豆状带绦虫六钩蚴Tp-dUTPase基因的表达及血清学诊断效果  

Prokaryotic expression of Tp-dUTPase gene of Taenia pisiformis oncospheres and establishment of dot-ELISA using the expressed protein

作　　者：陈林[1] 杨德英[1] 孙家刚[2] 周英姿[3] 农向[1] 谢跃[1] 古小彬[1] 杨光友[1] 

机构地区：[1]四川农业大学动物医学院,四川雅安625014 [2]四川省苍溪县畜牧食品局,四川苍溪628400 [3]四川省眉山市畜牧局,四川眉山620032

出　　处：《中国兽医学报》2014年第7期1100-1105,共6页Chinese Journal of Veterinary Science

基　　金：教育部长江学者和创新团队发展计划资助项目(IRT0848)

摘　　要：采用PCR方法从豆状带绦虫六钩蚴中扩增得到Tp-dUTPase基因编码区,构建了pET32a-Tp-dUTPase原核表达载体,以重组蛋白dUTPase作为包被抗原建立的兔豆状囊尾蚴病Dot-ELISA诊断方法对226份兔血清进行了检测。结果显示,Tp-dUTPase基因编码区长447bp,编码148个氨基酸,表达的重组蛋白相对分子质量为36 200,该重组蛋白能与家兔豆状囊尾蚴病阳性血清特异性结合。以重组蛋白Tp-dUTPase作为包被抗原建立的Dot-ELISA诊断方法显示有较高的特异性（85.7%~92.9%）和敏感性（86.4%~88.0%）,与其他种类兔寄生虫病阳性血清有较低的交叉反应。结果表明,纯化的Tp-dUTPase重组蛋白可作为兔豆状囊尾蚴病的诊断抗原。In order to establish diagnostic method of Cysticercus pisiformis,the purified recombinant protein was used for development of an dot-ELISA for detection of anti-Cysticercus pisiformis antibodies.The complete coding region of Tp-dUTPase gene was firstly amplified from the cDNA of activated oncospheres of Taenia pisiformis by RT-PCR.The expression vector of pET32a-Tp-dUTPase was successfully constructed and expressed in Escherichia coli BL21（DE3）.226sera samples of rabbit were tested.The open reading frame of Tp-dUTPase consisted of 447bp and encoding 148amino acids.The recombinant protein was about 36 200.The positive serum of rabbit infceted with Taenia pisiformis larvae could specifically bound to Tp-dUTPase recombinant protein through western blotting.This purified recombinant fusion protein,rTpdUTPase,was probed by dot-ELISA with sera from rabbits infected with Taenia pisiformis larvae and with other parasites.Results showed that the dot-ELISA assay had both high sensitivity（85.7%-92.9%）and specificity（86.4%-88.0%）to detect Taenia pisiformis larvae infections,indicating that it can be used as diagnostic antigen.

关 键 词：豆状带绦虫 Tp-dUTPase 免疫诊断 DOT-ELISA 

分 类 号：S852.7[农业科学—基础兽医学]