细粒棘球绦虫AgB8/1重组抗原表达系统的构建  

Establishment of Echinococcus granulosus AgB8/1 recombinant protein expression cystem

作　　者：古力帕丽.麦曼提依明 马海梅[1] 陈洁[1,2] 陈璐[1,2] 吾拉木.马木提 

机构地区：[1]新疆医科大学基础医学院病原学教研室,新疆乌鲁木齐830011 [2]新疆医科大学第一附属医院包虫病重点实验室,新疆乌鲁木齐830011

出　　处：《新疆医科大学学报》2011年第3期246-250,共5页Journal of Xinjiang Medical University

基　　金：国家自然科学基金项目(30760229);新疆维吾尔自治区高校科研计划(XJEDU2008S31)

摘　　要：目的构建pET32a-AgB8/1原核表达载体,并对其重组蛋白进行原核细胞表达。方法从细粒棘球绦虫原头蚴中提取总RNA,反转录生成cDNA,以此cDNA为模板用基因特异性引物扩增EgAgB8/1基因编码其分泌型多肽的片段,经测序后,以此基因片段为依据,人工合成合成EgAgB8/1抗原编码核酸序列,将其克隆至pUCm-T载体,测序鉴定其正确性。通过对pUCm-T/AgB8/1重组质粒进行双酶切,将获得的AgB8/1抗原编码核酸序列用定向克隆技术克隆至原核表达质粒pET32a上,测序鉴定插入片段正确性后,转化至E.coli BL21(DE3)Lys S,IPTG初步诱导表达pET32a-AgB8/1重组蛋白。用SDS-PAGE电泳分析鉴定重组蛋白的表达水平。结果测序表明,AgB8/1抗原编码核酸序列正方向插入至pET32a质粒。SDS-PAGE电泳分析显示,IPTG诱导后重组蛋白得到成功表达,在相对分子量约28 kDa处有表达条带。结论本研究成功构建了pET32a-AgB8/1原核表达质粒,并初步诱导表达出AgB8/1重组蛋白,为进一步研究其免疫学特性奠定了基础。Objective To construct pET32a-AgB8/1 antigen prokaryotic expression recombinant plasmid and expression of its recombinant protein.Methods Total RNA was extracted from protoscoleces of Echinococcus granulosus and reverse transcribed into cDNA.The cDNA encoding mature form of EgAgB8/1 antigen was amplified by PCR using gene specific primers,after sequence confirmation,based on the gene fragments,a nucleotide sequence encoding EgAgB8/1 antigen was artificially synthetized.The synthetized nucleotide sequence encoding the EgAgB8/1 antigen was conformed by sequencing after cloning into pUCm-T vector,then the target sequence was directionally ligated into pET32a plasmid after duoble digestion with restriction enzymes for prokaryotic expression,and the constructed recombinant plasmid pET32a-AgB8/1 was transformed into E.coli BL21（DE3） LysS,and the recombinant protein expression was induced by isopropyl-1-thio-β-galactopyranoside（IPTG）.The recombinant protein expression was analyzed by SDS-PAGE.Results Sequence analysis revealed that the nucleotide sequence encoding for AgB8/1 recombinant protein was directionally cloned into pET32a plasmid.SDS-PAGE analysis confirmed that the recombinant protein AgB8/1 fused with Trx was succesfully expressed in E.coli BL21,with its relative molecule mass of expressed product of about 28 kDa.Conclusion The recombinant plasmid pET32a-AgB8/1 is successfully constructed and the recombinant protein EgAgB8/1 is expressed.The results obtained in this study will provide a foundation for further study on its immune characteristics in the future.

关 键 词：细粒棘球绦虫 AgB8/1重组抗原 原核表达质粒 

分 类 号：R383.3[医药卫生—医学寄生虫学]