| 基于重组猪囊虫18 ku抗原的间接ELISA方法的建立 |
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| 引用本文： | 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). |
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| 作者姓名： | 吴国华 郑亚东 贾万忠 张少华 景志忠 骆学农 刘石泉 才学鹏 |
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| 作者单位： | 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 |
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| 基金项目： | 国家高技术研究发展计划"863"项目 |
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| 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.
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| 关 键 词： | 猪囊尾蚴 ku蛋白 大肠杆菌 表达 重组蛋白ELISA |
Development of an Indirect ELISA for Detecting Antibodies against Cysticercus cellulosae Based on Its Recombinant 18 ku Protein Expressed in E. coli |
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| 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). |
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| Authors: | WU Guo-hua ZHENG Ya-dong JIA Wan-zhong ZHANG Shao-hua JING Zhi-zhong LUO Xue-nong LIU Shi-quan CAI Xue-peng |
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| 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. |
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| Keywords: | 18 Cysticercus cellulosae 18 kDa protein E coli expression recombinant protein ELISA |
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