羊痘野毒感染与弱毒免疫iELISA鉴别诊断方法的建立  

Development of iELISA for differentiation of natural infection of capripox from vaccinated animals

作　　者：李慧霞[1] 朱学亮[1] 刘振勇[1] 窦永喜[1] 李辉[1] 骆学农[1] 才学鹏[1] 

机构地区：[1]中国农业科学院 兰州兽医研究所 家畜疫病病原生物学国家重点实验室 农业部兽医公共卫生重点开放实验室 甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2013年第7期701-706,共6页Chinese Veterinary Science

基　　金：甘肃省农业生物技术研究与应用开发项目(GNSW-2010-08)

摘　　要：为建立一种方便、灵敏、有效的检测羊痘野毒感染的iELISA方法,以山羊痘病毒基因组DNA为模板,PCR扩增获得ORF95和ORF103基因的完整ORF,连接至克隆载体pMD18-T。测序正确后,将两基因片段分别与表达载体pET-30a(+)连接,构建原核表达载体pET-95和pET-103,转入BL21(DE3)感受态细胞,经IPTG诱导,SDS-PAGE分析后,用纯化的两种重组蛋白进行iELISA方法研究。结果显示,ORF95和ORF103大小分别为483bp和570bp,重组菌经1mmol/L IPTG诱导5h后,分别在30ku和35ku处有高水平的融合表达,且均以包涵体形式存在。两种目的蛋白以10ng+20ng混合作为包被抗原时,该iELISA方法检测羊痘自然感染血清、弱毒疫苗免疫羊血清和阴性血清的D450nm有明显差异,且与口蹄疫病毒、小反刍兽疫病毒、羊口疮病毒无交叉反应。因此,该方法能有效排除羊痘弱毒疫苗免疫造成的干扰,可特异性检测羊痘野毒感染的血清样品。In order to develop an indirect enzyme-linked immunosorbent assay（iELISA） for detection of the antibodies against goatpox virus（GTPV） and sheeppox virus（SPPV）,ORF95 and ORF103 genes were amplified from GTPV genomic DNA by PCR. Sequence analysis showed that the two ORFs consisted of 483 bp and 570 bp, respectively. The genes were subcloned into pET-30a（＋） expression plasmid and ex- pressed in Escherichia coli BL21（DE3） by IPTG induction. SDS-PAGE analysis showed that the fusion protein had molecular weight of 30 ku and 35 ku, respectively, and mainly existed in a form of inclusion bodies. The mixture of the two purified proteins（10 ng＋20 ng） was used to detect antibodies to GTPV and SPPV by iELISA. The results showed that values at D450 nm between the sera of infected animals and those of animals immunized by attenuated vaccine were different significantly. The analysis of specificity demon- strated that this method was specific for GTPV and SPPV and had no cross-reactions with sera from foot- and-mouth disease virus,peste des petits ruminants virus and orf virus infected animals.

关 键 词：山羊痘病毒 绵羊痘病毒 ORF95基因 ORF103基因 间接酶联免疫吸附试验 

分 类 号：S852.659.1[农业科学—基础兽医学]