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 fines, 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 Sal I and BamH I , the fragment was sub-cloned into pDsRed-Nl vector. After confirmed by restriction enzyme digestion, the recombinant plasmid was named as pDsRed-Nl-LBP. Two different regions of LBP mRNA were selected as the RNAi target sitesj 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 pSicoRGFP. The recombined vector pSicoR-GFP-shLBP-774/1212/1864(N. C) were confirmed by PCR and sequencing. The fusion expression vector (pDsRed-Nl-LBP) was co-transfected into 293 cells together with shRNA lentiviral expression vector (pSicoR-GFP-shLBP) by using Lipofectamine?2000. 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 recom-binant fusion expression vector pDsRed-Nl-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 'A, 29. 27% respectively detected by QRT-PCR. Therefore,the valid shRNA sequences targeting the buffalo LBP gene named shLBP-774, shLBP12!2 were successfully screened, which layeii a foundation for further study on the biological function of LBP in LPS/ TLR4 signal transduction pathway.%试验旨在构建水牛脂多糖结合蛋白(LBP)基因融合蛋白表达载体,探讨其在293细胞中的表达情况；筛选获得可抑制水牛LBP基因表达的shRNA干扰片段.采用RT-PCR方法,以水牛肝脏cDNA为模板,扩增水牛LBP开放阅读框(ORF),将其连接到pMD18-T载体中.序列测定并分析正确后,采用Sal I和BamHI进行双酶切,将水牛LBP基因编码区定向克隆至pDsRed-N1载体中,构建其融合蛋白表达载体pDsRed-N1-LBP.设计2个针对LBP靶基因序列的shRNA(774-792、1212-1231),同时设计无关序列1864作为阴性对照.合成好的shRNA序列先退火连接到pUC 57载体上,再亚克隆到慢病毒表达载体pSicoR-GFP中,将重组质粒命名为pSicoR-GFP-shLBP774/1212/1864(N.C),采用PCR和测序方法鉴定阳性克隆.将LBP融合蛋白表达载体和其shRNA慢病毒表达载体经脂质体共转染293细胞,48 h后观测荧光蛋白表达情况.收集共转染细胞样品,采用实时定量PCR(QRT-PCR)检测LBP基因的表达,筛选有效抑制LBP基因表达的shRNA干扰序列.结果表明,成功构建水牛LBP融合蛋白表达载体pDsRed-N1-LBP,并在293细胞中瞬时表达.PCR和测序结果均证实所构建的shRNA慢病毒表达载体pSicoR-GFP-shLBP774/1212/1864(N.C)为阳性克隆.共转染48 h后观测,红色和绿色荧光蛋白均有表达.QRT-PCR检测结果显示,shRNA-774和shRNA-1212对293细胞中LBP基因mRNA的表达均有抑制效果,抑制效率分别为49.53％、29.27％.因此,设计合成的2条shRNA序列能有效抑制水牛LBP基因的表达,这为进一步探讨LBP基因在LPS诱导的革兰氏阴性菌跨膜机制和信号转导中的作用机理研究奠定了基础.
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