| 铜绿假单胞菌外毒素A全基因的克隆与分泌性表达 |
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| 引用本文： | 赵志强,方悦群,杜琳,谢贵林.铜绿假单胞菌外毒素A全基因的克隆与分泌性表达[J].微生物学免疫学进展,2006,34(1):22-26. |
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| 作者姓名： | 赵志强 方悦群 杜琳 谢贵林 |
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| 作者单位： | 兰州生物制品研究所,兰州,730046 |
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| 摘 要： | 利用PCR技术从铜绿假单胞菌PA103株DNA中扩增到铜绿假单胞菌外毒素A(EPA)全基因,选择合适位点插入pBV221 PLPR启动子下游,构建分泌性表达载体;转化宿主E.coliDH5α、JM109后,热诱导表达;SDS-PAGE分析表明表达产物占菌体总蛋白量的17%左右,分子量69kD左右;分离细胞组分蛋白发现仅有少量重组蛋白以成熟毒素形式分泌到宿主菌的周质间隙,并能检测到Vero细胞毒活性,其余大部分以包涵体形式存在。免疫印迹检测显示,表达产物与兔抗EPA多抗有特异性反应。此项工作为重组EPA的研制建立了有用的技术方法。
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| 关 键 词： | 铜绿假单胞菌外毒素A 分泌性表达 Vero细胞毒性 |
| 文章编号： | 1005-5673（2006）01-0022-05 |
| 收稿时间： | 2005-10-10 |
| 修稿时间： | 2005-12-05 |
Cloning and secreting expression of PA103 EPA in E. coli |
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| ZHAO Zhi-qiang, FANG Yue-qun, DU Lin,et al..Cloning and secreting expression of PA103 EPA in E. coli[J].Progress In Microbiology and Immunology,2006,34(1):22-26. |
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| Authors: | ZHAO Zhi-qiang FANG Yue-qun DU Lin |
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| Institution: | Lanzhou rtstitute of Biological Products, Lanzhou ,730046, China |
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| Abstract: | The full length(include signal peptide sequence) EPA gene of PA103 strain was amplified with PCR.The gene were purified and cloned into expression vector pBV221.The expression was induced in E.coli DH5α and JM109 at 42℃,under optimized condition,the target proteins were approximatly 17% of total proteins of host cell.Partial recombinant proteins were secreted into periplasma of bacteria and converted into mature toxin with Vero cytotoxicity.SDS-PAGE indicated that the molecular weight of target protein was about 69kD.The recombinant protein could also react specifically with anti-EPA polyclonal sera.It laid a foundation for development of rEPA gene engineer system. |
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| Keywords: | Pseudomonas aeruginosa exotoxin A Screction expression Vero cytotoxin |
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