山羊Toll样受体基因TLR2和TLR4及TLR9的荧光定量PCR检测方法的建立

Development of real-time RT-PCR assays based on SYBR GreenⅠ for detection of goat Toll-like receptors

根据山羊Toll样受体（TLR2、TLR4和TLR9）及管家基因β-actin的保守序列设计特异性引物,分别建立不同的荧光定量PCR标准曲线及直线回归方程,并对方法的灵敏度、重复性和特异性进行检验。结果表明：山羊TLR2、TLR4、TLR9及管家基因β-actin的荧光定量RT-PCR标准曲线相关性R2大于0.984;特异性强,出现特异性熔解峰;重复性好,组内与组间的变异系数均小于2%。应用所建立的方法对N1蛋白缺失的重组山羊痘病毒（△N1L株）感染山羊外周血淋巴细胞TLR2和TLR9 mRNA表达水平进行了检测,△N1L株可以显著提高机体固有免疫应答水平。本研究建立的山羊天然免疫因子相关受体荧光定量PCR方法可应用于山羊痘新型基因工程疫苗的评价。

For detection of the three goat Toll-like receptors TLR2,TLR4 and TLR9 and the housekeeping gene β-actin,a series of primers were designed according to the sequences.The recombinant plasmids containing the target genes were used to optimize assay condition of developing a SYBR Green Ⅰ real-time RT-PCR.The results showed that the Ctof TLR2,TLR4,TLR9 and β-actin cytokines had a good linear relationship（R20.984）.These assays were single specific melting peak for every cytokine.The coefficients of variation were lower than 2 percent for both intra-and inter-assay.The established assays were successfully used to detect TLR2 and TLR9 mRNA expression levels in peripheral blood mononuclear cells（PBMCs） in goats experimentally infected with recombinant goatpox virus（△ N1 L strain）,indicating△N1 L strain could significantly increase the level of innate immune response in vivo.The established real-time RT-PCR could be applied to the development of a genetic vaccines for goatpox.