| 猪日本脑炎病毒NS3蛋白的基因克隆、原核表达及纯化 |
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| 引用本文： | 韩秀杰,张保新,赵凡凡,余风艳,沈红霞,王晓杜.猪日本脑炎病毒NS3蛋白的基因克隆、原核表达及纯化[J].动物医学进展,2012(2):5-8. |
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| 作者姓名： | 韩秀杰 张保新 赵凡凡 余风艳 沈红霞 王晓杜 |
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| 作者单位： | 浙江农林大学动物科技学院 |
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| 基金项目： | 浙江省自然科学基金(Y3110124);浙江农林大学人才启动基金(2010FR080) |
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| 摘 要： | 提取猪日本脑炎病毒(JEV)上海分离株的基因组RNA,反转录合成cDNA,RT-PCR扩增JEV的NS3基因片段,亚克隆到原核表达载体pET-28(a)上,酶切鉴定和PCR鉴定重组原核表达质粒pET-28(a)-JEV-NS3,转化大肠埃希菌BL21(DE3)菌株,IPTG诱导表达重组His-JEV-NS3蛋白,SDS-PAGE分析重组蛋白的表达,his-band试剂盒纯化重组蛋白。获得了1.8kb NS3基因片段,成功构建重组原核表达质粒pET-28(a)-JEV-NS3。IPTG诱导获得分子质量为64ku的His-JEV-NS3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30%以上,纯化后获得高纯度重组蛋白,其占总蛋白比例达70%以上。
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| 关 键 词： | 日本脑炎病毒 NS3 原核表达 纯化 |
Cloning,Expression and Purification of Nonstructural Protein-3 of Japanese Encephalitis Virus from Pig |
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| HAN Xiu-jie,ZHANG Bao-xin,ZHAO Fan-fan,YU Feng-yan,SHEN Hong-xia,WANG Xiao-du.Cloning,Expression and Purification of Nonstructural Protein-3 of Japanese Encephalitis Virus from Pig[J].Progress In Veterinary Medicine,2012(2):5-8. |
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| Authors: | HAN Xiu-jie ZHANG Bao-xin ZHAO Fan-fan YU Feng-yan SHEN Hong-xia WANG Xiao-du |
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| Institution: | (College of Animal Science and Technology,Zhejiang Agricultural and Forestry University,Lin’an,Zhejiang,311300,China) |
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| Abstract: | NS3 is associated with viral RNA packaging and replication,viral pathogen.In this study,the cDNA of JEV were synthenized from viral genome by RT-PCR.The NS3 gene was cloned from cDNA by PCR and subcloned into pET-28(a) plasmid.The recombinant plasmid pET-28(a)-JEV NS3 was verified by PCR and enzyme digestion.pET-28(a)-JEV NS3 was transformed into E.coli.BL21(DE3),and the recombinant JEV NS3 protein was expressed by IPTG induction and purified by his-band kit.Expression and purification of His-JEV NS3 were analyzed by SDS-PAGE.In this paper,JEV NS3 product is 1.8 kb,the recombinant plasmid pET-28(a)-JEV-NS3 was constructed.His-JEV NS3 molecular weight is 64 ku.The protein was mainly expressed in the form of inclusion bodies and was more than 30% of the total cell protein.A large number of high-purity recombinant proteins were obtained by purification and the proportion reached more than 70% after purification.This study laid a foundation for JEV NS3 function and virus pathogen mechanism. |
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| Keywords: | Japanese encephalitis virus NS3 prokaryotic expression purification |
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