大肠杆菌中表达外源重组蛋白的研究  

The Expression Analysis of Recombinant Protein in Escherichia coli.

作　　者：吕素芳[1,2] 刘峥[2] 郭广君[1,2] 蔡永萍[1] 邱德文[1] 

机构地区：[1]安徽农业大学生命科学学院,合肥230036 [2]中国农业科学院植物保护研究所,北京100081

出　　处：《科学技术与工程》2006年第18期2872-2876,共5页Science Technology and Engineering

基　　金：国家重点基础发展计划(973计划);(2003CB114204)资助

摘　　要：通过聚合酶链式反应(PCR),扩增出交链孢菌激活蛋白辅助蛋白基因p42A,并将该基因按正确阅读框克隆到表达载体pGEX-KG中谷胱甘肽转移酶(GST)基因的下游。重组质粒转化大肠杆菌BL21,通过建立重组菌生长时间与OD600值的关系,及对菌液密度、诱导剂浓度、诱导时间和温度等条件的摸索,根据SDS-PAGE电泳检测结果判断融合蛋白的最佳表达条件。实验结果表明,最适诱导时期为重组菌生长对数中期;IPTG的最佳浓度为1.0mmol/L;37℃下诱导培养4h时产物表达量最高。超声波破碎菌体细胞,离心及电泳结果表明,表达产物为可溶性蛋白。Activator auxiliary protein gene 1M2A in genomic DNA of A hernaria sp. was amplified by polymerase chain reaction （PCR）. PCR product was cloned into pGEX-KG according to the right open reading frame （ORF）. The expressing of GSF-P42A fusion protein was studied in detail on many factors including thalli density, IPTG, time, temperature. The curve of OD600 and the growing time of the recombinant bacteria was also established, which is helpful to find the optimal inducing time. GST-P42A fusion protein was identified by SDS- PAGE. These results showed that the best inducing time is logarithmic growth phase and the best concentration of IPTG is 1.25 mmol/L, the expressing quantity of GST-P42A fusion protein has reached its expressing quantity of maximum by culturing for six hours in 28℃, that the GST-P42A fusion protein is soluble was confirmed by ultrasonic slacking thalli, high speed centrifugation and electrophoresis.

关 键 词：交链孢菌 p42A基因 融合蛋白 可溶性蛋白 

分 类 号：Q559[生物学—生物化学]