金黄色葡萄球菌肠毒素B编码基因的克隆及原核表达

发布时间：2018-01-03 13:03

本文关键词：金黄色葡萄球菌肠毒素B编码基因的克隆及原核表达　出处：《青岛大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的构建金黄色葡萄球菌肠毒素B(Staphylococcal Enterotoxin B,SEB)编码基因的原核表达克隆,并在大肠杆菌中表达,同时对表达蛋白的生物学活性进行研究。方法提取产肠毒素B的金黄色葡萄球菌临床分离菌株的DNA,DNAstar软件辅助设计引物,PCR扩增出肠毒素B(SEB)成熟肽前体蛋白基因,将其插入到质粒pGEX-4T-2编码谷胱肽转移酶(GST)读码框架下游的多克隆位点(MCS),构建原核表达克隆PGEX-4T-2/SEB。重组质粒经双酶切、测序鉴定后,以TSS法转入大肠杆菌DH5α,用终浓度为1mmol/L的IPTG诱导融合蛋白表达,SDS-PAGE电泳分析表达蛋白图谱,Western blot鉴定表达蛋白。结果经酶切和测序分析表明,克隆获得了金黄色葡萄球菌肠毒素B(SEB)的编码基因,且插入片段读码框架正确,无碱基错配,pGEX-4T-2/SEB原核表达克隆构建成功。经IPTG诱导,重组质粒转化的大肠杆菌表达出相对分子质量(Mr)为58×10~((3)) Daltons的融合蛋白。Western blotting证实该蛋白能与抗SEB抗体发生特异性结合反应,具有抗原活性。结论金黄色葡萄球菌肠毒素B(SEB)编码基因原核表达克隆的构建成功,并能在大肠杆菌中高效表达,为进一步研究肠毒素B(SEB)的生物学功能和临床应用奠定了基础。
[Abstract]:Objective to construct the prokaryotic expression clone of Staphylococcus aureus enterotoxin B (Staphylococcal Enterotoxin B (SEB) encoding gene, and express it in E.coli, and study the biological activity of the expressed protein at the same time.
Method for extraction of Staphylococcus aureus enterotoxin B clinical isolates DNA, DNAstar aided design software primer PCR amplified enterotoxin B (SEB) mature peptide precursor protein gene inserted into the plasmid pGEX-4T-2 encoding glutathione transferase (GST) multiple cloning sites downstream of the reading frame (MCS). Construction of prokaryotic expression clone of PGEX-4T-2/SEB. recombinant plasmid by restriction enzyme digestion and sequencing, the TSS method into Escherichia coli DH5 alpha fusion protein expression of 1mmol/L IPTG with the final concentration of induction, analysis of protein expression of SDS-PAGE blot protein electrophoresis, the expression of Western.
The results of restriction enzyme digestion and DNA sequencing showed that the cloned staphylococcal enterotoxin B (SEB) encoding gene, and insert the correct reading frame, no base mismatch, pGEX-4T-2/SEB prokaryotic expression plasmid was constructed successfully. After IPTG induction, the recombinant plasmid was transformed into Escherichia coli expression of the relative molecular mass (Mr) 58 x 10~ ((3)) Daltons fusion protein.Western blotting confirmed that the protein with anti SEB antibody specific binding reaction with antigen activity.
Conclusion the prokaryotic expression clone of Staphylococcus aureus enterotoxin B (SEB) encoding gene is successfully constructed, and can be highly expressed in E.coli, which laid the foundation for further research on the biological function and clinical application of enterotoxin B (SEB).

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378.11

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