环氧化酶-2基因shRNA重组腺病毒的构建及鉴定

Construction and identification of recombinant adenovirus with COX-2-shRNA

目的构建携带增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)标签的环氧化酶-2(cyclooxyge-nase-2,COX-2)基因shRNA重组腺病毒,并观察其对肝癌细胞SMMC-7721增殖的影响。方法将前期构建的真核表达质粒pGenesil-1-COX-2-shRNA及阴性对照质粒pGenesil-1-HK的表达启动子U6及shRNA序列亚克隆至腺病毒穿梭质粒pAdTrack中,构建重组腺病毒穿梭质粒pAdTrack-U6-COX-2-shRNA-EGFP和pAdTrack-U6-HK-EGFP,酶切及测序鉴定正确后,经PmeⅠ线性化,转化感受态AdEasier,构建重组腺病毒质粒pAd-U6-COX-2-shRNA-EGFP和pAd-U6-HK-EGFP,经PacⅠ线性化,转染AD293细胞,包装重组腺病毒Ad-U6-COX-2-shRNA-EGFP和Ad-U6-HK-EGFP,经3轮扩增后,测定滴度。RT-PCR和Western blot法检测感染细胞中COX-2基因mRNA的转录及蛋白的表达,MTS法观察重组腺病毒对肝癌细胞SMMC-7721增殖的影响。结果重组腺病毒穿梭质粒pAdTrack-U6-COX-2-shRNA-EGFP和pAdTrack-U6-HK-EGFP及重组腺病毒质粒pAd-U6-COX-2-shRNA-EGFP和pAd-U6-HK-EGFP经酶切和测序鉴定均构建正确,并成功转染AD293细胞,经包装和3轮扩增后,重组腺病毒Ad-U6-COX-2-shRNA-EGFP和Ad-U6-HK-EGFP的滴度分别为1.4×1012和2.0×1012pfu/ml。重组腺病毒感染的SMMC-7721细胞中COX-2基因mRNA、蛋白相对表达量及细胞增殖能力均明显低于空白对照组及阴性对照组(P均<0.05)。结论成功构建了COX-2基因shRNA重组腺病毒Ad-U6-COX-2-shRNA-EGFP,且可显著抑制肝癌细胞SMMC-7721的增殖,为进一步研究COX-2作为肝癌基因治疗靶点及机制奠定了基础。

Objective To construct and identify the recombinant adenovirus with a short hairpin RNA（shRNA）targeting cyclooxygenase-2（COX-2）,with a tag of enhanced green fluorescent protein（EGFP）,and observe its effect on the proliferation of liver cancer SMMC-7721 cells.Methods The U6 expression promoter and shRNA sequences of previously constructed eukaryotic expression vector pGenesil-1-COX-2-shRNA and negative control plasmid pGenesil-1HK were subcloned into adenovirus shuttle plasmid pAdTrack.The constructed recombinant adenovirus shuttle plasmids pAdTrack-U6-COX-2-shRNA-EGFP and pAdTrack-U6-HK-EGFP were identified by enzyme digestion and DNA sequencing,then linearized with Pme Ⅰ and transformed to competent AdEasier.The obtained recombinant adenovirus plasmids pAd-U6-COX-2-shRNA-EGFP and pAd-U6-HK-EGFP were linearized with PacⅠ and transfected to AD293 cells for packaging.The obtained recombinant adenoviruses Ad-U6-COX-2-shRNA-EGFP and Ad-U6-HK-EGFP were subjected to three cycles of amplification and determined for titer.The mRNA transcription and protein expression of COX-2 in infected cells were determined by RT-PCR and Western blot.The effect of Ad-U6-COX-2-shRNA-EGFP on proliferation of SMMC-7721 cells was observed by MTS method.Results Recombinant adenovirus shuttle plasmids pAdTrack-U6-COX-2-shRNA-EGFP and pAdTrack-U6-HK-EGFP,as well as recombinant adenovirus plasmids pAd-U6-COX-2-shRNAEGFP and pAd-U6-HK-EGFP,were constructed correctly as proved by enzyme digestion and sequencing,and successfully transfected to AD293 cells.The titers of recombinant adenoviruses Ad-U6-COX-2-shRNA-EGFP and Ad-U6-HK-EGFP were 1.4 × 10 12 and 2.0 × 10 12 pfu / ml respectively.The mRNA transcription and protein expression of COX-2 as well as proliferation ability of SMMC-7721 cells infected with Ad-U6-COX-2-shRNA-EGFP were significantly lower than those in blank and negative control groups（both P 0.05）.Conclusion Recombinant adenovirus Ad-U6-COX-2-shRNA-EGFP was constructed successfully,which inhibited the proliferation of SMMC-7721 cells significantly.The study solved the problems in clinical application and laid a foundation of research on therapy of liver cancer as well as the relevant molecular mechanism.