一种新的广州管圆线虫候选诊断抗原基因的克隆、表达及初步鉴定

Cloning,expression,and primary identification of a new candidate antigen for diagnosis of Angiostrongylus cantonensis

目的克隆广州管圆线虫Ⅴ期幼虫候选免疫诊断抗原基因,进行体外表达及免疫学鉴定。方法根据本室构建的广州管圆线虫Ⅴ期幼虫cDNA文库的免疫筛选结果 ,选择文库中表达具有免疫反应性Ac11蛋白基因,进行生物信息学(在线Blast)分析,PCR扩增后克隆入pET28a(+)载体中,进行诱导表达及免疫学鉴定。结果推断Ac11基因为亚硫酸盐氧化酶基因。构建的重组质粒pET28a(+)-Ac11经IPTG透导,表达以包涵体形式存在的重组融合蛋白,分子质量单位约为30 kd。Western blot显示纯化的重组蛋白具有良好的免疫反应性。结论制备了具有良好免疫反应性的广州管圆线虫Ⅴ期幼虫亚硫酸盐氧化酶重组融合蛋白,为广州管圆线虫病的免疫诊断研究奠定了基础。

Objective A specific candidate antigen of stage V larvae of Angiostrongylus cantonensis was cloned,expressed,and identified.Methods Based on the results of screening the stage V larvae of A.cantonensis cDNA library using immuno-probes,the Ac11 gene was immunoreactive.The Ac11 gene was amplified and submitted for BLAST analysis online.Then,it was subcloned into the expression vector pET28a（＋） and expressed in E.coli.The recombinant protein Ac11 was purified and identified immunologically.Results The Ac11 gene was predicted to encode sulfite oxidase.The recombinant plasmid pET28a（＋）-Ac11 was successfully constructed.The recombinant protein Ac11 with a molecular weight of about 30 kd was highly expressed in the form of inclusion bodies according to SDS-PAGE analysis.It was also highly immunoreactive in Western blot.Conclusion The recombinant protein Ac11,which is highly immunoreactive,was obtained in vitro and may provide a basis for further immunodiagnostic study of angiostrongyliasis.