| 细粒棘球绦虫重组pET30a-EgA31疫苗的构建和诱导表达 |
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| 引用本文： | 贾海英,马海梅,丁剑冰,曹春宝,吾拉木·马木提,马秀敏,张海涛,朱明,吕国栋,温浩.细粒棘球绦虫重组pET30a-EgA31疫苗的构建和诱导表达[J].科技导报(北京),2009,27(8). |
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| 作者姓名： | 贾海英 马海梅 丁剑冰 曹春宝 吾拉木·马木提 马秀敏 张海涛 朱明 吕国栋 温浩 |
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| 作者单位： | 贾海英,马海梅,丁剑冰,曹春宝,吾拉木·马木提,马秀敏,张海涛,JIA Haiying,MA Haimei,DING Jianbing,CAO Chunbao,MAMUTI Wulamu,MA Xiumin,ZHANG Haitao(新疆医科大学第一附属医院包虫病重点实验室,乌鲁木齐,830011;新疆医科大学基础医学院免疫学教研室,乌鲁木齐,830054);朱明,ZHU Ming(新疆医科大学基础医学院免疫学教研室,乌鲁木齐,830054);吕国栋,温浩,LU Guodong,WEN Hao(新疆医科大学第一附属医院包虫病重点实验室,乌鲁木齐,830011)
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| 基金项目： | 国家自然科学基金，教育部春晖计划项目 |
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| 摘 要： | 克隆细粒棘球绦虫EgA31抗原基因,构建pET30a-EgA31原核表达载体,并在大肠埃希菌宿主系统中表达EgA31重组蛋白,对表达产物进行SDS-PAGE分析.从细粒棘球绦虫成虫组织中提取总RNA,反转录生成cDNA,以此cDNA为模板RT-PCR 克隆获得EgA31抗原基因,将其克隆至pUCm-T载体,测序确定其正确性.利用定向克隆技术将EgA31抗原基因片段克隆至原核表达质粒pET30a上,转化E coli DH5α,根据选择标记的卡那霉素抗性基因筛选到阳性克隆,通过PCR分析和酶切鉴定筛选出阳性克隆.IPTG初步诱导和表达pET30a-EgA31重组蛋白,SDS-PAGE电泳检测,并经凝胶图像分析确定目的蛋白的表达水平.测序表明选取的pET30a-EgA31阳性克隆均为正确连接EgA31抗原基因的重组质粒.经IPTG诱导后重组蛋白得到成功表达,在相对分子量约为31 kDa处有表达条带,表达量约占菌体总蛋白质的26%.成功克隆并构建了pET30a-EgA31原核表达质粒.初步诱导表达出EgA31重组蛋白,为进一步研究其免疫特性奠定了基础.
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| 关 键 词： | 细粒棘球绦虫 EgA31抗原基因 原核表达质粒 |
Construction of the Recombinant Plasimid pET30a-EgA31 of Echinococcus Granulosus and Its Induced Expression in E. coli |
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| JIA Haiying,MA Haimei,DING Jianbing,CAO Chunbao,MAMUTI Wulamu,MA Xiumin,ZHANG Haitao,ZHU Ming,LU Guodong,WEN Hao . Hydatid Disease Key Laboratory,The First Affiliated Hospital,Xinjiang Medical University,Urumqi ,China.Construction of the Recombinant Plasimid pET30a-EgA31 of Echinococcus Granulosus and Its Induced Expression in E. coli[J].Science & Technology Review,2009,27(8). |
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| Authors: | JIA Haiying MA Haimei DING Jianbing CAO Chunbao MAMUTI Wulamu MA Xiumin ZHANG Haitao ZHU Ming LU Guodong WEN Hao Hydatid Disease Key Laboratory The First Affiliated Hospital Xinjiang Medical University Urumqi China |
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| Affiliation: | JIA Haiying1,2,MA Haimei1,DING Jianbing1,CAO Chunbao1,MAMUTI Wulamu1,MA Xiumin1,ZHANG Haitao1,ZHU Ming2,LU Guodong1,WEN Hao1 1. Hydatid Disease Key Laboratory,The First Affiliated Hospital,Xinjiang Medical University,Urumqi 830011,China 2. Department of Immunology of School of Basic Medical Science,Urumqi 830054,China |
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| Abstract: | This paper reports our work in cloning EgA31 antigen gene and constructing prokaryotic expression plasmid pET30a - EgA31, then expressing the recombinant protein EgA31 in E. coli and making analyses by SDS-PAGE. Total RNA was extracted from adult worms of Echinococcus granulosus, the cDNA encoding EgA31 antigen was amplified by RT-PCR from the Eg adult cDNA.The acquired EgA31 cDNA was cloned into pUCm -T vector to construct the recombinant plasmid pUCm-T/EgA31. Prokaryotic expressed plasmid pET30a was chose... |
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| Keywords: | echinococcus granulosus EgA31 antigen gene prokaryotic express plasmid |
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