摘要
目的:构建截短的泡球蚴18(Em18)基因的原核表达质粒,获得高效表达、有生物活性的重组蛋白,并对其进行初步鉴定。方法:DNAman软件设计引物,PCR法扩增并构建截短的pET41a-Em18．1原核表达质粒,测序鉴定插入序列正确性。异丙基硫代-β-D-半乳糖苷(IPTG)诱导、表达和纯化rEm18．1-GST重组蛋白,采用SDS-PAGE 电泳及Western免疫印迹法对其进行初步鉴定。结果:成功构建出截短的pET41a-Em18．1,SDS-PAGE检测表明rEm18．1-GST重组蛋白得到成功表达,在相对分子量为41 kDa处有表达条带。Western blot结果显示 rEm18．1-GST重组蛋白能被AE病人阳性血清识别。结论:截短的pET41a-Em18．1原核表达质粒表达的 rEm18．1-GST重组蛋白具有良好的抗原性,为Em18抗原表位的分析奠定基础。
Objective. To clone, construct and express truncated pET41a-Em18.1 recombinant plasmid and to study its primary antigenicity. Methods： The primers of truncated Em18 were designed by DNAman biosoftware and the truncated fragment was amplified by PCR from pMD18-T/Em18, and was cloned into prokaryotic expression plasmid pET41a to construct the pET41a-Em18-1. The recombinant plasmid was analyzed by sequenceing. The rEm18.1-GST fusion protein was expressed by induction with IPTG and purified using His-tag column, and then was detected by SDS-PAGE and Western Blot. Results： The pET41a-Em18-1 positive clone was the exact recombinant plasmid and the expressed rEm18.1-GST recom- binant protein can be detected as a band of 41KDa by SDS-PAGE and Western blot which confirmed that the recombinant protein could specifically react with the serum samples from patients with alveolar echinococcosis （AE）. Conclusion： The truncated pET41a-Em18.1 was constructed successfully and the rEm18.1- GST recombinant protein is expressed and shows a good antigenicity. This work has potential for use in the research of epitope analysis of Em18 antigen.
出处
《新疆医科大学学报》
CAS
2006年第4期283-285,288,共4页
Journal of Xinjiang Medical University
基金
国家自然科学基金(No.30360097)
新疆重点实验室开放课题基金资助项目(XJDX0202-2003-01)
关键词
泡球蚴
Em18基因
重组蛋白
Echinococcus multilocularis
Em18 recombinant protein
detection
