猪抗病毒蛋白基因重组质粒实时荧光定量PCR标准曲线的建立  

Establishment of standard curves for detection of porcine antiviral protein genes recombinant plasmids using Real-time PCR

作　　者：温立斌[1] 何孔旺[1] 杨汉春[2] 倪艳秀[1] 吕立新[1] 张雪寒[1] 茅爱华[1] 

机构地区：[1]江苏省农业科学院兽医研究所,南京210014 [2]中国农业大学动物医学院,北京100193

出　　处：《中国农业大学学报》2009年第2期1-4,共4页Journal of China Agricultural University

基　　金：国家“973”计划前期研究专项(2007CB116308);江苏省自然科学基金(BK2008351)

摘　　要：根据GenBank中的猪抗病毒蛋白PKR、OAS/RNase L及Mx核苷酸序列设计特异性引物,经RT-PCR技术从猪外周血单个核细胞中扩增和克隆了PKR、OAS/RNase L及Mx的101 bp核苷酸片段。测序鉴定后,将重组质粒10倍系列稀释后作为标准模板,通过实时荧光定量PCR方法,建立了抗病毒蛋白PKR、OAS/RNase L及Mx标准曲线及其直线回归方程;该方法具有线性关系好,特异性、敏感性、重复性高等特点;建立的荧光定量PCR方法可检测抗病毒蛋白PKR、OAS/RNase L及Mx mRNA水平。为猪体内抗病毒蛋白PKR、OAS/RNase L及Mx的mRNA水平的定量检测提供必要的技术。The current study is to provide powerful tools for the quantification of antiviral proteins （AVP ： PKR, OAS/ RNase L and Mx） in pigs. Primers were designed and synthesized based on the nucleotide sequences of PKR, OAS/ RNase L and Mx genes available in GenBank. 101 bp fragments were amplified by RT-PCR from the porcine peripheral blood mononuclear cells （PBMC）, and then the fragments were cloned and sequenced. Recombinant plasmids were diluted by 10-fold serial and used as the Real-time PCR standard templates. Their standard curves and the corresponding linear regression equations were obtained. Results indicated that the standard curves were shown to be of high linearity, specificity, sensitivity and reproducibility. The mRNA level of PKR, OAS/RNase L and Mx can be detected by established Real-time PCR assay.

关 键 词：抗病毒蛋白 外周血单个核细胞 实时荧光定量PCR 重组质粒 

分 类 号：S85[农业科学—兽医学]