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细粒棘球绦虫TPx基因的克隆及序列分析
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李航1，李文卉2，苟惠天2，李永光2，王艳华2，张德林2，贾万忠2，史大中1，付宝权2*
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(1．兰州大学 基础医学院,甘肃 兰州 730000；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 甘肃省动物寄生虫病重点实验室，甘肃 兰州 730046)
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摘要：
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从自然感染病羊肝内收集细粒棘球绦虫(Eg)原头蚴，分别提取总RNA和基因组DNA。根据GenBank数据库中的细粒棘球绦虫硫氧还蛋白过氧化物酶(EgTPx)基因序列设计1对引物，以总RNA为模板，采用RT-PCR技术扩增出EgTPx基因片段，同时以基因组DNA为模板扩增出对应的基因组序列。PCR产物经纯化后连接到pMD18-T载体，转化至大肠杆菌DH5α后，进行序列测定和生物信息学分析。结果表明，成功扩增到中国青海株细粒棘球绦虫EgTPx基因片段，其开放阅读框为582bp，编码193个氨基酸，与已知的EgTPx基因核苷酸序列及其编码蛋白氨基酸序列的同源性均为99%，有3个碱基发生变异，分别在245，306，318位由C变为T；引起1个氨基酸变异，由Ala82变为Val82。EgTPx具有典型的2-Cys型过氧化物氧还蛋白催化性位点Cys48和Cys169，以及周围保守的FVCP和VCPA序列。EgTPx基因的开放阅读框对应的基因组序列包含2个外显子和1个69bp的内含子。 |
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关键词：
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细粒棘球绦虫；硫氧还蛋白过氧化物酶；基因克隆；序列分析 |
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中图分类号：S 852.734：Q 785 文献标识码：
A 文章编号：1673-4696(2008)03-0191-05 |
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Cloning and sequence analysis of thioredoxin peroxidase gene
of Echinococcus granulosus |
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LI Hang1，LI Wen-hui2，GOU
Hui-tian2，LI Yong-guang2，WANG Yan-hua2，ZHANG
De-lin2，
JIA Wan-zhong2,SHI Da-zhong1，FU Bao-quan2 |
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(1.College of Basic Medical Sciences,Lanzhou University,Lanzhou 730000,China;2.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) |
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Abstract: Protoscoleces were recovered from the liver of sheep naturally infected with Echinococcus granulosus(Eg),and total RNA and genomic DNA were extracted from the protoscoleces,respectively.One pair of specific primers based on published thioredoxin peroxidase gene of Eg(EgTPx) sequence in GenBank were designed and used to amplify cDNA sequence of EgTPx by RT-PCR from the total RNA and to obtain genomic sequence of EgTPx by PCR from the genomic DNA.The amplified products were purified and cloned into pMD18-T vector and sequenced.Sequence analysis indicated that the EgTPx gene of E.granulosus isolated from Qinghai,China,was successfully cloned with an ORF of 582bp encoding 193 amino acids which shared 99% identity with the published EgTPx mRNA sequence(AF478688) and EgTPx(AAL84333),with three bases changed from T to C respectively and one amino acid changed from Ala82 to Val82.EgTPx presented two highly conserved motifs(FVCP and VCPA) around the catalytic sites Cys48 and Cys169 for the typical 2-Cys peroxiredoxin.Comparison of cDNA and genomic sequences of EgTPx gene revealed the existence of one intron of 69bp and 2 exons. |
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Key words: Echinococcus granulosus；thioredoxin peroxidase(TPx)；gene cloning；sequence analysis |
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