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目的 建立日本血吸虫线粒体相关蛋白原核表达产物的纯化方法 ,获得理想的表达产物。方法 菌体经冻融、超声破菌及提取包涵体后 ,用分子筛层析纯化蛋白质并使之复性 ,再用Western -blot方法对纯化蛋白的抗原性进行分析。经动物免疫后采用dot-ELISA方法检测兔血清中抗体滴度 ,分析纯化蛋白的抗原活性。结果 经分子筛纯化后的融合蛋白rSj3 3 8/ 2 6GST经SDS -PAGE电泳鉴定达电泳纯 ,Westernblot显示纯化蛋白能够被日本血吸虫重感染兔血清及rSj3 3 8/2 6GST免疫兔血清识别。dot-ELISA法检测结果表明经分子筛纯化并复性的rSj3 3 8/ 2 6GST融合蛋白免疫兔血清中特异性抗体的滴度最高 ,为 1∶5 12 0 0。结论 纯化后的融合蛋白rSj3 3 8/ 2 6GST具有较高的纯度及较强的免疫活性 ,可望作为日本血吸虫病疫苗候选分子进一步深入研究。
Objective To establish a method for the purification of the prokaryotic expression product of Schistosoma japonicum mitochondria related protein and to obtain the ideal expression product. Methods After frozen and thawed bacteria, sonication and extraction of inclusion bodies, the protein was purified by molecular sieve chromatography and renatured. The antigenicity of purified protein was analyzed by Western-blot. The antibody titer of rabbit serum was detected by dot-ELISA after animal immunization, and the antigenic activity of the purified protein was analyzed. Results The fusion protein rSj3 3 8/2 6GST purified by molecular sieve was identified by SDS-PAGE electrophoresis. Western blot showed that the purified protein could be recognized by rabbit sera infected with Schistosoma japonicum and rSj3 3 8/2 6 GST. The results of dot-ELISA assay showed that the titer of specific antibody in rabbit serum immunized with rSj3 3 8/2 6GST fusion protein purified by molecular sieve and refolded was the highest, which was 1: 5 12 0 0. Conclusion The purified fusion protein rSj3 3 8/2 6GST has high purity and strong immunogenicity and is expected to be further studied as a vaccine candidate for schistosomiasis japonica.