Screening Of Shrna Sequences Related To Buffalo CD14Gene And Their Effects On The Expression Of Related Genes

The purpose of this study was to screen shRNA sequences related to buffalo CD14gene, and examine their effects on the expression of CD14gene in buffalo peripheral blood monocye/macrophage and related genes after stimulating by LPS, so as to establish a foundation for further studying the role of CD14gene in the inflammatory reaction induced by Brucella and find a new way to prevent brucellosis for livestock.1. Five CD14shRNA (319/421/755/970/1041) sequences and a negative control sequence (NC-1864) were designed, synthesized and used to construct lentiviral recombinant plasmids pSicoR-GFP-shRNA. Lentiviral recombinant plasmids pSicoR-GFP-shRNA and fusion expression vector pDsRed-N1-buffalo CD14were co-transfect-ed into HEK293using liposome. At72h after transfection, the interference effects of each shRNA sequence were detected by QRT-PCR and FACS methods. The results showed that the expression of exogenous buffalo CD14mRNA was reduced at different level for all shRNA plasmids. Among the five CD14shRNA sequences, shRNA-1041had the higest interfering efficiency (P<0.01).2. Lentivirus of CD14shRNA and NC-1864was obtained by packaging three plasmids into293T cells using calcium phosphate precipitation method, and the titer of virus was determined. Then, buffalo peripheral blood monocye/macrophage was infected by the lentivirus. On7d after infection, the cells were stimulated by1μg/mL LPS for3hours. After that, the mRNA expression level of CD14、 TLR4、IL-6�'�TNF-a transcripts in the cells were dectected by QRT-PCR analysis. The results showed that the expression of endogenous CD14was reduced by CD14shRNA-1041which had hightest interfering efficiency. After stimulating by LPS, CD14mRNA level of infected cells were significantly reduced in comparison with control group. The mRNA expression level of TLR4、IL-6and TNF-a genes were also reduced significantly.In conclusions, the five interference sites (319,421,755,970and1041) can inhibit the expression of buffalo CD14gene, in which the1041site has the highest efficiency. Not only lentivirus of CD14shRNA-1041can inhibit the expression of CD14mRNA on Buffalo peripheral blood monocye/macrophage, but also down-regulate the mRNA expression of CD14, TLR4, IL-6and TNF-a genes, reduce the signal conductive function of TLR4after stimulating by LPS.