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 1.5×10

12

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.