p50PIK基因重组腺病毒的构建及对Hela细胞的影响

目的 构建携带P50基因的重组腺病毒载体,观察其感染宫颈癌Hela细胞后对p-AKT(Thr308)的影响.方法 以PcDNA3.1-p50PIK质粒为模板,构建带有p50PIK基因的重组腺病毒质粒Ad-p50PIK-GFP,酶切鉴定后经PacI线性化后转染HEK293包装细胞,观察细胞内绿色荧光蛋白(GFP)表达情况,并进行3轮扩增,收获带有目的 片段的腺病毒.将重组腺病毒Ad-p50PIK-GFP感染Hela细胞并通过Western blot技术检测其对p-AKT的影响.结果 将Ad-p50PIK-GFP经Xho I和Kpn I双酶切鉴定和琼脂糖电泳,在1400bp附近有目的 条带,送测序结果与GeneBank报道的序列完全一致,表明重组腺病毒Ad-p50PIK-GFP构建成功;然后将其转染HEK293细胞,绿色荧光表明Ad-p50PIK-GFP在HEK293包装细胞中成功表达;用SDS-PAGE电泳方法检测p50对Akt磷酸化的影响,结果表明高表达蛋白p50明显促进AKT的磷酸化(Thr308).结论 成功构建重组腺病毒Ad-p50PIK,p50在宫颈癌Hela细胞株中的过表达可使p-AKT表达升高.

Abstract：

Objective To construct recombinant adenovirus carrying p50PIK gene and examine the effect on cervix cancer Hela cell lines after transfection with Ad-p50PIK. Methods p50PIK cDNA was amplified by polymerase chain reaction (PCR), with the template PcDNA3. 1-p50PIK, then cloned into the shuttle plasmid pAdTrack-CMV. The plasmid pAdTrackCMV-p50 was linearized by PmeI, followed by homologous recombination with bone plasmid pAdEasy-1 in BJ5183, then identified by enzyme digestion.After linearized by PacI and transfection into HEK293 cells, recombinant adenovirus Ad-p50PIK were obtained in HEK293 cells then amplified by 3 circles. The green fluorescence in HEK293 cells was observed.Ad-p50PIK was transfected into Hela cells. The phosphorylation of Akt was detected by Western blotting.Results After double restriction enzyme digestion and agarose gel electrophoresis of The Ad-p50PIK-GFP,the 1400 bp purpose band was sequenced, Results was exactly the same with the sequence GeneBank had reported,indicating that the recombinant adenovirus Ad-p50PIK-GFP were successfully constructed; Then transfected into HEK293 cells, green fluorescence shows that Ad-p50PIK-GFP in HEK293 packaging cells successfully expressed; by SDS-PAGE electrophoresis was used to detect p50 on Akt phosphorylation, the Results show that high expression of p50 protein significantly increased AKT's Phosphorylated (Thr308).Conclusion Recombinant adenovirus Ad-p50PIK had been successfully constructed. Overexpression of p50PIK in Hela cell lines can promote phosphorylation of AKT.

Construction of recombinant adenoviruses carrying p50 and Its effect on Hela cells

Objective To construct recombinant adenovirus carrying p50PIK gene and examine the effect on cervix cancer Hela cell lines after transfection with Ad-p50PIK. Methods p50PIK cDNA was amplified by polymerase chain reaction (PCR), with the template PcDNA3. 1-p50PIK, then cloned into the shuttle plasmid pAdTrack-CMV. The plasmid pAdTrackCMV-p50 was linearized by PmeI, followed by homologous recombination with bone plasmid pAdEasy-1 in BJ5183, then identified by enzyme digestion.After linearized by PacI and transfection into HEK293 cells, recombinant adenovirus Ad-p50PIK were obtained in HEK293 cells then amplified by 3 circles. The green fluorescence in HEK293 cells was observed.Ad-p50PIK was transfected into Hela cells. The phosphorylation of Akt was detected by Western blotting.Results After double restriction enzyme digestion and agarose gel electrophoresis of The Ad-p50PIK-GFP,the 1400 bp purpose band was sequenced, Results was exactly the same with the sequence GeneBank had reported,indicating that the recombinant adenovirus Ad-p50PIK-GFP were successfully constructed; Then transfected into HEK293 cells, green fluorescence shows that Ad-p50PIK-GFP in HEK293 packaging cells successfully expressed; by SDS-PAGE electrophoresis was used to detect p50 on Akt phosphorylation, the Results show that high expression of p50 protein significantly increased AKT's Phosphorylated (Thr308).Conclusion Recombinant adenovirus Ad-p50PIK had been successfully constructed. Overexpression of p50PIK in Hela cell lines can promote phosphorylation of AKT.