Position: Home > Articles > Stable Expression and Purification of Rabies Virus Nucleoprotein in Escherichia coli
Journal of Jilin Agricultural University
2006,28
(5)
113-117
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
作 者：
于志凤;张守峰;刘晔;张菲;扈荣良
单 位：
军事医学科学院军事兽医研究所
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
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。
译 名：
Stable Expression and Purification of Rabies Virus Nucleoprotein in Escherichia coli
作 者：
YU Zhi-feng~(1,2),ZHANG Shou-feng~1,LIU Ye~(1,2),ZHANG Fei~1, HU Rong-liang~21.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)
关键词：
Rabies virus N gene;E.coli;inducing temperature;strain density;IPTG concentration;inducing time
摘 要：
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.
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