OjCHS原核表达载体的构建及重组蛋白的纯化  

Prokaryotic expression vector construction and recombinant proteins purification of OjCHS

作　　者：孙威[1] 林建 申欢 彭贵 鞠志刚 马玲 乙引[1,3] SUN Wei;LIN Jian;SHEN Huan;PENG Gui;JU Zhigang;MA Ling;YI Yin(Key Laboratory of Plant Physiology and Development Regulation,School of Life Science,Guizhou Normal University,Guiyang 550025;Key Laboratory of State Forestry Administration on Biodiversity Conservation in Karst Mountain Area of Southwest of China,Guiyang 550025;Medicine College,Guiyang University of Chinese Medicine,Guiyang 550025)

机构地区：[1]贵州师范大学生命科学学院植物生理发育调控重点实验,贵阳550025 [2]贵阳中医学院药学院,贵阳550025 [3]西南喀斯特山地生物多样性保护重点实验室,贵阳550025

出　　处：《安徽农业大学学报》2018年第6期1102-1106,共5页Journal of Anhui Agricultural University

基　　金：国家自然科学基金(31760076);贵州省教育厅青年科技人才成长项目(黔教合KY字[2017]122);贵州省联合基金项目(黔科合LH字[2016] 7211号;黔科合LH字[2017] 7358号);贵州省中医药管理局中医药;民族医药科学技术研究课题(S20160829000);贵州省重点实验室建设项目(黔科合计Z字[2011]4005)共同资助

摘　　要：查尔酮合酶(chalconesynthase,CHS)是类黄酮生物合成过程中的第一个关键酶,也是限速酶,它直接影响并限制类黄酮的合成与积累。在克隆得到日本蛇根草CHS基因(Oj CHS)基础上,将该基因插入原核表达载体pET-28a,完成重组表达质粒pET28a-CHS的构建。随后,利用冻融法将上述质粒转入大肠杆菌BL21感受态细胞中用于重组蛋白的表达。选择不同IPTG浓度、诱导温度及诱导时间对重组菌进行诱导,确定了可溶性重组蛋白的最佳诱导表达条件。最后,通过洗脱、纯化、透析与浓缩完成重组蛋白的纯化。结果显示,成功构建原核表达载体pET28a-CHS,可溶性重组蛋白最佳诱导条件为:IPTG浓度0.45 mmol·L-1,25℃下诱导12 h。最后大量制备并纯化获得符合预期大小的可溶性蛋白,为深入研究Oj CHS的生物学功能奠定了基础。Chalcone synthase(CHS) is the first and rate-limiting enzyme in the biosynthesis of flavonoids, and also plays important role during the biosynthesis and accumulation of flavonoids. In this study, we inserted OjCHS into pET-28 a vector to obtain recombinant pET28-CHS on the basis of the sequence of OjCHS. Then the recombinant plasmid was transfected into E.coli competent cell BL21 using a freeze-thaw method for the expression of recombinant proteins. Subsequently, the optimally induced expression condition was confirmed including IPTG concentration, inducing temperature and inducing time. Then a large number of soluble recombinant proteins were obtained through eluting with Ni column purification, further dialysis and concentration. The results showed that recombinant pET28-CHS vector was constructed and the optimally induced expression condition was 25 ℃ for 12 h by 0.45 mmol·L^-1 IPTG induction. Finally, we obtained abundant soluble recombinant protein which was consistent with the expected size, which would lay a foundation for the further study of the biological functions of OjCHS.

关 键 词：日本蛇根草 查尔酮合酶基因 原核表达载体 蛋白表达与纯化 

分 类 号：Q786[生物学—分子生物学] Q512