Screening Valid Shrna Sequences Of Buffalo TREM1and LBP Gene

In the immune responses elicited by gram-negative bacteria, silencing of triggering receptor on myeloid cell1(TREM1) will affect CD14gene expression in the TLR4-mediated signaling pathway, inhibiting lipopolysaccharide-binding protein (LBP) expression will block LPS/CD14pathway in LPS recognition/signaling. To explore the signal transduction mechanisms of brucellosis and finally study the feasibility of using RNAi technology to produce genetically anti-brucellosis animals. In this study, the shRNA sequences targeting buffalo TREM1and LBP were screened respectively.Research1. Screening of valid shRNA sequence targeting buffalo TREM1and isolation of buffalo macrophages1. The buffalo and bovine TREM1mRNA ORF sequences were cloned respectively, their homology was analyzed. The fusion expression vector of buffalo TREM1was constructed, then transfected into293cells. The expression level of TREM1in293cells was detected by RT-PCR method. The results showed that the homology between the buffalo and bovine TREM1mRNA ORF sequences was97.2%. The buffalo TREM1GenBank accession number was JQ390222. The buffalo TREM1recombinant plamid was sucessfully expressed in HEK293cells.2. Five shRNAs targeting buffalo TREM1were designed, recombinant shRNA expressing vectors named as pSicoR-shTREM-96/194/294/578/643respectively were constructed. Non-silencing sequence named pSicoR-GFP-1864(N.C) was used as negative control. After confirmed by restriction enzyme digestion and sequencing methods, recombinant fusion expression vector of buffalo TREM1(pDsRed-N1-TREM1) and short hairpin RNA (shRNA) lentiviral expressing vectors targeting TREM1(pSicoR-shTREM-96/194/294/578/643/N.C) were co-transfected into HEK293cells at the ratio of1:1and1:3using LipofectamineTM2000, respectively. Expression of TREM1was detected by FACS and QRT-PCR methods. The results showed that when the co-transfection dose ratio was1:1, the inhibition effects of shTREM-194, shTREM-294and shTREM578were53.86%,49.27%and40.10%, respectively. While the dose ratio was1:3, their knockdown results were63.07%,79.24%and31.14%, respectively.3. Buffalo macrophages were isolated from peripheral blood. They were infected by high-titer lentivirals which expressed the valid shRNAs. The results showed that TREM1and CD14expressed in buffalo macrophages detected by QRT-PCR analysis. The virus infection efficiency in macrophages was low.Research2. Screening of valid shRNA sequence targeting buffalo LBP1. The buffalo and bovine LBP mRNA ORF sequences were cloned respectively. The fusion expression vector of buffalo LBP was constructed and transfected into293cells. The expression of LBP gene was detected by QRT-PCR method. The results showed that the homology between the buffalo and bovine LBP mRNA ORF sequences was98%. GenBank accession number of buffalo LBP gene was JQ390223. The buffalo LBP recombinant plamid was successfully expressed in HEK293cells.2. Two shRNAs targeting buffalo LBP were designed, recombinant shRNA expressing vectors named as psicoR-shLBP-774/1212were constructed, respectively. After confirmed by PCR and sequencing methods, recombinant fusion expression vector of buffalo LBP (pDsRed-N1-LBP) and short hairpin RNA (shRNA) lentiviral expressing vectors targeting LBP (pSicoR-shLBP-774/1212/N.C) were co-transfected into HEK293cells respectively at the ratio of1:3using LipofectamineTM2000. Expression of LBP gene was detected by QRT-PCR method. The results showed that the inhibitive efficiency of pSicoR-shLBP-774/1212were49.53%and29.27%, respectively.In conclusion, the three shRNA sequences named shTREM-194, shTREM-294and shTREM-578can effectively inhibit the expression of buffalo TREM1, in which the inhibition efficiency of shTREM-194and shTREM-294was63.07%and79.24%. And the two shRNAs named shLBP774and shLBP1212can effectively down-regulated the LBP expression, in which the inhibition efficiency of shLBP774is arrived at49.53%. Buffalo macrophages are isolated and cultured successfully from peripheral blood.