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目的提取、纯化牛心肌肌钙蛋白T(Cardiac troponin T,cTnT),并制备其单克隆抗体。方法采用组织匀浆、蛋白层析技术提取、纯化牛cTnT,紫外分光光度法检测其浓度;SDS-PAGE检测其纯度;间接ELISA法检测其反应特性。将纯化的牛cTnT免疫小鼠,进行细胞融合,间接ELSIA法筛选阳性杂交瘤细胞株,采用小鼠体内法制备腹水。结果纯化的两批牛cTnT浓度分别为0.246和0.336 mg/ml;相对分子质量约为37 000,纯度分别为86.02%和93.77%;纯化的cTnT蛋白可与鼠抗牛cT-nT单克隆抗体结合。共获得5株分泌抗牛cTnT单克隆抗体的杂交瘤细胞株,其中,阳性杂交瘤细胞株3B10腹水抗体效价达1∶10 240。结论已成功提取、纯化了牛cTnT,并建立了稳定分泌抗牛cTnT单抗的杂交瘤细胞株,为cTnT检测试剂盒的国产化奠定了基础。
Objective To extract and purify cardiac troponin T (cTnT) and prepare its monoclonal antibody. Methods Tissue homogenate and protein chromatography were used to extract and purify bovine cTnT. The concentration of bovine cTnT was determined by ultraviolet spectrophotometry. The purity of bovine cTnT was detected by SDS-PAGE and the reaction was detected by indirect ELISA. The purified bovine cTnT mice were immunized, and the cells were fused. The positive hybridoma cells were screened by indirect ELSIA and the ascites was prepared by the mouse in vivo method. Results The cTnT concentrations in two batches of bovine serum were 0.246 and 0.336 mg / ml, respectively. The relative molecular mass was about 37 000 with purity of 86.02% and 93.77%, respectively. The purified cTnT protein could bind to mouse anti-cT-nT monoclonal antibody . Five hybridoma cell lines secreting anti-cTnT monoclonal antibodies were obtained, of which the titer of the ascites antibody of the positive hybridoma cell line 3B10 was 1:10 240. Conclusion The bovine cTnT has been successfully extracted and purified, and a hybridoma cell line stably secreting anti-cTnT mAb has been established, which lays the foundation for the localization of cTnT kit.