葡萄球菌肠毒素B突变体基因表达和功能的初步研究  

Study on expression and functions of staphylococcal enterotoxin B mutants

作　　者：俞红[1] 钱利生[1] 

机构地区：[1]上海医科大学基础医学院微生物学教研室,200032

出　　处：《中华微生物学和免疫学杂志》2001年第2期200-203,共4页Chinese Journal of Microbiology and Immunology

摘　　要：目的　应用PCR致变及基因克隆技术 ,获得抗原性保留 ,但超抗原毒性明显下降的葡萄球菌肠毒素B(SEB)突变体 ,用作新型淋巴细胞激活分子和超抗原疫苗的研究。方法　用PCR和PCR致变技术 ,从SEB标准株扩增SEB(SEB N)和SEB突变基因 (SEB M) ,分别与原核表达质粒pTrc99A重组 ,转化大肠杆菌JM10 9,经筛选获得重组质粒pTrcNb和pTrcMb ,用双脱氧链终止法作序列分析。表达的SEB N和SEB M蛋白 ,经双向免疫扩散试验作抗原性鉴定后 ,刺激小鼠脾细胞并由ELISA法检测培养上清中IL 2的水平。结果　SEB N 15 0位苏氨酸 (密码子ACT)非定向突变为丙氨酸(密码子GCT) ,SEB M 2 3位天冬酰胺 (密码子AAT)定向突变为丝氨酸 (密码子AGT)。两种突变基因表达的蛋白均与抗SEB形成明显沉淀线 ,与天然SEB刺激小鼠脾细胞产生IL 2水平相比 ,SEB N和SEB M突变体蛋白分别下降 12 .5倍和 40倍。结论　获得了能表达SEB N和SEB M突变体蛋白的 2株工程菌。表达的突变体蛋白具有良好的免疫反应性 ,但刺激小鼠脾细胞产生白细胞介素 2的超抗原生物学活性明显下降。Objective To obtain staphylococcal enterotoxin B (SEB) mutant with normal antigenicity but low toxicity. Methods Using PCR technique, normal SEB (SEB N) gene which was amplified from S. aureus S 6B . SEB mutant gene (SEB M) was prepared from the same strain, but one nucleotide in SEB gene was changed from asparagine (N23) to serine (S23). SEB N and SEB M were cloned into procaryotic expression vector pTrc99A then and transferred into E. coli JM109. SEB N and SEB M which were cloned into plasmid were sequenced directly by dideoxynucleotide method. The crude expressed proteins were identified by double agar immunodiffusion. The level of IL 2 in supernatants of mouse splenocytes stimulated by crude expressed proteins was determined by ELISA. Results SEB N and SEB M were obtained through PCR. The sequence of SEB N was changed with non site directed mutagenesis, threonine at the residue 150 of SEB N was replaced with alanine (ACT→GCT, T 150 A). As being expected, at the residue 23 of SEB M, serine substituted for asparagine (AAT→AGT, N 23 S) with site directed mutagenesis. Double agar immunodiffusion showed obvious precipitin line with anti SEB by both crude SEB N and SEB M mutant proteins could produce, but not by non recombinant strain. ELISA demonstrated that the level of IL 2 in supernatant of mouse splenocytes stimulated by natural SEB protein (containing equal amount of JM109P crude protein) was 40 times as much as that stimulated by SEB M and 12.5 times as much as that stimulated by SEB N. Conclusions We obtained two recombinant strains which produced T 150 A and N 23 S mutant SEB protein. The mutant proteins showed binding ability to anti SEB as the normal protein. However, their biological activity as superantigen decreased sharply. We consider that it is promising for further study of molecular adjuvant or superantigen vaccine.

关 键 词：葡萄球菌肠毒素B 突变体蛋白 超抗原 白细胞介素2 

分 类 号：R378[医药卫生—病原生物学]