摘要
目的构建重组pET-30a-SEB原核表达载体,转化感受态大肠杆菌BL-21(DE3),诱导表达超抗原金黄色葡萄球菌肠毒素B(staphylococcalenterotoxinB,SEB)。方法利用PCR技术从产SEB的金黄色葡萄球菌标准菌株CMCC-26075基因组DNA中克隆SEB全长序列,将其克隆到pGEM-TEasy载体中并进行测序。构建pET-30a-SEB原核表达质粒,转化感受态大肠杆菌BL21(DE3),异丙基硫代β-D半乳糖苷(isopropyl-beta-D-thiogalactopyranoside,IPTG)诱导表达,经镍离子螯合亲和层析纯化后免疫鉴定。结果PCR获得超抗原SEB基因片段,与克隆载体连接后经测序与文献报道的基本一致;成功构建了pET-30a-SEB原核表达质粒且成功诱导表达出相对分子质量(Mr)约31×103的蛋白。结论成功克隆了seb基因序列,并进行了原核表达和鉴定,获得了SEB蛋白,为后续对超抗原SEB的进一步研究奠定了基础。
Objective To clone the seb gene and construct the SEB. After being transformed into competent E. coli BL-21（DE3）, the protein SEB was induced by IPTG and identified by ELISA and Western blot. Methods PCR was used to done seb gene from standard staphylococcal aureus CMCC-26075. The gene was cloned into clone vector pGEM-T Easy and sequenced. By identification of correct sequence, we constructed the prokaryotic expression plasmid pET-30a-SEB. E. coli BL-21 （DE3） transformed with plasmid pET-30a-SEB was induced by IPTG and identified by Western blot and ELISA . Results We cloned the seb gene by PCR and it＇s sequence was the same as the sequence reported in literature. E. coli BL-21 （DE3） containing plasmid pE330a-SEB can express a Mr31×10^3 protein induced by IPIG. Conclusion We successfully cloned and expressed the gene seb in E. coli, and obtained protein.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2006年第5期418-421,共4页
Chinese Journal of Microbiology and Immunology
