人S100A8重组腺病毒质粒的构建及鉴定

Construction and Identification of Recombinant Adenovirus Vector Expressing Human S100A8

目的构建人S100A8(hS100A8)重组腺病毒质粒,为hS100A8的深入研究奠定基础。方法从pGST-hS100A8中扩增hS100A8片段,亚克隆至穿梭质粒pAdTrack-TOX,构建重组穿梭质粒pAdTrack-TOX-hS100A8。经酶切、PCR及测序鉴定,再经PmeⅠ酶切线性化后电转化感受态AdEasier细胞,获得重组腺病毒质粒pAdhS100A8,经PacⅠ酶切后转染至HEK293细胞进行包装,制备重组腺病毒,扩增后进行病毒滴度测定及RT-PCR和Western blot鉴定。结果重组穿梭质粒pAdTrack-TOX-hS100A8及腺病毒质粒pAdhS100A8经鉴定均构建正确;重组腺病毒AdhS100A8在HEK293中成功包装,扩增后病毒滴度为1011 IU/ml;hS100A8在HEK293细胞中成功表达。结论成功构建了hS100A8重组腺病毒质粒,为深入研究hS100A8奠定了基础。

Objective To construct recombinant adenovirus vector expressing human S100A8（hS100A8） and lay a foundation of further study on hS100A8.Methods From plasmid pGST-hS100A8,hS100A8 gene fragment was amplified and subcloned into shuttle plasmid pADTrack-TOX.The constructed recombinant shuttle plasmid pADTrack-TOX-hS100A8 was identified by restriction analysis,PCR and sequencing,then linearilized with Pme Ⅰ and transformed to competent AdEasier cells.The obtained recombinant adenovirus vector pAdhS100A8 was digested with Pac Ⅰ and transfected to HEK293 cells for packaging,and the prepared recombinant adenovirus was amplified for titration and identification by RT-PCR and Western blot.Results Both recombinant shuttle plasmid pADTrack-TOX-hS100A8 and recombinant adenovirus vector pAdhS100A8 were constructed correctly.Recombinant adenovirus AdhS100A8 was successfully packaged in HEK293 cells,reached a titer of 1011 IU / ml after amplification,and successfully expressed in HEK293 cells.Conclusion The recombinant adenovirus vector expressing hS100A8 was successfully constructed,which laid a foundation of further study on hS100A8.