牛心肌肌钙蛋白T的提取、纯化及其单克隆抗体的制备

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。

Extract and Purification of Bovine Myocardial Troponin T and Preparation of Monoclonal Antibody

Objective To extract and purify bovine cardiac troponin T(cTnT) and prepare its monoclonal antibody(McAb).Methods Bovine cTnT was extracted and purified by tissue homogenation and protein chromatography,then determined for concentration by UV spectrophotometry,for purity by SDS-PAGE and for reaction character by ELISA.Mice were immunized with purified bovine cTnT for cell fusion,and positive hybridoma cell strains were screened by indirect ELISA for preparation of McAb in ascites.Results Both the relative molecular masses of two batches of purified bovine cTnT were about 37 000,while the concentrations were 0.246 and 0.336 mg / ml,and the purities were 86.02% and 93.77%,respectively.The purified cTnT showed specific binding to mouse anti-bovine cTnT McAb.A total of five hybridoma cell strains secreting McAbs against bovine cTnT were obtained,of which 3B10 strain secreted the McAb with a titer of 1 ∶ 10 240.Conclusion Bovine cTnT was successfully extracted and purified,and a hybridoma cell strain stably secreting McAb against bovine cTnT was established,which laid a foundation of production of domestic detection kit for cTnT.