超抗原SEA(D227A)的构建及其免疫活性鉴定  

Construction of superantigen SEA (D227A) and its immunological identification

作　　者：宋宏萍[1] 隋延仿[1] 叶菁[1] 李增山[1] 陈广生[1] 张秀敏[1] 

机构地区：[1]第四军医大学基础部病理学教研室,陕西西安710032

出　　处：《免疫学杂志》2004年第5期357-360,共4页Immunological Journal

基　　金：国家自然科学基金 (39770 82 7);全军医药卫生科研基金 (0 1Z0 84)重点资助项目

摘　　要：目的　构建SEA(D2 2 7A)基因的原核表达载体 ,并进行表达、分离纯化与免疫活性鉴定。方法　采用PCR技术从产SEA的葡萄球菌标准株中克隆SEA基因 ,通过下游引物上的点突变 ,使SEA基因第 6 80位碱基由A突变为C ,致使SEA成熟蛋白的第 2 2 7位氨基酸残基由天冬氨酸 (GAT)突变为丙氨酸 (GCT) ,构建pGEX SEA(D2 2 7A)重组表达质粒 ,在大肠杆菌DH5α中原核表达 ,再通过淋巴细胞增殖实验对其免疫活性进行测定。结果　构建的SEA(D2 2 7A)基因经测序证实与设计的序列一致 ;成功地构建pGEX SEA(D2 2 7A)重组表达质粒 ,转化感受态大肠杆菌DH5α ,通过IPTG诱导、GSTrapFF柱分离纯化及凝血酶切除GST后 ,得到Mr 为 2 70 0 0的目的蛋白。淋巴细胞增殖实验显示 ,10 0ng mL重组SEA(D2 2 7A)可明显刺激淋巴细胞增殖。结论　成功地构建了SEA(D2 2 7A)基因原核表达载体 ,并在大肠杆菌中得到高效表达 ,所得重组SEA(D2 2 7A)能够刺激淋巴细胞增殖 ,但作用与野生型SEA相比较温和。该结果为寻找有效、毒副作用低的超抗原提供了线索。Objective To construct the prokaryotic expression vector of SEA mutant gene SEA (D227A). The gene was expressed in E. coli, and the induced protein was purified and identified. Methods The SEA gene was cloned by PCR from Staphylococcus aureus strain FRI 100. D227A was introduced by changing the Asp encoded by GAT into Ala (GCT) in the primer. The expression plasmid pGEX- SEA (D227A) was constructed and transformed into DH5α. Immunological competence of the induced protein was identified through stimulating lymphocytes proliferation. Results The nucleotide sequence of SEA (D227A) was found to be identical to the designed sequence. The prokaryotic expression vector pGEX-SEA (D227A) was successfully constructed, and then transforming into host strain DH5α for expression. A M r 27 000 protein was obtained by IPTG inducing, GST system purifying, and thrombin digesting. 100 ng/mL SEA (D227A) could significantly stimulate lymphocytes proliferation. Conclusion The SEA (D227A) gene is constructed and expressed successfully, and the induced protein has a lower toxicity compared with the wild-type SEA. The study dose gave a clue to the research of low-toxic superantigen.

关 键 词：葡萄球菌肠毒素A 超抗原 原核表达 点突变 

分 类 号：R392.11[医药卫生—免疫学]