牛支原体HB0801株VspX蛋白单克隆抗体的制备与鉴定

发布时间：2019-05-18 08:28

【摘要】：为获得牛支原体(Mycoplasma bovis,M.bovis)VspX蛋白单克隆抗体,将编码该蛋白的基因克隆、表达并纯化,作为免疫原,以QuickAntibody-Mouse 5W为免疫佐剂,免疫BALB/c小鼠。经3次免疫后,将小鼠脾细胞与SP2/0骨髓瘤细胞融合,经3次亚克隆筛选后,共获得5株能稳定分泌抗VspX蛋白抗体的杂交瘤细胞株,分别命名为1A8、3A3、3C12、3H9及4D11。亚型鉴定表明,3C12重链为IgG2b,其余4株为IgG1,轻链均为κ链。间接ELISA结果表明,5株细胞培养上清的抗体效价在1∶1×104~1∶2×105,腹水效价在1∶1×105~1∶8×105。选其中两株杂交瘤细胞株3H9和4D11的腹水纯化,进行亲和力测定,解离常数分别为6.3×109和7.8×109,属高亲和力抗体。Western blotting结果显示,5株单抗均能与牛支原体发生特异性反应,而单抗4D11与羊无乳支原体标准株PG2和丝状支原体丝状亚种标准株PG3均不反应。流式细胞术结果表明,单抗4D11与牛支原体表面的VspX的结合呈剂量依赖性。间接免疫荧光结果表明,单抗4D11可以识别黏附到胚胎牛肺细胞上的重组VspX蛋白。本试验成功制备的单克隆抗体为VspX蛋白功能的研究奠定基础。
[Abstract]:In order to obtain monoclonal antibody against bovine mycoplasma (Mycoplasma bovis,M.bovis) VspX protein, the gene encoding the protein was cloned, expressed and purified as immunogen. QuickAntibody-Mouse 5W was used as immune adjuvant to immunize BALB/c mice. After three times of immunization, the mouse spleen cells were fused with SP2/0 myeloma cells. After three subcloning and screening, five hybridoma cell lines, named 1A8, 3A3, 3H9 and 4D11, which could stably secrete anti-VspX protein antibodies, were obtained. The identification of subtypes showed that the 3C12 heavy chain was IgG2b, and the other four strains were IgG1, light chain. The results of indirect ELISA showed that the antibody titers of 5 cell culture smears were 1: 1 脳 10 ~ 4 ~ 1: 2 脳 10 ~ 5, and those of ascitic fluid were 1: 1 脳 10 ~ 5 ~ 1:8 脳 10 ~ 5. The ascitic fluid of two hybridoma cell lines 3H9 and 4D11 was purified and their affinity was determined. the dissociation constant was 6.3 脳 109 and 7.8 脳 109, respectively, which belonged to high affinity antibody. Western blotting results showed that the five McAbs could react specifically with Mycoplasma bovis. However, the monoclonal antibody 4D11 did not react with the standard strain PG2 and the standard strain PG3 of Mycoplasma filamentum. The results of flow cytometry showed that the binding of monoclonal antibody 4D11 to VspX on the surface of Mycoplasma bovis was dose-dependent. The results of indirect immunofluorescence showed that monoclonal antibody 4D11 could recognize the recombinant VspX protein attached to embryonic bovine lung cells. The monoclonal antibodies successfully prepared in this experiment laid a foundation for the study of the function of VspX protein.
【作者单位】：华中农业大学动物医学院农业微生物国家重点实验室;
【基金】：国家自然科学基金(31302111、31272587) 中央高校基本科研业务费专项资金(2013QC001)
【分类号】：S852.62

【相似文献】