磷脂酰丝氨酸合成酶基因的克隆及在枯草芽孢杆菌中的表达  

Cloning of the Phosphatidylserine Synthase Gene and Its Secretive Expression in Bacillus subtilis

作　　者：张业尼[1] 路福平[1] 李玉[1] 王建玲[1] 李静文[1] 

机构地区：[1]天津市工业微生物重点实验室天津科技大学生物工程学院,天津300457

出　　处：《中国生物工程杂志》2008年第9期56-60,共5页China Biotechnology

基　　金：国家科技基础平台项目"工业发酵微生物资源的标准化整理整合"资助项目(2005DKA21204-10)

摘　　要：应用PCR技术从Escherichia coli K12 Sgal-(ExPASy P23830)中扩增到大小为1350bp编码磷脂酰丝氨酸合成酶的DNA片段,将其插入枯草芽孢杆菌诱导型表达载体pBES,获得重组质粒pBES-pss后转化Bacillus subtilis DB104。经蔗糖诱导后,该磷脂酰丝氨酸合成酶在Bacillus subtilis DB104(pBES-pss)中获得胞外分泌表达,SDS-PAGE分析发现目的蛋白分子量约为52kDa,酶联比色法检测酶活力为1.50U/ml,提高了磷脂酰丝氨酸合成酶的表达产量,为工业化发酵生产磷脂酰丝氨酸合成酶奠定了良好的基础。The gene encoding the phosphatidylserine synthase in Escherichia coli K12 Sgal- （ExPASy P23830） was amplified by PCR. After DNA sequence analysis, it was inserted into the inducible expressive shuttle vector pBES of Bacillus subtilis, which was constructed in the lab, and the recombinant plasmid pBES-pss was transformed into competent cells of the Bacillus subtilis strain DB104. The positive transformant DB104 （pBES-pss） was grown on Bacillus subtilis common fermentation medium, which contained 30ug/ml kanamycin. After 2 hours cultivation, sucrose was added and increased to the final concentration of 2% for induction and this phosphatidylserine synthase was secreted into the medium. The result of SDS-PAGE showed that the molecular weight of the protein was 52kDa and the result of enzyme coupling colorimetric method showed that the enzyme activity was 1.50U/ml. The recombinant Bacillus subtilis has increased the yield of phosphatidylserine synthase which will be used for industrial biosynthesis of phosphatidylserine.

关 键 词：磷脂酰丝氨酸合成酶 枯草芽孢杆菌 异源表达 酶联比色法 

分 类 号：Q786[生物学—分子生物学]