Aim: To construct a siRNA plasmid to knockdown Coronin-1.
Methods: The cDNA of coronin-1 was amplified by RT-PCR from the total RNA of macrophage, and then inserted into pSEB-HUS vector to generate pSEB-HUS-C plasmid. Three synthesized siRNAs targeting Coronin-1 were cloned into pSEB-HUS-C respectively, resulting in the pSEB-HUS-C1, pSEB-HUS-C2 and pSEB-HUS-C3 plasmids. These plasmids were transiently transfected into A549, and the Coronin-1 level was detected by RT-PCR, Real time PCR and Western blot.
Results: All plasmids were successfully constructed as confirmed by restriction enzyme digestion and DNA sequencing. The pSEB-HUS-C3 vector had the most significant knockdown effect on Coronin-1, with 75.9% inhibition at mRNA level and 75.1% inhibition at protein level.
Conclusion: A siRNA plasmid targeting Coronin-1 was successfully constructed and validated for its knockdown effect, which will serve as a loss-of-function tool for the further mechanistic study of Coronin-1 in tuberculosis pathology.