大鼠Hes1腺病毒表达载体构建及功能鉴定

目的 构建高滴度大鼠Hes1腺病毒过表达载体(Ad-Hes1).方法 以大鼠cDNA文库为模板,PCR法扩增Hes1,通过定向克隆构建pShuttle-CMV-Hes1穿梭质粒,再以pShuttle-CMV-Hes1为基础,构建pAdeno-Hes1病毒质粒,将pAdeno-Hes1转染293细胞,包装Ad-Hes1,利用改进TCID50法进行病毒滴度测定.Ad-Hes1感染H9c2心肌细胞,Western blot检测Hes1表达.结果 pShuttle-CMV-Hes1穿梭质粒、pAdeno-Hes1病毒质粒构建成功,总滴度为1.6×1011 PFU,Ad-Hes1可在H9c2心肌细胞内正常表达,其MOI值为30.结论 Ad-Hes1包装成功,为进一步研究Hes1的心肌保护作用奠定实验基础.

Construction and functional identification of rat Hes1 adenovirus expression vector

Objective to construct the high titers rat Hes1 adenovirus expression vector (Ad-Hes1).Methods With the rat cDNA as a template,the Hes1 fragment was amplified by PCR,which constructed pShuttle-CMV-Hes1 shuttle plasmid by directly clone.Based on pShuttle-CMV-Hes1,pAdeno-Hes1 virus plasmid was constructed,pAdeno-Hes1 was transfected into 293 cells to package Ad-Hes1,virus titers were determined by modified TCID50.Hes1 was detected by Western blot after Ad-Hes1 infected with H9c2 myocardial cells.Results pShuttle-CMV-Hes1 shuttle plasmid and pAdeno-Hes1 plasmid were constructed successfully,with a general titer of 1.6 × 1011 PFU,Ad-Hes1 can be expressed in H9c2 myocardial cells,and its MOI value was 30.Conclusion Ad-Hes1 is successfully constructedand packaged,thus provide basis for further research on the protection effect of Hes1 on myocardium.