动物双歧杆菌肌球交叉反应抗原MCRA酶学功能的研究  

Study of the Enzymatic Function of Myosin Cross Reactive Antigen from Bifidobacterium animalis

作　　者：杨波[1] 陈海琴[1] 宋元达[1] 张灏[1] 陈卫[1] 

机构地区：[1]江南大学食品学院食品科学与技术国家重点实验室,无锡214122

出　　处：《中国生物工程杂志》2012年第12期30-36,共7页China Biotechnology

基　　金：国家自然科学基金(21276108);"十一五"国家"863"计划(2007AA100402);"十二五"国家"863"计划(2011AA100905)资助项目

摘　　要：目的:将肌球蛋白交叉反应抗原基因(Myosin cross reactive antigen,MCRA)在毕赤酵母中重组表达,对其酶学功能进行研究。方法:利用动物双歧杆菌BB-12(Bifidobacterium animalissubsp.lactis BB-12)的肌球交叉反应抗原(MCRA)基因序列特征设计引物进行PCR扩增后克隆至毕赤酵母表达载体pPinkα-HC,电转化获得PichiaPinkTM重组菌。通过SDS-PAGE和Westernblot检测重组蛋白的表达情况,GC-MS分析重组菌的脂质组成情况,最终确定重组蛋白的活性及其催化特性。结果:SDS-PAGE及Western blot结果表明动物双歧杆菌BB-12的MCRA蛋白在重组毕赤酵母菌PichiaPinkTMpPinkα-HC-MCRA中成功表达且定位至细胞膜,大小为82 kDa。GC-MS结果显示,添加底物亚油酸培养基后,重组菌将亚油酸转变为羟基化衍生物,产物为10-羟基-顺-12-十八碳烯酸(10-HOE)。结论:这是首次对该类蛋白成功定位后对其酶学功能进行研究,动物双歧杆菌BB-12的MCRA基因首次在毕赤酵母中实现了活性表达,产物为10-羟基-顺-12-十八碳烯酸。Objective： To express the myosin cross reactive antigen （MCRA） from Bifidobacterium animalis subsp, lactis BB-12 and to determine its enzymatic function. Methods： MCRA gene was amplified by PCR from chromosome of B. animalis subsp, lactis BB-12, and then the gene was cloned into PichiaPinkTM expression vector pPinket-HC, and transformed into PichiaPinkTM wild strain. The recombinant MCRA protein was confirmed through SDS-PAGE and Western blot. The lipid profile of the recombinant PichiaPinkTM was analyzed via GC- MS. Results： It was indicated from the results of SDS-PAGE and Western blot that the MCRA protein was expressed successfully and targeted into the cell membrane in the recombinant PichiPinkTM, with a molecular weight of 82 kDa. With adding LA in the media, the recombinant PichiaPinkTM converted LA into 10-hydroxycisl2-octadecenoic acid （10-HOE）. Conclusion： This is the first report of expressing the MCRA gene with successful target and function. The MCRA of B. animalis subsp, lactis BB-12 was successfully and actively expressed in Pichia pastoris, and the product is 10-HOE.

关 键 词：肌球蛋白交叉反应抗原 亚油酸 毕赤酵母表达系统 重组表达 

分 类 号：Q786[生物学—分子生物学]