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当前位置: 首页 > 文章 > 多头带绦虫TPx基因片段的克隆及原核表达 中国兽医科学 2009,39 (3) 251-256
Position: Home > Articles > Cloning and prokaryotic expression of thioredoxin peroxidase gene fragment of Taenia multiceps Chinese Veterinary Science 2009,39 (3) 251-256

多头带绦虫TPx基因片段的克隆及原核表达

作  者：
李永光;李文卉;李航;盖文燕;王鸿盛;姚菊霞;王艳华;张德林;贾万忠;Radu Biaga;付宝权
单  位：
甘肃农业大学动物医学院;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;College
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
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。
译  名：
Cloning and prokaryotic expression of thioredoxin peroxidase gene fragment of Taenia multiceps
作  者：
LI Yong-guang1,2,LI Wen-hui2,LI Hang2,GAI Wen-yan2,WANG Hong-sheng2,YAO Ju-xia2,WANG Yan-hua2,ZHANG De-lin2,JIA Wan-zhong2,Radu Blaga3,FU Bao-quan2(1.College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;2.Key Laboratory of Animal Parasitology of Gansu Province/Key Laboratory of Veterinary Public Health of the Ministry of Agriculture/State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;3.College of Veterinary Medicine,University of Agricultural Sciences and Veterinary Medicine,Cluj-Napoca 400372,Romania)
关键词：
Taenia multiceps;coenurus;thioredoxin peroxidase;recombinant protein;immunogenicity
摘  要：
Protoscoleces of cyst were recovered from the brain of sheep naturally infected with coenurus 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.

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