F15L和K207E突变与Borrelia螺旋菌磷脂酰胆碱合成酶的生物活性

F15L和K207E突变与Borrelia螺旋菌磷脂酰胆碱合成酶的生物活性 Replacement of F15 and K207 by Lysine and Glutamate Does Not Impair Activity of Borrelia Phosphatidylcholine Synthase 王行国 WANG Xing-guo 1 2 first-author 宣文静 XUAN Wen-jing 1 2 常玲 CHANG Ling 1 2 黄俊 HUANG Jun 1 2 湖北大学生命科学学院 湖北大学生命科学学院 School of Life Science,Hubei University School of Life Science,Hubei University 湖北大学生命科学学院,湖北,武汉,430062 湖北大学生命科学学院,湖北,武汉,430062 磷脂酰胆碱合成酶是磷脂酰胆碱合成酶（PCS）途径中的关键酶.根据Borrelia burgdorferiBB0249ORF的DNA序列设计引物对,从B.afzelii总DNA中扩增出一段660 bp DNA.序列分析发现扩增的DNA片段编码的蛋白中仅两个氨基酸（L15和E207）不同于B.burgdorferiBB0249 ORF编码的蛋白（F15和K207）.将克隆的基因插入pET表达载体,并在大肠杆菌中表达.在活体中进行的活性分析发现,克隆的DNA片段编码的蛋白质能利用外源的胆碱和大肠杆菌细胞内的CDP-二酰基甘油合成磷脂酰胆碱（PC）,表明螺旋菌磷脂酰胆碱合成酶15位的苯丙氨酸和207位赖氨酸残基分别被亮氨酸和谷氨酸替代并不影响磷脂酰胆碱合成酶的活性. Phosphatidylcholine synthase（PCS） catalyzing the condensation of choline and CDP-diacylglyceride to form Phosphatidylcholine is the key enzyme in the PCS pathway.The 660 bp DNA fragment was amplified from B.afzelii total DNA by PCR.Amino acid alignment reveal that 99.7% amino acids encoded by the cloned DNA fragment are identical to those of B.burgdorferi PCS.Difference occurs at positions 15 and 207（L15 and E207 for B.afzelii） and（F15 and K207 for B.burgdorferi）.The cloned DNA fragment was inserted into pET21b expression vector and transformed into E.coli BL21 CodonPlus(DE 3 )-RIL.TLC showed that the transformed E.coli cells synthesized phosphatidylcholine when the cells grew in LB medium containing 0.4% choline and 0.5 mmol/L IPTG,but the control group did not under the same condition.Our results demonstrated that Borrelia phosphatidylcholine synthase,in which phenylalanine at position 15 and lysine at position 207 were replaced by lysine and glutamate in nature respectively,remained full enzyme activity. 螺旋菌 磷脂酰胆碱合成酶 磷脂酰胆碱 phosphatidylcholine phosphatidylcholine synthase amino acid substitution Q936 国家自然科学基金资助项目（30570009）;湖北省教育厅重大项目（2004Z001）;湖北大学重点基金资助项目（080-095152） 2005-01-01 2021-04-01 S2