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

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.