Construction of conditionally replicative adenovirus expressing proliferation cell nuclear antigen siRNA, human telomerase reverse transcriptase siRNA with proliferation cell nuclear antigen promoter regulation

Abstract

Objective To construct conditionally replicative adenovirus (CRAds) expressing proliferation cell nuclear antigen (Ki-67) siRNA and human telomerase reverse transcriptase (hTERT) siRNA with Ki-67 promoter regulation.Methods (1) The hTERT siRNA sequence was synthesized and cloned into pSilencer3.1,resulting in the plasmid pShTERT siRNA.(2) Both pZKi-67 and pZD55-Ki-67 siRNA plasmid were digested with Xba I/EcoRV,the fragment containing Ki-67 promoter and the fragment containing Ki-67siRNA were connected,resulting in the plasmid pZKi-67-ZD-Ki-67siRNA.(3) The hTERTsiRNA was amplified from pShTERT siRNA and cloned into pZKi-67-ZD-Ki-67siRNA,resulting in the plasmid pZKi-67-ZD-Ki-67siRNA-hTERT siRNA.(4) pZKi-67-ZD-Ki-67 siRNA-hTERT siRNA was mixed with adenovirus packaging plasmid pBHGE3 and then co-transfected into HEK293 cells.The viral plaques appeared 9-12 days after infection.To verify the sequences of ZKi-67-ZD-Ki-67siRNA-hTERTsiRNA,DNA was extracted from purified viruses and analysed by polymerase chain reaction (PCR).Results Confirmed by PCR assay and sequence assay,a dual-RNA interferenced oncolytic adenovirus,which carried the Ki-67-siRNA and hTERT-siRNA and synchronously was regulated by both Ki-67 promoter was constructed.Conclusion We successfully constructed a dual-RNA interferenced oncolytic adenovirus and provide a new platform for cancer gene therapy. Key words: Conditionally replicative adenovirus; Proliferation cell nuclear antigen promoter; Proliferation cell nuclear antigen; Human telomerase reverse transcriptase; RNA interference