多头带绦虫TPx基因片段的克隆及原核表达  

Cloning and prokaryotic expression of thioredoxin peroxidase gene fragment of Taenia multiceps

作　　者：李永光[1,2] 李文卉[2] 李航[2] 盖文燕[2] 王鸿盛[2] 姚菊霞[2] 王艳华[2] 张德林[2] 贾万忠[2] Radu Blaga 付宝权[2] 

机构地区：[1]甘肃农业大学动物医学院 [2]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 [3]College of Veterinary Medicine,University of Agricultural Sciences and Veterinary Medicine,Cluj-Napoca 400372,Romania

出　　处：《中国兽医科学》2009年第3期251-256,共6页Chinese Veterinary Science

基　　金：国家"十一五"科技支撑计划项目(2007BAD40B04);中央级公益性科研院所基本科研业务费专项项目(BRF080303);中国-罗马尼亚政府间科技合作项目(39-18)

摘　　要：从自然感染的病羊脑内采集多头蚴原头节,提取总RNA,采用RT-PCR技术扩增多头带绦虫硫氧还蛋白过氧化物酶(TmTPx)基因片段,PCR产物连接到pMD18-T载体,转化至大肠杆菌DH5α后测序,发现克隆到的TmTPx基因片段开放阅读框为453 bp,编码150个氨基酸。对TmTPx基因片段进行限制性酶切后连接到表达载体pGEX-4T-1,构建重组表达质粒pGEX-4T-TmTPx,转化大肠杆菌DH5α后筛选阳性克隆,经限制性酶切分析、PCR及测序鉴定后,转化大肠杆菌BL21,以IPTG进行诱导表达,用SDS-PAGE分析表达产物。结果显示,成功表达了大小约为43 ku的融合蛋白。对表达产物纯化后免疫家兔,采集血清用ELISA测定抗体效价,发现兔抗TmTPx重组蛋白的抗体能够与多头蚴原头节抗原发生特异性反应,说明该重组蛋白具有较好的免疫原性。Protoscoleces of cyst were recovered from the brain of sheep naturally infected with coenu- rus of Taenia multiceps and total RNA was extracted. T. multiceps thioredoxin peroxidase （TmTPx） cDNA fragment was amplified by RT-PCR from the total RNA, ligated into pMD18-T vector and sequenced. TmTPx cDNA consisting of 453 bp encoding 150 amino acids was digested with restriction enzymes and ligated into pGEX-4T-1 vector. The recombinant plasmid was transformed into Escherichia coli BL21 and induced by IPTG for expression. SDS-PAGE analysis showed that a 43 ku fusion protein was successfully expressed. The purified recombinant protein was used to immunize rabbits and the antibodies in serum were detected with ELISA. The specific rabbit antiserum against the purified TmTPx recombinant protein could specifically bind to the coenurus protoscoleces antigen,suggesting that the TmTPx recombinant protein had good immunogenicity.

关 键 词：多头带绦虫 多头蚴 硫氧还蛋白过氧化物酶 重组蛋白 免疫原性 

分 类 号：S852.734[农业科学—基础兽医学] Q786[农业科学—兽医学]