负载PCA3启动子及CD-TK自杀基因腺病毒载体的构建及包装

目的 构建负载PCA3启动子联合CD-TK基因的腺病毒载体,并对其进行包装及滴度测定.方法 将PCA3启动子无缝克隆到pHBAd-U6-GFP上,取代原有U6启动子形成pHBAd-PCA3-GFP,AgeI单酶切该重组载体,将CD-TK基因片段无缝克隆至线性化pH-BAd-PCA3-GFP上,抽提质粒抗性筛选后经PCR及测序鉴定为pHBAd-PCA3-CD-TK载体.取上述重组载体质粒及骨架质粒pHBAd-BHG,用LipofiterTM转染试剂进行转染HEK293细胞包装成病毒,大量扩增并测定病毒感染性滴度.结果 经PCR及测序验证证实成功构建负载PCA3启动子及CD-TK自杀基因的重组腺病毒pHBAd-PCA3-CD-TK并包装扩增,病毒滴度为1×1010PFU/mL.结论 构建负载PCA3启动子及CD-TK自杀基因的重组腺病毒,为进一步体内外对前列腺癌细胞的杀伤效应研究奠定了基础.

Construction and packaging of Adenovirus vector containing PCA3 promoter and CD-TK suicide gene

Objectives To construct and package that adenovirus vector containing PCA3 promoter and CD-TK suicide gene,and to determine the titer.Methods pHBAd-PCA3-GFP was created by replacing U6 with PCA3 promoter.After digest with Age I,CD-TK suicide gene was inserted into pHBAd-PCA3-GFP.The positive clones were identified by PCR and sequencing.pHBAd-PCA3-GFP-CD-TK and pHBAd-BHG were co-transfected into HEK293 cells with Lipofiter TM to package into adenovirus.The titer of the lentivirus was measured cells with the application of Li-pofiter TM to package the adenovirus.The title of the lentivirus was measured.Results PCR and sequencing results showed that pHBAd-PCA3-GFP-CD-TK plasmid was constructed successfully.The titer of the virus was 1 × 1010 PFU/mL.Conclusions The recombinant adenovirus vectors with PCA3 promoter and CD-TK suicide gene is successfully constructed,which can provide foundation further research of killing effect on prostate cancer cells.