人胆固醇酯水解酶重组腺病毒载体的构建

Construction of human CEH recombinant adenovirus vector

目的构建人胆固醇酯水解酶(hCEH)基因重组腺病毒载体,并在人胚肾细胞(HEK293)中扩增。方法将hCEH基因整合入腺病毒穿梭质粒pAdTrack-CMV中,构建重组穿梭质粒pAdTrack-CMV-hCEH,线性化后与pAdeasy-1在大肠杆菌BJ5183中进行同源重组,获得含目的基因的重组质粒pAd-hCEH,酶切后用脂质体转染HEK293,包装成重组体腺病毒Ad-hCEH,经扩增和纯化得到高滴度重组病毒。采用PCR法进行鉴定,绿色荧光蛋白(GFP)追踪,荧光显微镜下观察并计算Ad-hCEH病毒滴度。结果重组穿梭质粒及其载体经鉴定后,电泳结果与预期相符。Ad-hCEH-EGFP感染HEK293后,随时间增加,HEK293表达绿色荧光的量也增加。hCEH碱基序列经测序与Genebank中已知序列完全吻合。Ad-hCEH电泳结果显示1 700 bp附近有一条带,与目的片段相符合。最终测得Ad-hCEH滴度为1.2×1010 pfu/ml。结论成功构建了携带hCEH基因的重组腺病毒载体。

Objective To construct hCEH recombinant replication deficient adenovirus vector pAd-hCEH amplify in HEK293 cells.Methods hCEH gene was cloned into shuttle vector pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-hCEH,and the resultant plasmid was linearized and was subsequently homogenous recombination with pAdeasy-1 in E.coli BJ5183 cells.Recombinants were screened and were identified by observation of green fluorescent protein（GFP） and PCR.The recombinant plasmid was transfected into HEK293 to package the adenovirus,and the hCEH recombinant adenovirus was observed by fluorescent microscope.Results The hCEH recombinant adenoviral vector was constructed successfully,which was confirmed by restriction enzyme and GFP expression.High titer recombinant adenoviral vector was constructed.Conclusion The hCEH recombinant adenovirus is constructed successfully.