Construction And Selection Of SiRNA Plasmid Vector For Suppressing Coronin-1 Gene Expression

Objective: To construct the plasmid vectors of siRNA which can specifically suppress the expression of coronin-1 gene and select the most effective one for further study.Methods: The cDNA of coronin-1 was amplified from the total RNA of macrophage by RT-PCR, then it was inserted into pSEB-HUS vector. After identified by restriction digestion and DNA sequencing, the constructed recombinant plasmids were named pSEB-HUS-C. 3 synthesized siRNAs were cloned into pSEB-HUS-C respectively, in order to construct the pSEB-HUS-C1, pSEB-HUS-C2 and pSEB-HUS-C3 plasmids, which were designed for Coronin-1 as target gene. After these plasmids was transiently transfected into A549, the inhibition level of Coronin-1 in A549 cells was detected by RT-PCR, Real time PCR and Western blot.Result: The recombinant plasmids were confirmed by double-enzyme digestion and DNA sequencing. The expression of Coronin-1 mRNA and Coronin-1 protein of pSEB-HUS-C3 group decreased significantly and the inhibition rate was 75.9% and 75.1% respectively.Conclusion: Specific siRNA interference plasmid vector targeted to coronin-1 gene mRNA is successfully constructed, which provides a good basis for further research on the function of Coronin-1 against tuberculosis...