日本血吸虫果糖二磷酸醛缩酶的重组表达及其在血吸虫生活史各阶段的表达分析

目的 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 方法 以日本血吸虫成虫cDNA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 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 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。

Recombinant expression of Schistosoma japonicum fructose-1, 6-bisphos-phate aldolase and its expression in different developmental stages of S. japonicum

Objective Objective To clone，express and purify Schistosoma japonicum fructose?1， 6?bisphosphate aldolase（SjFBPA） in E. coli and observe its expression in different developmental stages of S. japonicum. Methods Methods FBPA gene was amplified from S. japonicum adult worm cDNA by using PCR. The amplified product was recombined into pET28a plasmid，and inducibly expressed with IPTG in E. coli BL21. SDS?PAGE and Western blotting were employed to analyze and identify the recombinant protein SjFBPA（rSjFBPA） . Then，rSjFBPA was purified by chromatographic purification and its purity was analyzed by SDS? PAGE. The protein concentration of rSjFBPA purified was measured by the BCA method. Furthermore，SjFBPA mRNA was ana? lyzed in different developmental stages of S. japonicum by RT?PCR. Results Results SjFBPA was successfully amplified by using PCR and identified by restriction enzyme digestion and sequencing. The Western blotting analysis confirmed that the recombinant pro? tein could specifically reactive to the anti ?His? tag monoclonal antibody. The concentration of the purified recombinant protein was about 4 mg/ml. The result of RT?PCR showed that SjFBPA mRNA was expressed in cercaria，schistosomulum，adult worm and egg of S. japonicum. Conclusion Conclusion SjFBPA is successfully recombined and expressed in a prokaryotic system，and SjFBPA mRNA is expressed in cercaria， schistosomulum，adult worm and egg of S. japonicum.