检测猪分子伴侣Jiv基因的SYBR GreenⅠ荧光定量RT-PCR方法的建立  

Establishment of a real-time RT-PCR based on SYBR GreenⅠfor detecting pig molecular chaperone Jiv gene

作　　者：谭学超[1] 郭抗抗[1] 宁蓬勃[1] 刘伟[1] 曹伟伟[1] 徐磊[1] 武宁[1] 林青[1,2] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]省部共建国家重点实验室培育基地青海省高原放牧家畜营养与生态重点实验室,青海西宁810061

出　　处：《中国预防兽医学报》2013年第11期894-898,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：西北农林科技大学基本科研业务费(QN2011109、QN2012018);陕西省13115科技创新工程重大科技专项(2010ZDKG-71);青海省高原放牧家畜营养与生态重点实验室开放课题

摘　　要：Jiv蛋白是一种与猪瘟病毒（CSFV）复制密切相关的细胞分子伴侣，为检测CSFV感染细胞内分子伴侣Jiv基因表达变化，本研究以猪Jiv基因（DNAJCl4）为参考序列，建立了检测猪分子伴侣JiV基因的SYBRGreenI荧光定量RT．PCR方法。结果显示，所建方法的标准曲线相关系数为0．999，扩增效率为103．4％，在10‘～10’拷贝数范围内具有较好的线性关系；对Jiv基N最低可检测到10拷贝，该方法的批内变异系数和批间变异系数均在O．5％以下，具有较高的敏感性和重复性。应用建立的方法对猪多种组织及猪源传代细胞中Jiv基N的表达进行检测，结果显示Jiv基因在所检测的组织和细胞中均有表达，表明所建方法可以用于Jiv基因的检测；同时对CSFV感染猪和阴性猪脾脏中的Jiv基因表达水平进行检测，统计发现CSFV感染猪脾脏中的Jiv基因表达水平显著上调，提示Jiv基因的表达在CSFV复制中发挥着一定的作用。本研究建立的检测猪Jiv基因的荧光定量RT．PCR方法为进一步明确Jiv蛋白在CSFV致病中的作用提供方法支持。Jiv protein is a kind of molecular chaperone closely related to the replication of classical swine fever virus （CSFV）. In this study, a real-time RT-PCR based on SYBR Green I was develop for detecting the molecular chaperone Jiv gene expression with the primers designed according to the porcine Jiv gene （DNAJC14）. The results showed the real time PCR had a linear relationship （between 101 to 107 copys） and high amplification efficiency （103.4%）. The variation coefficients of intro- and inter-assay were both less than 0.5%. The Jiv gene expression were detected in various of swine organs and cell lines by the assay, and the results showed that Jiv gene expressed in all these samples. Furthermore, the expression of Jiv gene in CSFV-infected and CSFV-free spleens were also detected, and revealed a higher expression of Jiv gene in spleen in pigs infected with CSFV-infected than that in uninfected pigs, indicating that the Jiv gene might play a role in the process of CSFV infection and replication.Therefore, the developed real-time RT-PCR detecting the expression Jiv gene could be effective for further study on the role of Jiv in pathogenic mechanism of CSFV.

关 键 词：Jiv蛋白 猪瘟病毒 荧光定量RT-PCR SYBR GreenⅠ 

分 类 号：S852.4[农业科学—基础兽医学]