猪IL-1β与IL-18 SYBR Green Ⅰ荧光定量PCR检测方法的建立  

Development of SYBR GreenⅠ-based real-time PCR assays for detection of porcine IL-1β and IL-18

作　　者：汪伟[1,2] 倪艳秀[1] 温立斌[1] 吕立新[1] 周俊明[1] 俞正玉[1] 茅爱华[1] 张雪寒[1] 李彬[1] 郭容利[1] 王小敏[1] 范红结[2] 何孔旺[1] 

机构地区：[1]江苏省农业科学院兽医研究所,农业部兽用生物制品工程技术重点实验室,国家兽用生物制品工程技术研究中心,江苏南京210014 [2]南京农业大学动物医学院,江苏南京210095

出　　处：《江苏农业学报》2011年第6期1289-1294,共6页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省自然科学基金面上项目(BK2009327);江苏省自然科学基金重点项目(BK2010068);国家自然科学基金项目(31072155);江苏省农业科技自主创新探索性研究项目[CX(11)4040];国家“973”项目(2006CB504403)

摘　　要：IL-1β和IL-18是重要的促炎症细胞因子,在介导机体炎症反应中起重要作用。为了建立在体外定量检测猪细胞因子IL-1β和IL-18 mRNA表达水平的SYBR GreenⅠ荧光定量PCR方法,依据GenBank中登录的猪细胞因子基因IL-1β、IL-18及管家基因β-actin核苷酸序列,设计特异性引物,经RT-PCR从3D4猪肺泡巨噬细胞系中克隆和扩增了上述3个因子核苷酸片段,构建含有各自引物扩增序列的重组质粒作为阳性模板,建立了检测IL-1β、IL-18及β-actin SYBR GREEⅠ荧光定量PCR方法。该方法起始模板数与Ct值之间线性关系好,相关系数达到0.990以上;特异性强,扩增产物形成单一的特异性熔解峰;敏感性高,初始模板的检出下限达到了1μl 1.0×102拷贝;重复性好,组内变异系数均小于4%。The specific primers were designed and synthesized according to the nucleotide sequence of the porcine pro-inflammatory cytokines,i.e.IL-1β,IL-18 genes as well as housekeeping gene β-actin available in GenBank.IL-1β and IL-18 play important roles in mediating inflammatory response.The three fragments were amplified by RT-PCR from 3D4 porcine alveolar macrophages,cloned,and sequenced.The recombinant plasmids containing the target gene were constructed,and used as the real-time PCR standard templates.Real-time PCR assays based on SYBR Green Ⅰ for detection of IL-1β,IL-18 and β-actin were established.The results showed a good linear relationship between template copy number and circulation number,and the correlation coefficients（R2） of the standard curves for IL-1β,IL-18 and β-actin were over 0.990.Also,these assays were highly specific and there was single specific melting peak for every cytokine.Moreover,the assays were highly sensitive and had a detection limit of 1.0×102copies in 1 μl of initial templates.Finally,it was highly repeatable and had a coefficient of variation less than 4 percent for intra-assay.

关 键 词：猪 促炎症细胞因子 IL-1Β IL-18 荧光定量PCR 

分 类 号：S854.4[农业科学—临床兽医学]