Construction of a plasmid expressing siRNA aimed at the H-2K~d gene and expression of H-2Kd in mouse LAK cells

Objective To construct the siRNAs expressing plasmid aiming at the H-2Kd gene and to explore the H-2Kd expression in mouse LAK cells and function of H-2Kd with the RNA interference technology.Methods First,a target sequence of the H-2Kd gene was selected,then according to the sequence,two complementary oligonucleotides were lisated into psilencer 3.0-H1,which was transformed into DH5a bacteria,and then it was amplified and purified to get the plasmid.The purified plasmid was identified by gel electrophoresis and sequencing.H-2Kd-targeting siRNA plasmid was used in spleen lymphocytes of BALB/C mice.Flow cytometry(FCM) was used to determine the expression of H-2Kd on the transfected and control cells. Results Gel electrophoresis and sequencing results showed that the plasmid was about 2.8 kb which was identical with the positive control,and the sequence was identical with what had been inserted.Also siRNA treatment significantly inhibited the expression of the targeted protein. Conclusion siRNAs expression plasmids aiming at the H-2Kd gene have been successfully constructed and could inhibit the H-2Kd expression.