Abstract: The assay was aimed to construct the fusion expression vector of buffalo lipopolysaccharide-binding protein(LBP), investigate the effect of LBP short hairpin RNA (shRNA) lentiviral vector on buffalo LBP expression in 293 cell lines, and finally screen its valid shRNA sequence. The open reading frame of LBP was amplified from total RNA of buffalo liver by RT-PCR. The fragment of LBP was inserted into pMD18-T and identified by nucleotide sequencing. After digestion by restriction endonuclease SalⅠ and BamHⅠ, the fragment was sub-cloned into pDsRed-N1 vector. After confirmed by restriction enzyme digestion, the recombinant plasmid was named as pDsRed-N1-LBP. Two different regions of LBP mRNA were selected as the RNAi target sites: 774 to 792 bp, 1212 to 1231 bp, non-silencing sequence named 1864 as a negative control. These shRNAs were designed, annealed and synthesized into pUC57 vector. Then the shRNAs fragment was sub-cloned into lentiviral expression vector pSicoR-GFP. The recombined vector pSicoR-GFP-shLBP-774/1212/1864(N.C) were confirmed by PCR and sequencing. The fusion expression vector (pDsRed-N1-LBP) was co-transfected into 293 cells together with shRNA lentiviral expression vector (pSicoR-GFP-shLBP) by using Lipofectamine^TM2000. The expression of LBP was detected by real-time RT-PCR to investigate the optimal shRNA-LBP which had apparent knock-down capacity. The results showed that the recombinant fusion expression vector pDsRed-N1-LBP was successfully constructed. ShRNA lentiviral expression vector was constructed and it can inhibit the expression of LBP in 293 cells effectively. The level of LBP was reduced by 49.53%, 29.27% respectively detected by QRT-PCR. Therefore,the valid shRNA sequences targeting the buffalo LBP gene named shLBP-774, shLBP1212 were successfully screened, which layed a foundation for further study on the biological function of LBP in LPS/TLR4 signal transduction pathway.

Key words: LBP; shRNA; lentiviral expression vector; co-transfection