Aim: To construct recombinant vectors expressing siRNA that target CD59 gene and a stable-inhibit cell line A2780 in order to analyze the role of CD59 in the protection to complement-mediated cytolysis.
Methods: The 60 bp encoded targeting CD59 gene shRNA sequence was cloned into pSUPER vector with DNA recombinant technique. The ovary cancer cell A2780 was transfected with this recombinant plasmid using liposome and the stable strains was selected by using G418-medium, CD59 mRNA and protein level was detected by RT-PCR and Western blot, and its function was analysed by dye release assay.
Results: The pSUPER-siRNA expressing vector was successfully constructed. And a stable cell line A2780 was selected and was detected the expression of GFP. The siRNA vector effectively inhibited the CD59 gene expression from mRNA and protein level. Dye release assay suggested that CD59's protection to complement-mediated cytolysis decreased.
Conclusion: The siRNA vector targeting CD59 gene could consistently inhibit CD59 expression. Furthermore, it decreased CD59's protection against complement. These results may pave the way for studying the role of CD59 in the immune escape of tumors cells as well as in tumor therapy.