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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 a recombinant adenovirus vector containing PTEN gene and lay a foundation for the gene therapy of PTEN for lung adenocarcinoma. Methods After amplifying PTEN gene with PTEN primers VT415 and VT416, PCR-PTEN and PSUCMV were double-digested with EcoRⅠ + SalⅠ, respectively. A small fragment of PTEN cDNA and a large fragment of PSUCMV were recovered and purified, and the two were ligated to obtain recombinant PTEN gene Shuttle plasmid vector PSUCMV-PTEN; Recombinant adenovirus vector of PTEN gene was constructed by intracellular homologous recombination method. Plasmid PSUCMV-PTEN and plasmid Pbghe3 containing adenovirus type 5 right arm were co-transfected into 293 cells by Lipofectamine 2000, Identification, amplification of virus, cesium chloride density gradient centrifugation purification concentration, determination of virus titer. The results were identified by double enzyme digestion and PCR. The correct adenovirus was named as VSUCMV-PTEN. The virus titer was 1.0 × 10 ~ (10) pfu / ml by TCID_ (50) method. Conclusions The recombinant adenovirus Ad-PTEN containing PTEN gene was successfully constructed, which laid the foundation for further studies on the inhibitory effect of PTEN on lung cancer in vitro and in vivo.