金黄色葡萄球菌肠毒素B的基因克隆与表达  

Cloning and expression of Staphylococcal enterotoxin Bgene

作　　者：宋云扬[1] 王惠芳[1] 李艳军[1] 高川[1] 张靖[1] 张金平[1] 吴方晖[1] 

机构地区：[1]总装备部防化研究院第四研究所,北京102205

出　　处：《免疫学杂志》2006年第3期337-340,共4页Immunological Journal

摘　　要：目的克隆葡萄球菌肠毒素B(SEB)基因,构建其原核表达载体,表达并纯化基因重组SEB,并对其进行活性研究。方法采用PCR技术从金黄色葡萄球菌染色体DNA上扩增得到SEB基因,克隆到原核表达载体pET22b(+)上,转化大肠杆菌BL21(DE3)感受态细胞,全自动测序仪测序。IPTG诱导表达,SDS-PAGE电泳分析,用HiTrapTMchelating HP柱纯化重组表达的SEB,通过ELISA实验检验重组SEB的抗原性。用脂多糖和放线菌素D致敏小鼠模型检测重组SEB的毒性。结果所克隆的SEB基因核苷酸序列与报道的序列完全相同。表达的SEB在SDS-PAGE图谱的位置与预期相符,得到了SDS-PAGE电泳纯的基因重组SEB。ELISA试验结果表明,纯化的重组SEB与抗野生型SEB单克隆抗体呈现明显的阳性免疫反应。小鼠致死性试验表明,重组SEB与野生型SEB具有相同的毒性。结论在大肠杆菌中成功克隆及表达了金黄色葡萄球菌肠毒素B,为进一步分析SEB分子中相关活性位点及研究SEB发病机制打下了基础。Objective To obtain recombinant Staphylococcal enterotoxin B （SEB） by gene engineering and evaluate its biological activity, Methods The full-length of SEB gene amplified from DNA of S6 strain of Staphylococcus aureus by high-fidelity PCR was cloned into prokaryotic expression vector pET22b^＋ , and then the vector was transformed into BL21 （DE3） to construct a prokaryotic expression system. The sequence of SEB gene was confirmed by DNA sequencing and the recombinant SEB （rSEB） was induced by 1 mmol/L IPTG. The expressed product was identified by SDS-PAGE and the expressed rSEB was purified by HiTrap^TM chelating HP. The antigenicity of rSEB was tested by ELISA and the toxicity of rSEB was tested by using a lipopolysaccharide （LPS） and actinomycin D sensitized mouse model. Results The nucleotide sequence of the cloned SEB gene was the same as that of reported in Genebank. The relative molecular weight shown on SDS-PAGE profile was consistent with expected value. The purified rSEB was obtained. Positive immune reaction was occurred between the rSEB and the monoclonal antibody of anti-wild type SEB. The lethality assay in mice demonstrated that toxicity of rSEB was the same as that of wild type SEB. Conclusion The gene of SEB is successfully cloned into plasmid pET22b and expressed in E. Coli, which would provide a basis for analyzing the to-xicity-related active sites in SEB molecule and further studies on the pathogenesis of SEB.

关 键 词：金黄色葡萄球菌肠毒素B 克隆 多聚酶链试反应 原核表达 

分 类 号：R378.11[医药卫生—病原生物学] Q78[医药卫生—基础医学]