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目的扩增金黄色葡萄球菌肠毒素B(SEB)基因,构建其原核表达载体,并进行诱导、表达,为其应用研究奠定基础。方法根据GenBank中SEB的基因序列,设计一对分别含BamHⅠ、XhoⅠ酶切位点的特异性引物,以金黄色葡萄球菌基因组DNA为模板进行PCR扩增后,经BamHⅠ、XhoⅠ双酶切,并与做相应酶切的pET-28α(+)连接,转化大肠杆菌BL21,提取质粒进行双酶切鉴定及测序,用IPTG诱导表达融合蛋白,SDS-PAGE和Western blot印迹鉴定表达产物。结果成功扩增出SEB基因,基因大小为801bp,重组PET-28α(+)-SEB双酶切鉴定可见目的片段,测序结果显示SEB在正确读框中,序列比对分析显示其与相关报道核苷酸序列一致性达99%。经IPTG诱导后,pET-28α(+)-SEB/BL21在相应的相对分子质量(35×103)可见融合蛋白以包涵体形式表达,免疫印迹在相应分子量检测到目的蛋白。结论克隆了SEB基因,并成功在大肠杆菌BL21中以包涵体形式表达,为肠毒素B应用研究奠定了基础。
Objective To amplify the gene of Staphylococcus aureus enterotoxin B (SEB) and construct its prokaryotic expression vector, and to induce and express it, which lays the foundation for its application. Methods According to the gene sequence of SEB in GenBank, a pair of specific primers containing BamH Ⅰ and Xho Ⅰ restriction sites were designed respectively. After amplifying the DNA of S. aureus genomic DNA by PCR, the two fragments were digested with BamH Ⅰ and Xho Ⅰ. The recombinant plasmid pET-28α (+) was ligated with pET-28α (+). The recombinant plasmid was transformed into E. coli BL21. The plasmid was digested with restriction endonucleases and sequenced. The fusion protein was induced by IPTG. The expressed product was identified by SDS-PAGE and Western blot. Results The SEB gene was successfully amplified and the gene size was 801bp. The recombinant plasmid was identified by PET-28α (+) - SEB double digestion. The sequencing results showed that the SEB was in the correct reading frame. The nucleotide sequence identity of 99%. After IPTG induction, pET-28α (+) - SEB / BL21 expressed in the corresponding relative molecular mass (35 × 103) as inclusion body, and Western blotting detected the target protein at the corresponding molecular weight. Conclusion The SEB gene was cloned and successfully expressed in E. coli BL21 as inclusion body, which laid the foundation for the study of the application of enterotoxin B.