IFN-gamma, the IL-8 is an important neutrophil activating factor, and the IFN-gamma is a wide immunomodulatory factor. According to the research, the MbovP274 is considered to be an important pathogenic factor of mycoplasma bovis and can activate host immunity, 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 secretory protein MbovP274 identified for the first time by Mycoplasma bovis, which can induce bovine macrophages to highly express IL-8 and IFN-γ. 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 infect cattle of any age, and calves aged 2 to 6 months are most susceptible (Stipkovits et al 2000). It can cause various diseases in dairy and beef cattle. The main clinical symptoms of beef cattle after the onset are pneumonia and arthritis. Cows mainly present with mastitis. After Mycoplasma bovis infects the body, it is difficult to be cleared, and it often develops into chronic wasting disease in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569A61K39/02A61P31/04C07K14/30C12N15/31C12N15/70
CPCA61K39/0241A61P31/04C07K14/30C12N15/70C12N2800/22G01N33/56933G01N2333/30
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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