p50PIK基因重组腺病毒的构建及对Hela细胞的影响

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

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XhoI和KpnI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。

Objective To construct recombinant adenovirns carrying p50PIK gene and examine the effect on cervix cancer Hela cell lines after transfection with Ad-p50PIK. Methods p50PIK eDNA 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 adenovirns Ad-p50PIK had been successfully constructed. Overexpression of p50PIK in Hela cell lines can promote phosphorylation of AKT.