中慢生型天山根瘤菌中MrtR蛋白的融合表达研究及鉴定  

Recombinant Expression and Identification of MrtR Protein in Mesorhizobium tianshanense

作　　者：戴俊[1] 郑会明[1] 杨梦华[1] 钟增涛[1] 朱军[1] 

机构地区：[1]南京农业大学生命科学学院,农业部农业环境微生物工程重点开放实验室,南京210095

出　　处：《土壤》2008年第4期658-661,共4页Soils

基　　金：国家杰出青年基金项目(30325004);自然科学基金项目(30570011)资助

摘　　要：通过PCR扩增出中慢生型天山根瘤菌中群体感应调控蛋白MrtR的基因片段,克隆至表达载体pALEX中,从而构建了原核表达质粒pALEX-mrtR。将pALEX-mrtR转化至大肠杆菌BL2/DE3菌株感受态细胞中,经IPTG诱导,表达出58kD的GST-MrtR融合蛋白。用纯化好的GST-MrtR制备多克隆抗体,经Westernblot检测,该血清可与目的蛋白发生特异性反应,证明其具有良好的免疫原性。MrtR融合蛋白的成功表达及其多克隆抗体的制备为进一步研究MrtR蛋白的功能奠定了基础。The LuxR-LuxI-type quorum sensing systems have been shown to play a key role for rhizobia to form nodules on their plant hosts. The protein MrtR in Mesorhizobium tianshanense is a Lux-type regulator and regulates the LuxI-type synthesis, MtrI, in the quorum sensing system Of M.tianshanense. The mrtR coding sequence was fused to the T7 promoter of bacteriophage T7 by PCR amplification, using M.tianshanense genomic DNA as template. The resulting PCR product was cloned into pALEX, resulting in co-expressed GST-MrtR plasmid pALEX-mrtR. The resulting plasmid pALEX-mrtR was transformed into Escherichia coli strain BL21/DE3. High quantity of soluble GST-MrtR was obtained when the recombinant E.coli strain was incubated at 37℃ with IPTG inducer. The protein was purified by GST affinity chromatography and tested by SDS-PAGE. The polyclonal antibody against MrtR was obtained. The ELISA titer of antiserum against MrtR was about 1 ： 262000. Western blot analysis showed that the antiserum could bind to the expressed fusion protein specifically.

关 键 词：群体感应 中慢生型天山根瘤菌 MrtR 融合表达 多克隆抗体 

分 类 号：Q933[生物学—微生物学]