Mycoplasma bovis enolase and new applications of coding gene thereof

A kind of technology of enolase and Mycoplasma bovis, applied in the field of biomedicine

Inactive Publication Date: 2012-07-04
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2008, Mycoplasma bovis was isolated and identified for the first time in my country, but so far there is no statistical report of the economic loss caused by the disease

Method used

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  • Mycoplasma bovis enolase and new applications of coding gene thereof
  • Mycoplasma bovis enolase and new applications of coding gene thereof
  • Mycoplasma bovis enolase and new applications of coding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Cloning and expression of embodiment 1 Mycoplasma bovis enolase gene (P18)

[0036] 1. Materials

[0037] 1.1 Mycoplasma culture

[0038] Mycoplasma bovis Hubei isolate (HB) was isolated and identified by our laboratory (Vassalli J D, Sappino A P, and Belin D. The plasminogen activator / plasmin system [J]. J Clin. Invest. 1991.88 (4): 1067-1072). According to the literature (Radaelli E, Luini M. Bacteriological, serological, pathological and immunohistochemical studies of Mycoplasma bovis respiratory infection in veal calves and adult cattle at slaughter [J], Research in Veterinary Science 2008.85 (2): 282-290.) described Methods: Expand the strains in PPLO medium containing 20% ​​horse serum, 10% yeast extract, thallium acetate (0.125mg / ml) and penicillin (200IU / ml). After culturing at 37.0°C for 3-5 days, the cells were collected by centrifugation at 13,000 r / min for 30 minutes at 4°C, and the cells were washed with PBS for 3 times. Bacteria are stored in -70°C refrig...

Embodiment 2

[0057] Example 2 The binding activity of recombinant Mycoplasma bovis enolase and plasminogen

[0058] The purified recombinant Mycoplasma bovis enolase sample obtained in Example 1 was subjected to 10% SDS-PAGE electrophoresis, and then transferred to a cellulose acetate membrane. Block overnight at 4°C with PBS containing 5% fish gelatin, wash the membrane three times with PBST, and incubate at 37°C for 1 hour with PBS containing 1 unit / ml human plasminogen (containing 1% BSA and 25MmEDTA). Wash the membrane with PBST to wash away unbound plasminogen, and incubate overnight at 4°C with PBS containing 25ug / ml goat anti-human plasminogen antibody. After washing with PBST, the HRP-labeled rabbit anti-goat secondary antibody diluted 1:10000 (v / v) was incubated with cellulose acetate membrane at 37°C for 1 hour. DAB chromogenic detection.

[0059] The ability of Mycoplasma bovis enolase to bind plasminogen was detected by ligand experiment, and the results showed that the purif...

Embodiment 3

[0060] Example 3 Western blot detection of protein immunogenicity

[0061] The purified recombinant Mycoplasma bovis enolase sample was subjected to 10% SDS-PAGE electrophoresis, and then transferred to a cellulose acetate membrane. After blocking overnight at 4°C in PBS containing 5% fish gelatin, the bovine anti-Mycoplasma bovis hyperimmune serum (1:160) prepared and preserved in our laboratory was used as the primary antibody, and the HRP-labeled goat anti-bovine secondary antibody (1:8000). DAB chromogenic detection.

[0062] The immunogenicity of Mycoplasma bovis enolase was detected by Western blot experiment, and the experimental results showed that the purified Mycoplasma bovis enolase had immunogenicity and could react with Mycoplasma bovis positive serum ( Figure 5 ).

[0063] In this experiment, Mycoplasma bovis enolase was obtained through prokaryotic expression, and the protein was successfully soluble expressed by adjusting the induction conditions, and finall...

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Abstract

The invention discloses mycoplasma bovis enolase and new applications of a coding gene thereof. Specifically, the invention discloses the mycoplasma bovis enolase and applications of the coding gene of the mycoplasma bovis enolase in preparing reagents for detecting plasminogen and detecting or diagnosing infections caused by mycoplasma bovis. According to the mycoplasma bovis enolase disclosed by the invention, an enolase protein is obtained through prokaryotic expression, and soluble expression is successfully performed on the protein through regulating induction conditions, and further, through recombinant enolase protein and plasminogen ligand experiments and enolase protein and mycoplasma bovis antibody Western blot experiments, the protein is finally proved to have the binding activity and the immunogenicity of the plasminogen, therefore, the protein can be used for detecting the plasminogen and detecting or diagnosing the infections or diseases caused by the mycoplasma bovis. Moreover, a new path is also opened up for researching the adhesion mechanism and the pathogenicity of the mycoplasma bovis and developing vaccines in future.

Description

technical field [0001] The invention relates to a new application of mycoplasma bovis enolase and its encoding gene, in particular to the application of the mycoplasma bovis enolase and its encoding gene in preparing reagents for detecting plasminogen and detecting or diagnosing mycoplasma bovis infection. The invention belongs to the field of biomedicine. Background technique [0002] Mycoplasma bovis (Mycoplasma bovis) belongs to the genus Mycoplasma genus of the Mycoplasma family of the family Mollicula, Mollicula, Mycoplasma, and is the pathogen of Mycoplasma bovis-related diseases. It can cause calf pneumonia, mastitis, keratitis and arthritis. It is an important Respiratory pathogens, the mechanism by which Mycoplasma bovis adheres to host cells is still unclear. In 1961, Hale isolated the pathogen from the milk of a case of mastitis in the United States for the first time. At present, Mycoplasma bovis has become an important pathogen in North America, Europe and Asi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/56C12Q1/527C12N9/88C12Q1/68C12N15/60C12Q1/04C12R1/35
Inventor 辛九庆李媛刘洋
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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