| 猪日本乙型脑炎病毒NS5原核表达和多克隆抗体制备 |
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| 引用本文： | 余风艳,韩秀杰,沈红霞,张保新,赵凡凡,王晓杜.猪日本乙型脑炎病毒NS5原核表达和多克隆抗体制备[J].福建农林大学学报(自然科学版),2013,42(3). |
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| 作者姓名： | 余风艳 韩秀杰 沈红霞 张保新 赵凡凡 王晓杜 |
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| 作者单位： | 浙江农林大学动物科技学院,浙江临安,311300 |
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| 基金项目： | 浙江省自然科学基金资助项目，浙江农林大学人才启动基金项目 |
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| 摘 要： | 提取猪日本乙型脑炎病毒(JEV)上海分离株的基因组RNA,反转录合成cDNA,扩增JEV-NS5基因片段,亚克隆到原核表达载体pET-28(a)上,构建重组原核表达质粒pET-28(a)-JEV-NS5,转化大肠埃希氏菌BL21(DE3)菌株,IPTG诱导表达重组JEV-NS5蛋白,获得分子质量为103 ku的重组蛋白.该蛋白主要以包涵体形式表达,表达量占总菌体蛋白的35%以上,His-band Ni+纯化后,获得高纯度重组蛋白占总蛋白的比例达75%以上.以纯化的蛋白免疫小鼠,制备小鼠抗JEV-NS5抗体,抗体效价达到3×104,并能与病毒感染的样本反应,证明该蛋白具有较好的特异性.
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| 关 键 词： | 日本乙型脑炎病毒 非结构蛋白NS5 原核表达 纯化 |
Prokaryotic expression and polyclone antibody preparation of nonstructural protein 5 of Japanese encephalitis virus from pig |
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| YU Feng-yan
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HAN Xiu-jie
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SHEN Hong-xia
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ZHANG Bao-xin
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ZHAO Fan-fan
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WANG Xiao-du.Prokaryotic expression and polyclone antibody preparation of nonstructural protein 5 of Japanese encephalitis virus from pig[J].Journal of Fujian Agricultural and Forestry University,2013,42(3). |
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| Authors: | YU Feng-yan HAN Xiu-jie SHEN Hong-xia ZHANG Bao-xin ZHAO Fan-fan WANG Xiao-du |
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| Abstract: | The cDNA of Japanese encephalitis virus(JEV) was synthensized from viral genome by RT-PCR.The JEV-NS5 gene was cloned from cDNA by PCR.The JEV-NS5 gene was subcloned into pET-28(a) plasmid.The recombinant plasmid pET-28(a)-JEV-NS5 was transformed into E.coli.BL21(DE3),The recombinant JEV-NS5 protein was expressed by IPTG induction.SDS-PAGE results showed that its molecular weight is 103 ku.The protein was mainly expressed in the form of inclusion bodies.The recombinant JEV-NS5 was more than 35% of the total cell protein by TLC scanning analysis.A large number of high-purity recombinant proteins were obtained by his-band Ni +purification.The proportion of recombinant JEV-NS5 reached more than 75% after purification.The recombinant JEV-NS5 proteins were immunized into mouse,and mouse anti-JEV NS5 antibody was prepared.The antibody titer is 3×104.The specific of antibody was detected by western-blotting.This study was a tool and foundation of JEV NS5 function and virus replication mechanism. |
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| Keywords: | Japanese encephalitis virus nonstructural protein 5 prokaryotic expression purification |
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