广州管圆线虫ASP基因的克隆及原核表达

Cloning and prokaryotic expression of ASP gene from Angiostrongylus cantonensis

目的对广州管圆线虫ASP基因的完整开放读码框进行克隆、表达及重组蛋白的免疫性分析。方法以广州管圆线虫幼虫cDNA文库中含有ASP基因的质粒为模板,扩增目的基因,进一步将其克隆到原核表达质粒pET-30a(+)中,重组质粒转化大肠杆菌BL21(DE3),经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,Ni-IDA亲和层析纯化表达产物。免疫印迹(Western blotting)分析重组蛋白的免疫性。结果广州管圆线虫ASP基因的编码区含有366个碱基,编码121个氨基酸,相对分子量(Mt)为13398.26Da。重组质粒pET-30a(+)-ASP构建成功,IPTG诱导获得可溶性表达的重组蛋白,经亲和层析获得的纯化蛋白可被广州管圆线虫病人血清识别。结论广州管圆线虫ASP基因可在原核表达系统中获得具有免疫性的高效表达。

The purpose of this study was to clone and express the complete open reading frame of ASP gene from Angiostrongylus cantonensis and analyze the immunogenicity of the recombinant protein. The coding region of ASP gene was amplified from the larvae cDNA plasmid library by PCR. The PCR product was then cloned into the prokaryotic expression vector pET-30a （＋） and transformed into E.coli BL21 （DE3）. After that,the IPTG induced product was purified by Ni-IDA affinity chromatography,then detected by SDS-PAGE and identified its immunogenicity by Western blotting. Results showed that the coding sequence of ASP gene contains 366 base pairs encoding 121 amino acids,with a theoretical molecular weight of 13398.26 Da. PCR,double enzyme digestion and DNA sequencing assay confirmed that the recombinant plasmid pET-30a （＋）-ASP was constructed successfully. By IPTG inducing,the soluble expression of recombinant protein was obtained in E.coli BL21. The affinity chromatography purified recombinant protein which could be recognized by the serum of the angiostrongyliasis cantonensis patients with Western-blotting. It＇s concluded that the ASP gene has been efficiently expressed in prokaryotic expression system with immunogenicity in vitro.