Construction And Identification Of Rnai Lentiviral Vector On Human Sedl Gene

Objective:To construct the lentiviral RNA interference (RNAi) vector of human SEDL gene, and generating high titer recombinant lentiviral, effectively silence the SEDL gene expression of human Osteoblasts.Provide a vector tool for the further study of X-linked spondyloepiphyseal dysplasia tarda (SEDL) disease patho genes is.Method:1.Four target sequences were selectde according to SEDL mRNA sequence, synthesized and annealed to form double-stranded DNA, inserted into plasmid pLenOR-THM whose segments were digested by Restriction endonuclease MluI and Clal,and transformde in the E. coli competent cells. identified by enzyme cutting and gene screening.2.293T cell was trans fected by above postive recombined plasmid and lentiviral packing materials. Collected viral supernatant.3. Realtime fluores-cence quantitative PCR was used to detect the expression ation of SEDL, Fitlered the best interference target.4. Determined the virus titer by serial dilution assay. recombination plasmids targeting the SEDL gene were successfully constructed.2.The fluorescence microscopy observations confirmed that the lentivirus plasmid was transfected into the package cell293T successfully.3. When transferred to human osteoblasts, the recombined lentiviral express well, the third interference target has the highest inhibition effciency4. The titre of the recombinant ad eno virus was4.6x108pfu/ml.Conclusion:1.The RNA interference system targeting the SEDL gene were successfully constructed.2.successfully interfered the expression of SEDL gene in hFOB1.19cells,a set of vectors with the highest inhibition efficiency was filtered out. and a interference vector with higher inhibition effciency was screened.