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

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...

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

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.