The Inhibiting Of SIRNA On VP1 Expression In Human Cell

AIM: To obtain the SiRNA which can efficiently inhibit the expression of VP1 of EV71 virus steadily; and to transform it into attenuated Salmonella to construct a oral gene vaccine targeting to VP1 gene of EV71, then to provide the theoretical and experimental basis for targeting treatment of hand-foot-mouth disease with RNA interference technology。METHODS: By using molecular cloning technique, the specific SiRNA interference plasmids targeting to VP1 mRNA were constructed based on the pSEB-HUS vector which has EGFP maker, U6 and H1 promoters, which were confirmed by double-enzyme digestion and DNA sequencing. After these plasmids were transinfected into A549 cell respectively, the expression level of vp1 mRNA and protein was detected by Q-PCR, Western blot and fluorescence microscope, in order to screen the clones in which VP1 was steadily inhibited.RESULTS: The SiRNA interference plasmids (pSVsi1, pSVsi2 and pSVsi3) were successfully constructed. The expression level of VP1 mRNA and VP1 protein of pSVsi1 group decreased significantly and the inhibition rate were 90% and 89% respectively. The selected SiRNA plasmid pSsi1 was contructed and transformed into the attenuated Salmonella successfully.CONCLUSION: The specific SiRNA interference plasmids targeting to VP1 gene mRNA is successfully designed and constructed. The SiRNA which can efficiently inhibit the expression of VP1 of EV71 steadily is obtained, which provides the experimental models for developing EV71 vaccine in further study.