广州管圆线虫半乳凝素基因的克隆表达、蛋白纯化及免疫反应性研究

目的 构建在E.coli中高效表达广州管圆线虫半乳凝素蛋白的重组表达质粒,并对重组蛋白纯化条件进行优化及免疫反应性鉴定.方法 根据本室构建的广州管圆线虫幼虫cDNA文库EST测序结果,筛选出有诊断潜能的半乳凝素基因,经过PCR扩增其cDNA全长序列后克隆入pET32a( )载体进行诱导表达,包涵体经洗涤、变性、逐步透析复性,浓缩后进行聚丙烯酰胺凝胶电泳及免疫印迹.结果 本研究首次克隆出广州管圆线虫半乳凝素蛋白基因全长cDNA序列(GenBank收录编号为DQ384534).成功构建重组质粒pET32a( )-Ac GAL,通过IPTG诱导得到以包涵体形式表达的重组融合蛋白,Western-blot结果显示纯化的重组蛋白具有良好的免疫反应性.结论 克隆表达了广州管圆线虫半乳凝素基因,为广州管圆线虫病诊断研究奠定了基础.

Cloning, prokaryotic expression and immunoreactivity evaluation of Angiostrongylus cantonensis galectin

Objective To construct the recombinant plasmid for Angiostrongylus cantonensis (AC) galectin (GAL) cDNA and analyze the immunological activity of the recombinant protein. Methods AcGAL cDNA was screened from the cDNA library and amplified by PCR. The amplified fragment was subcloned into the expression vector pET32a( ) and expressed in E.coli. The inclusion body was washed, degenerated, refolded by dialysis, and condensed for SDS-PAGE and Western blot analysis of the protein. Results For the first time the full-length cDNA of AcGAL was cloned (GenBank GeneID: DQ384534). Restriction enzyme digestion indicated that the recombinant plasmid pET32a( )-AcGAL was successfully constructed. SDS-PAGE analysis confirmed high expression of the recombinant protein AcGAL in E.coil in the form of inclusion bodies, which possessed good immunoreactivity as shown by Western blot analysis. Conclusion The success in cloning and identification, the recombinant AcGAL may provide basis for further diagnostic study of angiostrongyliasis.