| 肠道病毒71型外壳蛋白VP3在Pichia.pastoris酵母中的表达 |
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| 引用本文： | 周世力,李琳琳.肠道病毒71型外壳蛋白VP3在Pichia.pastoris酵母中的表达[J].江汉大学学报(自然科学版),2006,34(3):41-43,52. |
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| 作者姓名： | 周世力 李琳琳 |
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| 作者单位： | 1. 江汉大学,医学与生命科学院,武汉,430056
2. 病毒基因工程国家重点实验室,北京,100052 |
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| 摘 要： | 利用逆转录聚合酶链式反应(RT-PCR)的方法克隆肠道病毒71型SHZH98株外壳蛋白VP3基因,连接T载体,经序列测定后,构建酵母分泌型表达质粒pPIC9K/VP3,经序列测定证实-factor信号肽和VP3的序列和阅读框正确后,用SacI酶切使之线性化,电转化法将目的基因整合到宿主菌GS115基因组上;经转化子表型筛选和PCR分析鉴定后,甲醇诱导,筛选到5株高效表达VP3蛋白的工程菌株.ELISA实验表明:重组蛋白VP3具有较好的免疫原性.
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| 关 键 词： | 肠道病毒71型 VP3 巴斯德毕赤酵母 GS115菌株 |
| 文章编号： | 1673-0143（2006）03-0041-03 |
| 收稿时间： | 2005-11-21 |
| 修稿时间： | 2005年11月21 |
Expression Antigen VP3 of Enterovirus 71 in Pichia. Pastoris |
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| ZHOU Shi-li,LI Lin-lin.Expression Antigen VP3 of Enterovirus 71 in Pichia. Pastoris[J].Journal of Jianghan University:Natural Sciences,2006,34(3):41-43,52. |
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| Authors: | ZHOU Shi-li LI Lin-lin |
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| Abstract: | The genes encoding the capsid protein VP3 were amplified by RT-PCR.After con-firmed by DNA sequence analysis,the VP3 was inserted into the Pichia.Pastoris expression vector pPIC9K.The sequence of ORF of-factor and VP3 were confirmed by DNAsequencing with primer 5'-AOX1 and 3'-AOX1.Then the pPIC9K/VP3 weretransformed into yeast GS115 by the of electro-transformation.After the PCR analysis and the screening for Mut+ phenotype of the transformants,Then multiple inserted transformants were fermented in flasks.After induction by methanol(96 hours),recombinant protein could be effectively secreted into media.SDS-PAGE showed that the gene VP3 could expressas 31kD protein as same as thenatural protein,ELISA analysisof the culture supernatants confirmed that they had the desired immunogenicity of EV71. |
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| Keywords: | VP3 |
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