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

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。

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

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 this lab, and the recombinant plasmid pBES-pss was then 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 30μg/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.