基于重组猪囊虫18 ku抗原的间接ELISA方法的建立

畜牧兽医学报 ›› 2009, Vol. 40 ›› Issue (12): 1788-1793.doi:

基于重组猪囊虫18 ku抗原的间接ELISA方法的建立

吴国华¹,郑亚东¹,贾万忠¹,张少华¹,景志忠¹,骆学农¹,刘石泉²,才学鹏¹*

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甘肃省动物寄生虫病重点实验室，农业部兽医公共卫生重点开放实验室，兰州 730046；2.江西农业大学动物科学技术学院，南昌 330045

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2009-12-24 发布日期:2009-12-24
通讯作者: 才学鹏

Development of an Indirect ELISA for Detecting Antibodies against Cysticercus cellulosae Based on Its Recombinant 18 ku Protein Expressed in E. coli

WU Guohua ¹, ZHENG Yadong¹, JIA Wanzhong ¹, ZHANG Shaohua¹, JING Zhizhong ¹, LUO Xuenong¹,LIU Shiquan², CAI Xuepeng ¹*

1. Key Laboratory of Veterinary Parasitology of Gansu Province, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046,China; 2. College of Animal Science and Technology，Jiangxi Agricultural University，Nanchang 330045，China

Received:1900-01-01 Revised:1900-01-01 Online:2009-12-24 Published:2009-12-24
Contact: CAI Xuepeng

摘要/Abstract

摘要： 利用反转录聚合酶链式反应(RTPCR)从猪囊尾蚴中克隆到18 ku蛋白基因，将扩增产物与pGEMT Easy载体连接后测序分析。将目的基因亚克隆至表达载体，构建重组质粒pGEXCE18，经转化大肠杆菌BL21（DE3）后诱导表达，用SDSPAGE和Westernblot分析表达产物。表达的目的蛋白纯化后作抗原建立检测猪囊虫抗体的重组蛋白间接ELISA方法。结果表明，18 ku 蛋白基因在大肠杆菌中成功表达，表达产物约为35 ku的融合蛋白，并能被猪囊虫感染血清识别。经薄层扫描分析，表达量占菌体蛋白总量的28%。与商品化ELISA试剂盒平行检测178份阳性血清样品，二者的符合率为 98.83%，说明建立的重组蛋白ELISA方法可用于猪囊虫病的诊断。

Abstract: A 18kDacoding gene of Taenia solium metacestodes was amplified by RTPCR and subcloned into pGEMT vector for sequencing. A recombinant plasmid pGEXCE18 was constructed and transformed into E. coli BL21 for in vitro expression. SDSPAGE and Westernblot were employed for analyzing the recombinant protein, which was used for development of an indirect ELISA for detection of anticysticercosis antibodies. The results showed that the interest protein was 35 kDa in size, accounting for 28% of total bacteria proteins, and could be recognized by positive sera against cysticercosis. Using the constructed indirect ELISA and a commercialized ELISA kit, paralleled analysis of 178 serum samples indicated that the concordant rate was 9883% and the ELISA showed good performance in specificity and sensitivity, supporting its application for cysticercosis diagnosis.

吴国华;郑亚东;贾万忠;张少华;景志忠;骆学农;刘石泉;才学鹏. 基于重组猪囊虫18 ku抗原的间接ELISA方法的建立[J]. 畜牧兽医学报, 2009, 40(12): 1788-1793.

WU Guohua;ZHENG Yadong;JIA Wanzhong;ZHANG Shaohua;JING Zhizhong;LUO Xuenong;LIU Shiquan;CAI Xuepeng . Development of an Indirect ELISA for Detecting Antibodies against Cysticercus cellulosae Based on Its Recombinant 18 ku Protein Expressed in E. coli[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2009, 40(12): 1788-1793.

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