北柴胡糖基转移酶基因BcUGT8的克隆、序列分析及其原核表达载体构建  

Cloning, sequence analysis, and prokaryotic expression vector construction of glycosyltransferase gene BcUGT8 from Bupleurum chinense

作　　者：徐洁森[1,2] 魏建和[1,2] 陶韵文[1,3] 孙晶[1,4] 隋春[1,2] 

机构地区：[1]中国医学科学院北京协和医学院药用植物研究所,北京100193 [2]中草药物质基础与资源利用教育部重点实验室,北京100193 [3]佳木斯大学药学院,黑龙江佳木斯154007 [4]东北林业大学生命科学院,黑龙江哈尔滨150040

出　　处：《中草药》2013年第17期2453-2459,共7页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(81072994);北京市自然科学基金资助项目(5102033);中医药行业科研专项(201107011)

摘　　要：目的克隆北柴胡Bupleurum chinense中可能参与柴胡皂苷生物合成的糖基转移酶基因BcUGT8,构建其原核表达载体,为通过体外表达和纯化蛋白的催化活性,分析验证其功能奠定基础。方法在Roche(454)GS FLX系统高通量测序已获得部分cDNA序列基础上,通过cDNA末端快速扩增技术(RACE)和长距离PCR扩增方法(LD-PCR)克隆全长cDNA。设计含有酶切位点的PCR引物,PCR扩增其开放阅读框(ORF),酶切后,插入到pET-28a(+)载体中,构建出原核表达载体。结果扩增到北柴胡1个糖基转移酶(UGT)基因全长cDNA,构建了这一基因的原核表达载体。结论通过BcUGT8基因的全长cDNA克隆、序列分析和原核表达载体的构建,为后续开展体外酶蛋白表达、纯化和催化活性分析实验,验证其生物功能奠定了基础。Objective To clone the full-length cDNA of the uridine diphosphate glycosyltransferase （UGT） gene in Bupleurum chinense （BcUGT8）, which may be involved with the saikosaponin biosynthesis, and to construct the prokaryotic expression vector. The work will provide the foundation for its further function verification by in vitro expression and activity analysis of the purified protein. Methods RACE and LD-PCR were used to clone the full-length cDNA of BcUGT8, on the basis of its partial cDNA sequence obtained from our previous high-flux sequencing by Roche （454） GS FLX system. The open reading form （ORF） was PCR cloned using primers with corresponding restriction enzymes cutting sites. The PCR products were digested with corresponding restriction enzymes and then were inserted in expression vector pET-28a （＋） to construct the recombinant expression vectors. Results The full-length cDNA of UGT gene was cloned from B. chinense, and the prokaryotic expression vector was obtained. Conclusion The fulMength cDNA cloning, sequence analysis, and prokaryotic expression vector construction provide a substantial foundation for follow-up bio-ftmction analysis of BcUGT8 through protein expression, purification, and activity analysis in vitro.

关 键 词：北柴胡 糖基转移酶基因 序列分析 原核表达载体 PCR 

分 类 号：R282.12[医药卫生—中药学]