基于rhaSR嵌合操纵子的构建及其在大肠杆菌中表达的研究  

Chimeric Operons Based on rhaSR and Their Expression in Escherichia coli

作　　者：关梅[1] 贾坤志[1] 李芳[1] 贺根和[1] 喻富根[1] 华子春[1] 

机构地区：[1]南京大学生命科学学院医药生物技术国家重点实验室,南京210093

出　　处：《微生物学通报》2007年第6期1129-1133,共5页Microbiology China

基　　金：国家自然科学基金资助(No.30370760)

摘　　要：通过PCR等重组DNA技术,构建了含rhaSR启动子表达调控元件、RhaR基因、报告基因gst(谷胱甘肽-S-转移酶)的两个嵌合操纵子,并插入大肠杆菌表达载体pALEX中构成pALEX-PR1和pALEX-PR2。其中pALEX-PR2的RhaR基因上游为原有的SD序列,而pALEX-PR1的RhaR基因上游则插入了增强的SD序列。把这两个重组表达质粒分别转入大肠杆菌BL21(DE3)中,报告基因gst能够在L-鼠李糖诱导下表达,其表达量是非诱导条件下的4～5倍,且pALEX-PR1的表达量是pALEX-PR2的3.14倍。以上结果表明,gst的表达既受L-鼠李糖诱导,同时又受RhaR的正调控。SDS-PAGE结果显示,GST占大肠杆菌培养物总可溶蛋白的5.41%(W/W),平均1L培养物可获得3.0mg纯化的GST。酶活性分析表明,所构建的嵌合操纵子表达的GST保持了正确的构型且具有很高的活性。Using gene recombinant techniques, two chimeric operons containing rhaSR promoter, RhaR gene, and reporter gene gst （glutathione Stransferase ）were constructed, and each was inserted into the E. coli expression vector pALEX to form pALEX-PR1 and pALEX-PR2. The pALEX- PR2 contained a native SD sequence in the upstream region of rhaR, while the pALEX-PR1 contained an enhanced SD sequence. Two plasmids were then transformed into E. coli BL21 （DE3）. The reporter gene gst within both chimeric operons expressed 4 to 5 folds higher with L-rhamnose induction than without the induction. Under the induction of L-rhanmose, the GST expression of the pALEX-PR1 was 3.14 folds than the pALEX- PR2. Our results suggested that the expression of gst was positively regulated by the induction of L-rhamnose and the RhaR expressed from the same chimeric operon. Furthermore, SDS-PAGE results showed that GST accounted for about 5.41% （W/W） of the total soluble proteins of the E. coli culture. An average of 3.0 mg purified GST was obtained from 1 L culture. The results of enzyme activity analysis showed that the GST expressed by reporter gene gst of our chimeric operons kept the correct configuration and high activity.

关 键 词：rhaSR 嵌合操纵子 SD序列 基因表达 

分 类 号：Q78[生物学—分子生物学]