| 狂犬病病毒核蛋白基因在大肠杆菌中稳定表达条件的优化及纯化 |
| |
| 引用本文： | 于志凤,张守峰,刘晔,张菲,扈荣良.狂犬病病毒核蛋白基因在大肠杆菌中稳定表达条件的优化及纯化[J].吉林农业大学学报,2006,28(5):581-585. |
| |
| 作者姓名： | 于志凤 张守峰 刘晔 张菲 扈荣良 |
| |
| 作者单位： | 1. 军事医学科学院军事兽医研究所,长春,130062;吉林大学畜牧兽医学院,长春,130062
2. 军事医学科学院军事兽医研究所,长春,130062 |
| |
| 基金项目： | 国家高技术研究发展计划(863计划) |
| |
| 摘 要： | 将狂犬病病毒SRV9株核蛋白基因按正确的读码框克隆至GST融合表达载体pGEX-4T-1中,转化至大肠杆菌Rosetta株,IPTG诱导表达。表达的融合蛋白经SDS-PAGE分析显示,相对分子量约为82 kD,与预期大小一致。Western-blot检测结果表明,融合蛋白能与多克隆阳性血清发生特异性反应。为获取大量ELISA包被用核蛋白,试验还借助SDS-PAGE方法对重组目的基因的表达条件进行了优化,比较了诱导温度、菌密度I、PTG浓度、诱导时间等参数对重组基因表达的影响,以确定最佳诱导表达条件。结果表明:27~32℃,OD5500.3~0.4,IPTG 0.06 mmol/L、诱导至OD550值不再增加时为最佳诱导表达条件。优化后经扫描分析显示,所表达融合蛋白占菌体总蛋白的20%以上。经包涵体纯化和亲和层析纯化,可获得纯度较高的GST融合蛋白。
|
| 关 键 词： | 狂犬病病毒核蛋白 大肠杆菌 诱导温度 菌密度 IPTG浓度 诱导时间 |
| 文章编号： | 1000-5684（2006）05-0581-05 |
| 收稿时间： | 2005-11-05 |
| 修稿时间： | 2005-11-052006-04-22 |
Stable Expression and Purification of Rabies Virus Nucleoprotein in Escherichia coli |
| |
| YU Zhi-feng,ZHANG Shou-feng,LIU Ye,ZHANG Fei,HU Rong-liang.Stable Expression and Purification of Rabies Virus Nucleoprotein in Escherichia coli[J].Journal of Jilin Agricultural University,2006,28(5):581-585. |
| |
| Authors: | YU Zhi-feng ZHANG Shou-feng LIU Ye ZHANG Fei HU Rong-liang |
| |
| Institution: | 1. Institute of Military Veterinary Science, Academy of Military Medical Sciences, Changchun 130062; China ; 2. College of Animal Husbandry and Veterinary Medicine, Jilin University, Changchun 130062, China |
| |
| Abstract: | By inserting the Rabies virus strain SRV9 N gene into the expressing vector pGEX-4T-1,the recombinant plasmid pGEX-RN was constructed.The pGEX-RN was then transformed into E.coli strain Rosetta and induced with IPTG.The expression was identified by SDS-PAGE and Western-blot analysis.A specific protein band of 82 kD was found as a fusion protein with glutathione transferase.In order to obtain massive N protein as ELISA coating antigen,expression optimization was studied and influence of inducing conditions such as temperature,density,IPTG concentration and time and so on was compared by SDS-PAGE method.The results of Densitometric scanning indicated that the expressed protein was more than 20% of total protein of Rosetta after the identification of the optimum expressing conditions. |
| |
| Keywords: | Rabies virus N gene E coli inducing temperature strain density IPTG concentration inducing time |
| 本文献已被 CNKI 维普 万方数据 等数据库收录！ |
|
