葡萄球菌肠毒素B突变体的构建  

Construction of Staphylococcal Enterotoxin B Mutants

作　　者：俞红[1] 钱利生[1] 李雪萍[1] 

机构地区：[1]上海医科大学基础医学院微生物学教研室,上海200032

出　　处：《上海医科大学学报》2000年第4期238-239,244,共3页Journal of Fudan University(Medical Science)

摘　　要：目的　应用重组和克隆技术在大肠杆菌中表达葡萄球菌肠毒素B(SEB)突变体。方法　利用PCR和PCR致变技术从产SEB的标准株扩增SEB基因 ,与原核表达载体 pTrc99A重组后引入大肠杆菌JM1 0 9。通过序列测定鉴定突变基因 ;用聚丙烯酰胺凝胶电泳 (SDS PAGE)和单向免疫扩散试验分别检测表达产物和表达量。结果　获得了 3株重组菌NJM1 0 9、M2 3JM1 0 9、M1 5 0JM 1 0 9。测序表明 ,SEB N序列与已报道的天然seb完全一致 ;SEB M 2 3第 2 3位天冬酰胺密码子AAT发生定向突变 ,即由丝氨酸密码子AGT替代 (N2 3S) ;SEB M1 5 0第1 5 0位氨基酸发生非定向突变 ,由苏氨酸密码子ACT突变为丙氨酸密码子GCT(T1 5 0A)。表达SEB M1 5 0和SEB M2 3的重组细菌裂解液经SDS PAGE ,在 2 80 0 0处可见有表达条带 ,非重组菌则无。单向免疫扩散试验和蛋白浓度测定表明 ,重组菌表达目的蛋白量为 5 μg/ml培养液 ,表达量占菌体总蛋白量的 3.5 %。 结论　获得了2株能表达SEB突变体的工程菌 。Purpose To clone and express staphylococcal enterotoxin B(SEB) mutants in E.coli . Methods The genes of natural SEB (SEB?N) and mutants SEB (SEB?M150 and SEB?M23) were amplified with PCR from SEB producing strain S 6B .The SEB?N,SEB?M150 and SEB?M23 genes were cloned into procaryotic expression vector pTrc99A.SEB?N and SEB?M cloned into plasmid were sequenced directly by dideoxynucleotide method.The expressed products were detected by SDS?PAGE and single agar immunodiffussion. Results Three recombinant strains NJM109,M23JM109,M150JM109 were obtained.The sequence of SEB?N was the same as that of previous report;threonine at the residue 150 of SEB?M150 was replaced with alanine (T150A);at the residue 23 of SEB?M23,serine substituted for asparagine(N23S) with site directed mutagenesis.SDS?PAGE and single agar immunodiffussion showed that the SEB?M150 and SEB?M23 were expressed in E.coli. JM109 and the expressed cytoplasmic SEB?M proteins was 5 μg/ml of broth,about 3.5 percent of total bacterial protein. Conclusions We obtained two recombinant strains which could produce T150A and N23S mutant SEB protein.These results have laid the foundation for further study of the bioactivity of SEB mutants.

关 键 词：葡萄球菌肠毒素B 突变体 克隆 细菌超抗原 

分 类 号：R392.11[医药卫生—免疫学]