摘要
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 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 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 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 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.
出处
《中国血吸虫病防治杂志》
CAS
CSCD
北大核心
2015年第3期277-281,共5页
Chinese Journal of Schistosomiasis Control
基金
安徽省自然科学基金(1308085MH124)
安徽省教育厅自然科学研究重点项目(KJ2011A205)
国家自然科学基金(81171606)
关键词
日本血吸虫
果糖二磷酸醛缩酶
克隆
表达
纯化
Schistosoma japonicum
Fructose-1 6-bisphosphate aldolase
Cloning
Expression
Purification
