| Ⅰ群禽腺病毒33K基因的原核表达与鉴定 |
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| 引用本文： | 罗思思,谢芝勋,刘加波,谢丽基,庞耀珊,邓显文,谢志勤,范晴,彭宜.Ⅰ群禽腺病毒33K基因的原核表达与鉴定[J].广西农业生物科学,2012,31(1):7-12. |
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| 作者姓名： | 罗思思 谢芝勋 刘加波 谢丽基 庞耀珊 邓显文 谢志勤 范晴 彭宜 |
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| 作者单位： | 广西兽医研究所,广西畜禽疫苗新技术重点实验室,南宁530001 |
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| 基金项目： | 广西特聘专家专项经费（2011B020）; 新世纪百千万人才工程国家级人选专项经费（945200603）; 广西科技攻关（0815009-3-6）; 广西自然科学基金（2010GXNSFA013090）共同资助 |
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| 摘 要： | 33K蛋白是Ⅰ群禽腺病毒（fowl adenovirus groupⅠ,FAVⅠ）的非结构蛋白,在感染的过程中起重要作用。为了构建原核表达载体pGEX-33K作为诊断抗原,本研究根据GenBank中FAVⅠ33K基因序列,设计1对特异性引物。用PCR方法扩增目的片段,将其克隆至原核表达载体pGEX-4T-1,构建了重组质粒pGEX-33K。将其转化大肠杆菌感受态细胞DH5α,经IPTG诱导和SDS-PAGE分析,pGEX-33K表达的分子量为36.7KD,表达产物主要以可溶性的形式存在于菌体中;最佳IPTG诱导浓度为0.75mmol/L,最佳诱导时间为4h;经Western Blot鉴定,pGEX-33K重组蛋白可与FAVⅠ阳性血清发生特异性反应,为FAVⅠ抗体ELISA鉴别诊断试剂盒的研究奠定基础。
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| 关 键 词： | Ⅰ群禽腺病毒 33K蛋白 原核表达 |
The Prokaryotic Expression and Identification of 33K Gene of Fowl Adeno-virus Group Ⅰ |
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| Luo Sisi Xie Zhixun Liu Jiabo Xie Liji Pang Yaoshan Deng Xianwen Xie Zhiqin Fan Qing Peng Yi.The Prokaryotic Expression and Identification of 33K Gene of Fowl Adeno-virus Group Ⅰ[J].Journal of Guangxi Agricultural and Biological Science,2012,31(1):7-12. |
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| Authors: | Luo Sisi Xie Zhixun Liu Jiabo Xie Liji Pang Yaoshan Deng Xianwen Xie Zhiqin Fan Qing Peng Yi |
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| Institution: | Luo Sisi Xie Zhixun Liu Jiabo Xie Liji Pang Yaoshan Deng Xianwen Xie Zhiqin Fan Qing Peng Yi Guangxi Veterinary Research Institute,Guangxi Key Laboratory of Animal Vaccines and New Technology,Nanning,530001 |
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| Abstract: | The non-structural protein 33K of fowl adenovirus group Ⅰ is important for virus infection.The purpose of this study is to construct prokaryotic expression vector pGEX-33K for the diagnosis antigen.According to the sequences of FAVⅠ 33K gene available in GenBank,a pair of primers was designed.The target sequence was amplified by PCR,and was ligated to prokaryotic vector pGEX-4T-1 to construct recombinant expression plasmid pGEX-33K.The recombinant plasmid was transformed into Escherichia coli DH5α,and then induced and expressed by IPTG induction.The pGEX-33K recombinant protein was analyzed by SDS-PAGE and Western Blot.The result showed that pGEX-33K was mainly expressed in the form of soluble proteins and the molecular weight of the fusion protein was about 36.7 KD.The optimal concentration of IPTG and the time was 0.75 mmol/L and 4 h,respectively.Furthermore,the result of the Western Blot demonstrated that the pGEX-33K could be specifically recognized by positive serum to FAVⅠ,which might be used as the coating antigen to develop a differential diagnosis kit against fowl adenovirus group Ⅰ. |
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| Keywords: | Fowl adenovirus groupⅠ 33K protein Prokaryotic expression |
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