| 猪戊型肝炎病毒ORF2-62的克隆、表达及间接ELISA方法的初步建立 |
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| 引用本文： | 王永霞,郝成武,马勋.猪戊型肝炎病毒ORF2-62的克隆、表达及间接ELISA方法的初步建立[J].中国兽医学报,2012,32(2):238-241. |
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| 作者姓名： | 王永霞 郝成武 马勋 |
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| 作者单位： | 石河子大学动物科技学院,新疆石河子,832003 |
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| 基金项目： | 国家自然科学基金资助项目(30760012);新疆生产建设兵团科技局博士基金资助项目(2007JC15);国际科技合作项目(2006DFA33740) |
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| 摘 要： | 为了获得具有反应原性的重组蛋白,以该蛋白建立初步的间接ELISA检测方法。本研究通过RT-PCR从猪粪便中克隆了戊型肝炎病毒（HEV）ORF2与急性期血清反应的抗原决定簇基因片段,将该片段插入pET-32a表达载体,命名为pET32a-ORF2-62,将重组表达质粒转化大肠杆菌BL21（DE3）,用1.0mmol/L IPTG进行诱导表达,通过Western-blotting检测其反应原性,并利用镍柱亲和层析纯化所获目的蛋白。结果,SDS-PAGE显示目的蛋白相对分子质量约为30 600,Western-blotting显示重组蛋白与HEV阳性血清反应,表明该蛋白具有良好的反应原性。经方阵滴定确定最佳包被浓度为7.7mg/L,血清最佳稀释比为1∶40。利用大肠杆菌表达的HEV重组蛋白建立了间接ELISA检测方法,此方法的建立为戊型肝炎早期诊断提供了一种快速简便的血清学诊断方法。
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| 关 键 词： | 戊型肝炎病毒 ORF2 原核表达 间接ELISA |
Clone,express and development of an indirect ELISA assay for detection of swine HEV ORF2-62 antibodies |
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| WANG Yong-xia,HAO Cheng-wu,MA Xun.Clone,express and development of an indirect ELISA assay for detection of swine HEV ORF2-62 antibodies[J].Chinese Journal of Veterinary Science,2012,32(2):238-241. |
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| Authors: | WANG Yong-xia HAO Cheng-wu MA Xun |
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| Institution: | *(College of Animal Science and Technology,Shihezi University,Shikezi,Xinjiang 832003,China) |
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| Abstract: | To obtain recombination protein which has reactionogenicity,then use it to construct the detection method about indirect ELISA.A fragment about forthcoming HEV ORF2 epitope with acute stage sero-reaction was cloned from swine feces by RT-PCR technology,after that it was inserted into pET-32a and named as pET32a-ORF2-62.This recombinant plasmid was transformed into DE3 and used 1.0 mmol/L IPTG final concentration to induce expression.SDS-PAGE showed its relative molecular mass was about 30 600.Western-blotting result displayed it had satisfactory reactionogenicity.The method of indirect ELISA was constructed by using the protein,The best peridium density of antigen was 7.7 mg/L by square matrix titration,the best dilution ratio of serum was 1∶40. |
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| Keywords: | HEV ORF2 prokaryotic expression indirect ELISA |
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