| 猪瘟病毒Npro基因的克隆表达 |
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| 引用本文： | 高斌,冯励,李永强,付晓萍.猪瘟病毒Npro基因的克隆表达[J].云南农业大学学报,2012,27(4):530-535. |
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| 作者姓名： | 高斌 冯励 李永强 付晓萍 |
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| 作者单位： | 云南农业大学 食品科学技术学院,云南 昆明 650201 |
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| 摘 要： | 为了得到一个表达Npro蛋白的真核表达载体,从而为下一步研究不同时间段细胞主要组织相容性复合体（MHC）表达的变化情况,从分子水平上说明Npro与MHC之间的关系,本研究应用反转录聚合酶链式反应（RT PCR）技术对猪瘟病毒石门毒株Npro基因进行了克隆和测序,结果得到了长度为592bp的目的片段。用BamHⅠ和XhoⅠ限制性内切酶分别对重组克隆质粒和转移载体pcDNA30进行双酶切,回收目的基因DNA片段,将目的基因Npro亚克隆至真核转移载体pcDNA30中,经双酶切和序列测定鉴定插入基因和连接方向正确,得到含完整Npro基因的重组载体质粒P Npro。构建成功的真核表达载体以脂质体介导的转染方法将此重组载体质粒转染PK 15细胞,转染24和48h后检测表达情况。经Western blot方法检测表明,此Npro蛋白在PK 15细胞中正确表达。
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| 关 键 词： | 猪瘟病毒 Npro基因 真核表达 |
Molecular Cloning and Expression of Npro Gene of the Classical Swine Fever Virus |
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| GAO Bin,FENG Li,LI Yong qiang,FU Xiao ping.Molecular Cloning and Expression of Npro Gene of the Classical Swine Fever Virus[J].Journal of Yunnan Agricultural University,2012,27(4):530-535. |
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| Authors: | GAO Bin FENG Li LI Yong qiang FU Xiao ping |
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| Institution: | College of Food Science and Technology, Yunnan Agricultural University,Kunming 650201, China |
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| Abstract: | In order to select an eukaryotic expression vector for Npro protein, and to study the relationship between Npro and major histocompatibility complex (MHC) at different time point, a full length Npro gene of classical swine fever virus (CSFV) of Shimen was amplified and cloned by RT PCR. The results showed that the Npro gene contained 592 bp. The recombinant plasmid and expression vector pcDNA30 were digested by the restriction enzymes Bam HⅠand Xho Ⅰ, after the amplified fragments was subcloned into the expression vector pcDNA30, the recombinant plasmid P Npro was obtained. The insert position,size and reading frame were determined by PCR, restriction digestion and the sequence analysis. The recombinant P Npro plasmid expressed Npro was used to transfect PK 15 cells in vitro by liposome infection protocol. The results showed that after 24h and 48h posttransfection, Npro protein was detected by Western blot analysis with anti His monoclonal antibody, and it was found that the Npro protein was located. |
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| Keywords: | Classical swine fever virus Npro gene eukaryotic expression |
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