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
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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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