Construction and identification of the replication defective adenoviral vectors carrying mouse SCp2shRNA / 中华肝胆外科杂志

ABSTRACT

Objectives It was constructed that the replication defective adenoviral vectors carried the short hairpin sequences of mouse SCP2.And we will make a further study of mechanism between SCP2 gene and cholesterol stone in gallbladder.Methods The short hairpin sequences of mouse SCP2 were cloned by two-step PCR,and connected together with the plasmid pDC312.The Admax Adenoviral Vector System was used to generate the replication defective adenoviral vectors,which were purified by CsCl method.The processes of TCID50 were applied to detect the titers of the adenoviral vectors.Furthermore,Protein levels of SCP2 were determined by Western blot analysis,and the levels of SCP2、CYP7A1、HMGCR mRNA from the hepa1-6 cell of mouse were measured by the usage of RT-PCR.Results SCP2mRNA and SCP2 protein were down-regulated by the replication defective adenoviral vectors carried the SCP2-shRNA.With the decreasing SCP2mRNA,the levels of HMGCRmRNA were down-regulated at same the time,while CYP7A1mRNA were up-regulated.Conclusions The replication defective adenoviral vectors carried SCP2-shRNA were constructed successfully.The lower levels of SCP2 could affect the activities of HMG-CoA reductase and CYP7-a enzyme,which caused the variations of cholesterol metabolism and then decreased the formation of cholesterol stone.