Hybridoma cell strain capable of secreting anti-mycoplasma bovis monoclonal antibody and application thereof

A technology of hybridoma cell lines and monoclonal antibodies, applied in anti-bacterial immunoglobulin, analytical materials, biochemical equipment and methods, etc., to achieve good application value, high specificity, and good sensitivity

Inactive Publication Date: 2016-06-22
GANSU AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the problems existing in detecting Mycoplasma bovis at present, and provides a hybridoma cell strain capable of producing an IgG1 subtype antibody that reacts with Mycoplasma bovis and its application

Method used

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  • Hybridoma cell strain capable of secreting anti-mycoplasma bovis monoclonal antibody and application thereof
  • Hybridoma cell strain capable of secreting anti-mycoplasma bovis monoclonal antibody and application thereof
  • Hybridoma cell strain capable of secreting anti-mycoplasma bovis monoclonal antibody and application thereof

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Experimental program
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Embodiment 1

[0027] The preparation of embodiment 1 mycoplasma bovis specific monoclonal antibody

[0028] 1 Experimental materials

[0029] 1.1 Plasmids, strains, cells and experimental animals

[0030] pET-28a(+) is preserved by the Preventive Veterinary Medicine Laboratory of Gansu Agricultural University College of Veterinary Medicine; Mycoplasma bovis Wuwei isolate and Lintao isolate (separated from diseased cattle herds in Wuwei City and Lintao County, Gansu Province respectively; Mycoplasma bovis Hubei-1 strain , PG45 and Mycoplasma agalactiae PG2 were donated by Huazhong Agricultural University; E.coliDH5α, BL21(DE3) were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; Serratia marcescens, Proteus, Staphylococcus aureus, Pseudomonas aeruginosa, Pasteurella, Escherichia coli, and Salmonella were preserved by our laboratory; SP2 / 0 myeloma cells were donated by Researcher Hu Rongliang's Laboratory of Military Veterinary Research Institute; New Zealand white rabbits, ...

Embodiment 2

[0116] The specific detection of embodiment 2 Mycoplasma bovis specific monoclonal antibody

[0117] 1.1 Indirect ELISA analysis of monoclonal antibodies

[0118] Escherichia coli BL21 (DE3) transformed with pET-28a (+) induced cell protein, Serratia, Proteus, Staphylococcus aureus, Pseudomonas aeruginosa, Pasteurella, Escherichia coli, Mycoplasma gallisepticum, no The bacterial proteins of Mycoplasma lactis and Salmonella bovis were used as antigens to coat the ELISA plate respectively, 5 μg / well, and the specificity of the monoclonal antibody was detected by indirect ELISA (the specific operation was the same as 2.4). ELISA results see Figure 5 .

[0119] 1.2 Monoclonal antibody westernblot analysis

[0120] The specific operation steps of Western blot detection are as follows:

[0121]1.2.1 Protein sample preparation: Take all the samples in 1.1, mix with 2×SDS loading buffer, heat in boiling water for 10 minutes, and centrifuge at 12000r / min for 15 minutes after cooli...

Embodiment 3

[0128] Example 3 Mycoplasma bovis monoclonal antibody is used to prepare detection kit

[0129] Dilute the monoclonal antibody obtained in step 2.10 with 0.01MPBST to 5000 times and apply indirect ELISA to detect Mycoplasma bovis, as follows:

[0130] 1) Antigen coating: Take the culture of Mycoplasma bovis in the middle and late stages of logarithmic growth, wash twice with 0.05M, pH9.6 carbonate buffer, and press 2×10 8 , 2×10 7 , 2×10 6 , 2×10 5 , 2×10 4 , 2×10 3 Coat the ELISA plate at a dilution of CFU / 100 μL, add 100 μL to each well, discard the solution in the well after overnight coating at 4°C, and wash with washing buffer (0.01MPBST) 3 times, 3 minutes each time;

[0131] 2) Blocking: Add 200 μL of 5% skim milk solution (0.01 MPBST dilution) to each well, 37° C. for 2 hours, spin dry and wash 3 times with washing solution;

[0132] 3) Primary antibody incubation: Add 200 μL of 1:5000 diluted purified monoclonal antibody to each reaction well, incubate at 37°C f...

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Abstract

The invention provides a hybridoma cell strain capable of secreting an anti-mycoplasma bovis monoclonal antibody and application thereof.The hybridoma cell strain can produce the anti-mycoplasma bovis monoclonal antibody and is used for detecting mycoplasma bovis.The preservation number of the hybridoma cell strain is CCTCC C2015183, and the preservation date is November 8, 2015.The hybridoma cell strain has the advantages that the hybridoma cell strain can specifically secrete the anti-mycoplasma bovis monoclonal antibody and has better sensitivity and higher specificity.The hybridoma cell strain further has the advantages that the antibody produced by the cell strain can be applied in a variety of ways and has a good application value during actual production.

Description

technical field [0001] The invention relates to a hybridoma cell line secreting a monoclonal antibody of mycoplasma bovis and its application. Background technique [0002] Mycoplasma (Mycoplasma), also known as mycoplasma, belongs to Molluscum class, Mycoplasma order, Mycoplasma family, its size is between bacteria and viruses, and it is the smallest prokaryotic microorganism known to be able to grow and reproduce in non-living medium , and can pass through a 0.22μm bacterial filter. Because mycoplasma lacks a cell wall, it is pleomorphic and resistant to cell wall-acting antibiotics. On solid media, mycoplasma colonies are dew-like, fried-egg-like, or granular. Since the French scientist Nocard et al. first isolated this type of microorganism from the lung tissue of cows suffering from respiratory diseases in 1898, such microorganisms derived from humans, animals, plants and insects have been isolated and identified one after another. In 1967, this type of microorganism...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/12G01N33/577G01N33/569
CPCC07K16/12
Inventor 包世俊邢小勇胡国明郝宝成胡永浩薛慧文伏小平温峰琴
Owner GANSU AGRI UNIV
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