茶毛虫核型多角体病毒ph基因抗体的制备与利用  

Preparation of the Antibody Against EupsNPV Polyhedrin and Its Utilization in Detection of the Viral Pesticide

作　　者：王礼中[1,2] 肖强[1] 张传溪[3] 

机构地区：[1]中国农业科学院茶叶研究所 [2]浙江大学应用昆虫研究所,浙江杭州310029 [3]浙江大学应用昆虫研究所

出　　处：《茶叶科学》2008年第4期260-266,共7页Journal of Tea Science

基　　金：国家科技支撑计划(2006BAD06B01);浙江省科技计划(2003C2003)

摘　　要：以茶毛虫核型多角体病毒(EupsNPV)的基因组DNA为模板,PCR扩增出全长为741bp的ph基因编码区片段。将Ep-ph编码区插入pET-28-a,构建了原核表达载体pET-28-a-Ep-ph,转化大肠杆菌BL21(DE3),在IPTG诱导下进行了高效的表达。以经表达纯化的目的融合蛋白作为抗原,免疫新西兰大白兔,制备了EupsNPV的ph抗体,测得的抗体效价为6.4×104。Western blotting检测表明制得的抗体对核型多角体病毒有良好的特异性。利用制备的抗体用间接ELISA方法对茶毛虫病毒样品进行了定量分析,得到线性回归方程为y=0.4152x-0.8299,相关系数r=0.9897(P<0.01)。间接ELISA方法表明该抗体可以用于核型多角体病毒生物农药的定量检测,从而为核型多角体病毒的检测农药制剂提供了一种准确有效的方法。The 741 bp coding region of polyhedrin gene （ph） was amplified from Euproctis pseudoconspersa nucleopolyhedrovirus （ EupsNPV ） genome and its prokaryotic expression vector pET-28-a-Ep-ph was constructed. Then the prokaryotic expression plasmid was transformed into Escherichia.coli. BL21 （ DE3 ） for expression. The expressed fusion protein was separated by SDS-PAGE and retrieved from the gel. New Zealand white rabbit was immunized with the purified fusion protein and the antiserum against ph with a titre of 6.4 × 10^4 was prepared. Western blotting indicated that the prepared polyclonal antibody was specific for polyhedrin. The EupsNPV was detected by indirect ELISA and its regression equation was obtained： y=0.4152x-0.8299 with correlation coeffient r=0.9897（P〈0.01）. Indirect ELISA detection showed that the antibody could be used for quantitative detection of the NPV in a commercial bio-pesticide and provided an accurate and effective method for detection.

关 键 词：茶毛虫核型多角体病毒 多角体蛋白基因 多克隆抗体 间接ELISA 检测 

分 类 号：S435.711[农业科学—农业昆虫与害虫防治]