旋毛虫成囊前期幼虫抗原结构基因TspE1的克隆与表达

目的　克隆和表达旋毛虫河南地理株成囊前期幼虫编码 3 1kDa抗原的结构基因 (TspE1)。　 方法　大鼠感染旋毛虫后第 17天收集成囊前期幼虫 ,提取虫体的总RNA。通过RT PCR特异性扩增目的基因 ,构建重组质粒pUC18 Ts HN3 ,将重组质粒 pUC18 Ts HN3中的目的基因亚克隆入原核表达载体pGEMEX 1,构建重组子 pGEMEX 1 Ts HN3 ,经IPTG诱导 ,在E .coliJM 10 9(DE3 )中表达。对表达产物进行SDS PAGE分析和Westernblotting鉴定。　 结果　SDS PAGE显示目的基因在大肠杆菌中获得高效表达 ,重组蛋白的分子量为 3 1kDa ,以诱导 4h时表达量最多。薄层凝胶光密度扫描分析结果显示 ,表达的融合蛋白量约占细菌总蛋白的 2 6%。Westernblotting证实 ,融合蛋白条带能被感染旋毛虫的大鼠血清及旋毛虫病患者血清识别。　结论　旋毛虫河南地理株成囊前期幼虫抗原结构基因TspE1克隆和原核表达成功。

Cloning and Expression of the Antigen Structural Gene TspE1 from Pre-encysted Larvae of Trichinella spiralis

Objective To clone and express the structural gene encoding a 31 kDa antigen of Trichinella spiralis (Henan isolate) pre encysted larvae (TspE1). Methods On the Day 17 after being infected with Trichinella spiralis ,pre encysted larvae were collected and total RNA of the larvae was obtained.The target gene in the recombinant plasmid (pUC18/Ts HN3) was sub cloned into the prokaryotic expression vector pGEMEX 1 and the recombinant pGEMEX 1/Ts HN3 was constructed. After IPTG induced incubation, the fusion protein was expressed in E.coli JM109(DE 3)competent cells, analysed by SDS PAGE and identified by Western blotting. Results The results of SDS PAGE demonstrated that the target gene was efficiently expressed and the level of expression peaked at 4 h post incubation. The molecular weight of the recombinant protein was 31 kDa. The portion of the fusion protein accounted for 26% of all the proteins by thin layer gel optical scanning. The fusion protein could be recognized by sera from rats infected with Trichinella spiralis and from patients with trichinellosis. Conclusion The gene encoding a 31 kDa antigen of Trichinella spiralis ( Henan isolate) pre encysted larvae ( TspE1) was cloned and expressed successfully in prokaryotic vector.