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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 and identify recombinant adenovirus AdE-SC-EGFP and its mutant carrying SH2-Caspase8 fusion gene and observe its inhibitory effect on the proliferation of K562 cells. Methods The SH2-Caspase8 fusion gene was amplified by RT-PCR and cloned into pAdTrack-CMV. The shuttle plasmid pAdT-SC-EGFP was constructed and sequenced. The recombinant plasmid pAdE-SC-EGFP and pAdE-SC-EGFP were transfected into AD293 cells by PacⅠ digestion, then transformed into pAdEasy-BJ5183 competent cells by homologous recombination in bacteria. The recombinant adenovirus AdE-SC-EGFP was identified by packaging, PCR and Western blot. The virus was amplified and tested for titer. Results The results of PCR, restriction enzyme digestion and sequencing showed that the recombinant adenoviral plasmid pAdT-SC-EGFP and pAdE-SC-EGFP were constructed successfully. The results of PCR and EGFP expression showed that the recombinant adenovirus AdE-SC-EGFP Success, amplification, the titer up to 1.5 × 1012pfu / ml. After infected with K562 cells, Western blot was used to detect the expression of target protein. MTT assay and semi-solid colony formation assay were used to detect the effect of K562 on the growth and proliferation of K562 cells. Conclusions Recombinant adenovirus AdE-SC-EGFP and its mutant carrying SH2-Caspase8 fusion gene were successfully constructed and identified. MTT assay and colony formation assay showed that recombinant adenovirus AdE-SC-EGFP had obvious effect on BCR-ABL positive K562 cells Inhibition of proliferation.