鸭Ⅰ型肝炎病毒VP1基因片段的克隆与表达载体的构建

Cloning of VP1 Gene Fragments of Duck Hepatitis Virus TypeⅠ and Construction of its Expression Plasmid

为研究鸭肝炎病毒VP1基因在大肠杆菌中的表达情况,根据鸭肝炎病毒核苷酸序列设计1对外引物和1对含有EcoRⅠ和SalⅠ酶切位点的内引物。以内外引物进行PCR扩增,将所得PCR产物回收,以相同的限制性内切酶酶切目的基因和表达载体pET-32a后构建重组表达载体,并转入宿主菌BL21和Rosseta,经酶切、PCR和测序鉴定,证明VP1基因正确插入了表达载体。用不同浓度的IPTG诱导VP1基因的表达,收集菌液进行SDS-PAGE电泳,结果表明,VP1基因片段在大肠杆菌Rosseta和BL21中均难以表达。

To study the expression of the duck hepatitis virus 1（DHV1） VP1 gene in E.coli,one outer primer pair and one inter primer pair containing EcoR Ⅰ and Sal Ⅰ according to complete genome of DHV1 were designed.PCR products were recycled by outer and inter primer pair.The PCR products and pET-32a vector were digested by EcoR Ⅰ and Sal Ⅰ to construct the recombinant plasmids.Then the recombinant plasmids were transfected into E.coli BL21 and Rosseta.After digestion,PCR and sequencing the target gene were inserted expression vector correctly.With different concentrations of IPTG induced bacteria express VP1 gene,the bacterias were collected and examined by SDS-PAGE.The results showed that the VP1 gene fragments were hard to express in E.coli Rosseta and BL21.