扩展莫尼茨绦虫DAZ基因保守结构域所在片段的克隆及原核表达  

Cloning and prokaryotic expression for the gene fragment from the conserved domain of DAZ gene in Moniezia expansa

作　　者：王涛[1,2] 夏党荣[1,2] 马勋[1] 薄新文[2] 王静梅[1] 

机构地区：[1]石河子大学动物科技学院,新疆石河子832000 [2]新疆农垦科学院新疆生产建设兵团绵羊繁育生物技术重点实验室,新疆石河子832000

出　　处：《黑龙江畜牧兽医》2012年第7期43-46,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金："973"国家重点基础研究发展计划前期研究专项(2006CB708512);国家绒毛用羊产业技术体系建设专项(CARS-40-11);兰州兽研国家重点实验室开放课题(SKLVEB2011KFKT008);兵团杰出青年科学基金项目(2011CD005)

摘　　要：为了克隆和表达编码扩展莫尼茨绦虫无精症缺失(DAZ)基因,试验根据GenBank中扩展莫尼茨绦虫DAZ基因cDNA序列(登录号为GH291478)设计1对特异性引物,以扩展莫尼茨绦虫组织总RNA为模板,RT-PCR扩增DAZ基因,并克隆到pMD19-T载体中进行序列测定,构建重组质粒pET28a-DAZ,转化BL21(DE3)大肠杆菌感受态细胞,以异丙基-β-D-硫代吡喃半乳糖苷(IPTG)进行诱导表达;利用非变性聚丙烯酰胺凝胶(SDS-PAGE)分析表达产物,通过螯合琼脂糖凝胶FF亲和层析柱对重组蛋白进行纯化。结果表明:重组DAZ基因在大肠杆菌中高效表达,表达的融合蛋白分子量约为14 ku。To clone and express the coding DAZ gene from Moniezia expansa,a pair of specific primers were designed by Moniezia expansa DAZ cDNA in GenBank（GH291478）.The total RNA extracted from was used as a template,and the DAZ gene was amplified by RT-PCR.The RT-PCR product was cloned into the pMD19-T vector and sequenced.The constructed recombinant plasmid pET28a-DAZ was transformed into E.coli BL21（DE3） and induced with IPTG.SDS-PAGE was used to analyze the recombinant protein,and the recombinant protein was purified by affinity chromatography on chelating Sepharose Fast Flow column.The results showed that the recombinant DAZ gene was expressed highly in E.coli,and its relative molecular weight of fusion protein was about 14 ku.

关 键 词：扩展莫尼茨绦虫(Moniezia expansa) 无精症缺失(DAZ)基因 克隆 原核表达 

分 类 号：Q785[生物学—分子生物学]