PTEN基因重组腺病毒载体的构建及鉴定

Construction and Identification of Recombinant Adenovirus Vector of PTEN Gene

目的构建含PTEN基因的重组腺病毒载体,为PTEN进行肺腺癌的基因治疗奠定基础。方法用PTEN引物VT415和VT416扩增PTEN基因后,分别用EcoRⅠ+SalⅠ双酶切PCR-PTEN和PSUCMV,回收并纯化PTEN cDNA小片断和PSUCMV的大片断,使两者连接获得含有PTEN基因的重组穿梭质粒载体PSUCMV-PTEN;采用细胞内同源重组方法构建PTEN基因重组腺病毒载体,将质粒PSUCMV-PTEN与含有5型腺病毒右臂的质粒Pbghe3通过Lipofectamine 2000共转染至293细胞,经PCR鉴定,扩增病毒,氯化铯密度梯度离心法纯化浓缩,测定病毒滴度。结果经双酶切和PCR进行鉴定,经鉴定正确的腺病毒命名为VSUCMV-PTEN,TCID_(50)法测定病毒滴度为1.0×10^(10)pfu/ml。结论成功构建了含PTEN基因的重组腺病毒载体Ad-PTEN,为下一步体内外实验证实PTEN对肺癌的抑制作用研究打下基础。

Objective To construct recombinant adenovirus vector carrying PTEN for lung cancer gene therapy.Methods After amplication of PTEN by primer VT415 ＋ VT416,PCR-PTEN and PSU CMV were cut off with endonuclease EcoR I and Sal I and then the cDNA fragment obtained from digestion was subcloned into PsuCMV to create a new shuttle vector PsuCMV-PTEN.The newly-recombinated PsuCMV-PTEN was tested by restriction endonulcease digestions.Then plasmid PSUCMV-PTEN and Pbhge3 were transfected by Lipofectamine2000 into adenovirus packaging cells,HEK293.The recombinant adenovirus Ad-PTEN,a non-proliferatable recombinant adenovirus,was obtained from the lysates of HEK293 pellet by repeating froze-melt procedure and the titer of which was estimated.Results The cloning sites and directions of insert of PTEN in the structure of PsuCMV-PTEN were exactly confirmed by digestion of two restrictive endonucleases.The exogenous PTEN gene could be transcripted and the titer of Ad-PTEN could reach 1.0×10^10 pfu/ml.Conclusion The recombinant adenovirus vector encoding PTEN gene has been constructed successfully,and confirmed the inhibition effect of PTEN on grouth of lung cancer in vitro and vivo for further.