华支睾吸虫成虫RPEF基因产物及其抗RPEF多克隆抗体的制备

目的 体外制备华支睾吸虫成虫RNA聚合酶Ⅱ延长因子(RPEF)基因产物及抗RPEF多克隆抗体.方法 亲和纯化试剂盒纯化表达蛋白pGEX-4T-1-RPEF,凝血酶酶切表达蛋白制备重组蛋白RPEF,将重组蛋白RPEF免疫小鼠,制备多克隆抗血清,检测抗体滴度和抗血清特异性.结果 体外制备的RPEF蛋白经SDS电泳呈单一条带;酶联免疫吸附结果显示,免疫过程抗体滴度呈连续上升趋势;免疫印迹结果显示,小鼠抗血清具有抗RPEF特异性.结论 获得较专一的华支睾吸虫RPEF蛋白和RPEF抗血清.

The preparation of RPEF gene products of Clonorchis sinensis and the anti-RPEF polyclonal antibodies

Objective To prepare RPEF Gene products of Clonorchis sinensis and the anti-RPEF polyclonal antibodies. Methods The expressed protein pGEX-4T-1-RPEF was purified by GST Bind Purification kit and was digested with thrombin. The experimental mice were inoculated with recombinant RPEF protein. The poly-clonal antisera were obtained from the immuned mice. The antisera collected were used to detect the titres of IgG antibodies and to identify anti-RPEF specificity. Results SDS-PAGE showed single band of the purified RPEF protein. ELISA analysis showed that IgG titer in the immuned mice was increased. The results of West-ern blot suggested that the mouse antir RPEF antisera were highly specific. Conclusion The RPEF gene products of Clonorchis sinensis and the anti-RPEF polyclonal antibodies were successfully prepared.