牛支原体HB0801株VspX蛋白单克隆抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2017.03.035

Abstract

Abstract:

To obtain the monoclonal antibody (McAb) against VspX protein of Mycoplasma bovis (M. bovis),VspX gene was amplified, expressed and purified. Then, BALB/c mice were immunized subcutaneously three times with the purified recombinant VspX (rVspX) mixed with QuickAntibody-Mouse 5W adjuvant. Three days after the last injection, spleen cells were collected aseptically, and fused with SP2/0 myeloma cells in the presence of polyethylene glycol. By the clone selection, five stable hybridomas against VspX protein were obtained, separately named as 1A8, 3A3, 3C12, 3H9 and 4D11. Antibody titers in cell supernatant were from 1:1×10⁴ to 1:2×10⁵, while from 1:1×10⁵ to 1:8×10⁵ in ascites of mice by indirect ELISA. The subtypes were determined to be IgG1 and IgG2b class, and all light chains were κ chain. The affinity constant of McAb 3H9 and 4D11 were 6.3×10⁹ and 7.8×10⁹, respectively, and they belonged to high-affinity antibodies. Western blotting results showed that all of five McAbs could specifically react with M.bovis, however, McAb 4D11 could not react with Mycoplasma arginini PG2 and Mycoplasma mycoides subsp. PG3. Flow cytometry showed that McAb 4D11 reacted with surface VspX of M. bovis in a dose-dependent manner. Indirect immunofluorescence assay demonstrated that 4D11 McAb was able to detect rVspX protein binding to embryonic bovine lung cells. In the present study, McAbs against rVspX protein had been successfully prepared, which provided a basis for future researches about the function of VspX protein and the pathogenesis of M. bovis.

Key words: Mycoplasma bovis; VspX; monoclonal antibody; specificity

CLC Number:

S852.62

CHEN Xi, Zhu Hong-mei, ZHAO Gang, HU Chang-min, CHEN Ying-yu, GUO Ai-zhen. Preparation and Identification of the Monoclonal Antibodies against VspX Protein in Mycoplasma bovis strain HB0801[J]. , 2017, 44(3): 868-878.

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Preparation and Identification of the Monoclonal Antibodies against VspX Protein in Mycoplasma bovis strain HB0801

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