Knocking down expression and function of human CD59 gene in heLa cells by retroviral vector-mediated RNAi

To construct recombinant retroviral vectors that target CD59 gene in order to analyze the role of CD59 in the immune escape of tumors.Three 60 bp sequences encoding CD59 gene shRNA were cloned into pSUPER vector with DNA recombinant technique, while control group was also prepared.The packaging cell Phoenix A was transfected with this recombinant plasmid using liposome;the virus supernants were harvested and used to infect HeLa cells.CD59 mRNA and protein levels were detected by RT-PCR and ELISA, while its function was analyzed by dye release assay.DNA sequencing demonstrated the pSUPER-siRNA expressing vectors were constructed successfully.RT-PCR and ELISA indicated that CD59 mRNA and protein levels were inhibited,while dye release assay suggested that anti-human complement effects of CD59 protection was decreased.pSUPER-siCD59 vectors were successfully constructed and identified, which could effectively and specially knock down the expression of CD59,even the anti-complement effects of CD59 protection.These results may pave the way for studying the role of CD59 in the immune escape of tumors,thus benefiting tumor therapy.