携SH2-Caspase8融合基因重组腺病毒的构建及对K562细胞增殖的影响

Construction of recombinant adenovirus carrying SH2-Caspase8 fusion gene and its effect on proliferation in K562 cells

目的构建并鉴定携SH2-Caspase8融合基因的重组腺病毒AdE-SC-EGFP及其突变体,观察其对K562细胞增殖的抑制作用。方法采用RT-PCR、重叠PCR扩增SH2-Caspase8融合基因,克隆至腺病毒穿梭载体pAdTrack-CMV中,构建穿梭质粒pAdT-SC-EGFP,进行酶切和测序鉴定。将PmeⅠ酶切后的穿梭质粒转化pAdEasy-BJ5183感受态,通过细菌内同源重组产生复制缺陷型重组腺病毒质粒pAdE-SC-EGFP及其突变体,PacⅠ酶切后将其转染AD293细胞进行包装,PCR和Western blot鉴定重组腺病毒AdE-SC-EGFP,验证后扩增病毒并测定滴度。结果 PCR检测、酶切和测序结果均表明腺病毒穿梭质粒pAdT-SC-EGFP和重组腺病毒质粒pAdE-SC-EGFP构建成功;PCR检测、EGFP表达检测结果证明重组腺病毒AdE-SC-EGFP包装成功,扩增后,滴度可达1.5×1012pfu/ml。感染K562细胞后,Western blot检测目的蛋白的表达,MTT和半固体集落形成实验检测其对K562细胞生长增殖的影响。结论成功构建并鉴定了携带SH2-Caspase8融合基因的重组腺病毒AdE-SC-EGFP及其突变体;MTT和克隆形成实验证明重组腺病毒AdE-SC-EGFP对BCR-ABL阳性的K562细胞有明显的增殖抑制作用。

Objective To construct the recombinant adenovirus carrying SH2-Caspase8 fusion gene and its mutant to observe its inhibitory effect on proliferation of K562 cells.Methods SH2-Caspase8 fusion gene was amplified by RT-PCR and splicing PCR,and cloned into the shuttle plasmid pAdTrack-CMV of adenovirus.The shuttle plasmid pAdT-SC-EGFP was constructed and identified by double-enzyme digestion and DNA sequencing,and transformed into ultra-competent pAdEasy-BJ5183 after PmeⅠdigestion.Defective replication recombinant adenovirus plasmid pAdE-SC-EGFP and its mutant were generated by homologous recombination and further transfected into AD293 cells to package recombinant adenovirus after PacⅠdigestion.Recombinant adenovirus plasmid AdE-SC-EGFP was identified by PCR and Western blotting,respectively,and amplified for the measurement of its titers.Results PCR,enzyme digestion and DNA sequencing showed that the shuttle and recombinant plasmids of adenovirus,pAdT-SC-EGFP and pAdE-SC-EGFP,were successfully constructed.PCR and EGFP expression confirmed that the recombinant plasmid of adenovirus,pAdE-SC-EGFP,was successfully packaged.After amplification,its titer was 1.5×1012 pfu/ml.Western blot analysis displayed the expression of target protein.MTT and colony formation test could inhibit the proliferation of K562 cells.Conclusion Recombinant adenovirus plasmid,AdE-SC-EGFP,carrying SH2-Caspase8 fusion gene and its mutant,has been successfully constructed,and can significantly inhibit the proliferation of BCR-ABL positive K562 cells in vitro.