泡球蚴18基因的克隆、原核表达质粒的构建及其初步表达的研究  

Cloning and Construction of pET41a-Em18 prokaryotic expression plasmid and its primary expression in Escherichia coli

作　　者：王俨[1] 林仁勇[1] 丁剑冰[2] 卢晓梅[1] 张静萍[1] 王星[1] 梁晓慧[1] 张亚楼[1] 张琰[1] 温浩[1] 

机构地区：[1]新疆医科大学第一附属医院新疆包虫病基础医学重点实验室,新疆乌鲁木齐830054 [2]新疆医科大学第一附属医院基础医学院免疫学教研室,新疆乌鲁木齐830054

出　　处：《新疆医科大学学报》2005年第1期18-20,共3页Journal of Xinjiang Medical University

基　　金：国家自然科学基金(No.30360097;No.30160081);新疆重点实验室开放课题基金(XJDX0202200301);自治区高校科研创新研究群体基金(XJEDU2004G10)资助项目

摘　　要：目的: 克隆、分析泡球蚴 18 (Em18)抗原基因,构建 pET41a Em18 原核表达质粒,并初步诱导表达Em18重组蛋白。方法: DNAman软件设计引物,RT PCR法克隆 Em18 cDNA并构建 pMD18 T/Em18 质粒,测序确定序列,利用DNAman和BLAST软件,对其进行基因序列分析。构建 pET41a Em18 原核表达质粒,测序鉴定插入序列正确性。IPTG初步诱导和表达 rEm18 GST重组蛋白,SDS PAGE电泳检测。结果: 测序结果显示Em18抗原基因长度为 486 bp,编码 161 个氨基酸。BLAST 比对分析表明为一新序列并被 GenBank 收录(AY513691)。构建的 pET41a Em18原核表达质粒,经 IPTG诱导后,SDS PAGE检测表明 rEm18 GST重组蛋白得到成功表达,在相对分子量为50 KDa处有表达条带。结论: 成功克隆并构建了 pET41a Em18 原核表达质粒,初步诱导表达出 rEm18 GST重组蛋白,为新一代包虫病诊断试剂盒的研制奠定基础。Objective: To clone and construct the pET41a-Em18 prokaryotic expression plasmid and to study its primary expression in Escherichia coli. Methods: The primers of Em18 were designed by DNAman biosoftware and the Em18 antigen cDNA was amplified by PCR from total RNA of Echinococcus multilocularis, and was cloned into prokaryotic expression plasmid pET41a to construct the pET41a-Em18. The recombinant plasimid was transformed into E.coli BL21(DE3)and the positive clones were screened by restriction endonuclease analysis, and then sequenced. The sequences were analyzed by DNAman and GenBank/Blast biosoftware. The rEm18-GST fusion protein was primary expressed by induction with IPTG and was detected by SDS-PAGE. Results: DNA sequence analysis of Em18 cDNA fragment indicated that the length of Em18 was 486bp and code 161aa and was accepted by Genbank as a new sequence (AY513691). The pET41a-Em18 positive clone was the exact recombinant plasmid and the rEm18-GST recombinant protein was primary expressed as a band of 50 KDa by SDS-PAGE detection. Conclusion: pET41a-Em18 was constructed successfully and the rEm18-GST recombinant protein was expressed primarily and has potential for use in the research of diagnosis for alveolar echinococcosis and the possible development of a diagnosis kit.

关 键 词：泡球蚴 Em18基因 重组蛋白 

分 类 号：R532.32[医药卫生—内科学]