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82kD，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、IPTG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℃，OD550 0．3～0．4，IPIG 0．06mmol／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。
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.
出处
《吉林农业大学学报》
CAS
CSCD
北大核心
2006年第5期581-585,共5页
Journal of Jilin Agricultural University
基金
国家"863"高技术研究发展计划基金资助项目(2001AA020304-1)
