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目的在大肠杆菌中原核表达、纯化日本血吸虫果糖二磷酸醛缩酶(rSjFBPA),观察其在血吸虫生活史各阶段的表达。方法以日本血吸虫成虫c DNA为模板扩增rSjFBPA基因,克隆至pET28a(+)质粒后,再转化入E.coli BL21。含重组质粒的菌株经IPTG诱导后,采用SDS-PAGE和Western blotting分析鉴定重组蛋白rSjFBPA是否表达,用层析法纯化rSjFBPA并用SDS-PAGE鉴定其纯度。同时,用RT-PCR方法分析SjFBPA在血吸虫尾蚴、童虫、成虫和虫卵各阶段的表达情况。结果经PCR扩增出目的基因,含目的基因的TA克隆质粒经双酶切和测序鉴定,证明插入片段与预期目的基因序列相符。Western blotting结果显示,表达后的重组蛋白可与His-tag单克隆抗体发生特异性反应。经镍亲和层析法制备了纯化的重组SjFBPA蛋白,纯化重组蛋白浓度达4 mg/ml。RT-PCR结果显示,SjFBPA在日本血吸虫尾蚴、童虫、成虫和虫卵阶段均有表达。结论 SjFBPA基因被成功克隆和表达,其在日本血吸虫尾蚴、童虫、成虫和虫卵各阶段均有表达。
Objective To prokaryotic express E. coli fructose diphosphate aldolase (rSjFBPA) in Escherichia coli and observe the expression of rSjFBPA in various stages of schistosomiasis life. Methods The rSjFBPA gene was amplified from c DNA of adult Schistosoma japonicum and cloned into pET28a (+) plasmid. The recombinant plasmid was transformed into E. coli BL21. The recombinant plasmids were induced by IPTG. The recombinant protein rSjFBPA was identified by SDS-PAGE and Western blotting. The purified rSjFBPA was purified by chromatography and identified by SDS-PAGE. At the same time, the expression of SjFBPA in various stages of schistosoma cercariae, schistosomula, adult and eggs was analyzed by RT-PCR. Results The target gene was amplified by PCR. The TA cloned plasmid containing the target gene was identified by double enzyme digestion and sequencing, which proved that the inserted fragment was consistent with the expected gene sequence. Western blotting results showed that the expressed recombinant protein could specifically react with His-tag monoclonal antibody. Purified recombinant SjFBPA protein was prepared by nickel affinity chromatography and the recombinant protein was purified to 4 mg / ml. RT-PCR results showed that SjFBPA was expressed in Schistosoma japonicum cercariae, schistosomula, adult and egg stages. Conclusion The SjFBPA gene was successfully cloned and expressed. It was expressed in all stages of Schistosoma japonicum, cercariae, adult worms and eggs.