The Construction and Identification of CD226 Molecule siRNA Lentiviral Vector

To construct a lentiviral vector of RNA interference (RNAi) of human CD226 gene,the overexpression vector pFlag—CD226 was confirmed.4 sequence of siRNA targeting human CD226 gene was designed,synthesizedand co-transfected into 293T cells to screen the effective one.The complementary DNA containing both sense and antisense Oligo DNA of the effective sequence siRNA—513 was cloned into the pLKO.1 vector.The resulting lentiviral vector containing CD226 shRNA was named pLKO—C226,and it was confirmed by restriction endonucleaseanalysis and sequencing.293T cells were co-transfected with lentiviral vector pLKO—C226,VSV—G and 8.9.All virus stocks were produced by calcium phosphate mediated transfection,and the supernatant of transfected 293T cells was collected to obtain the recombinant lentivirus.The interference effective of virus was tested according to the expression level of CD226 on Jurkat/E6 cells.It is resulted that PCR and DNA sequencing demonstrates that the lentivirus RNAi vector of CD226 producing shRNA is constructed successfully,and which can inhibit the expression of CD226 molecule on Jurkat/E6 cell effectively.conclused that the lentivirus RNAi vector of CD226 is constructed successfully.