金黄色葡萄球菌肠毒素A基因的克隆及原核表达

目的从分离培养的金黄色葡萄球菌中提取肠毒素A(SEA)基因,亚克隆入表达载体pET-28a(+),诱导融合蛋白的表达,为肠毒素A应用研究奠定基础。方法根据GenBank中SEA的序列,设计一对特异性引物,以金黄色葡萄球菌DNA为模板PCR扩增SEA,纯化DNA进行BamHⅠ、XholⅠ双酶切鉴定及测序鉴定,并与做相应酶切的pET-28a(+)连接,转化大肠杆菌BL21,并对质粒进行双酶切鉴定及基因序列分析,用IPTG诱导融合蛋白的表达,His标签单克隆抗体进行免疫印迹验证融合蛋白的表达。结果以金黄色葡萄球菌DNA为模板,成功扩增了SEA基因,基因大小为774bp,重组PET-28a(+)-SEA双酶切鉴定可见目的片段,测序结果显示SEA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99.9%。经IPTG诱导后,SDS-PAGE可见pET-28a(+)-SEA/BL21在相应分子量(约30000Mr)条带大量表达,免疫印迹能检测到目的蛋白条带,说明其与His标签融合表达。结论克隆了金黄色葡萄球菌SEA基因,并成功在大肠杆菌BL21中融合表达,为肠毒素A应用研究奠定了基础。

Amplification,cloning,and expression of staphyloccal enterotoxin A

Objective To amplify staphyloccal enterotoxin A(SEA) gene from Staphylococcus aureus,subclone into pET-28a(+) expression plasmid,analyze the sequence,and express as recombinant fusion protein for further research of Staphylococcus aureus SEA.Methods A pair of specific primer was designed according to SEA gene sequence in the GenBank for the amplification of the gene by PCR using Staphylococcus aureus DNA as template.The PCR product was digested by BamH I and Xhol I,was and then ligated with pET-28a(+) which was digested with same enzymes.The recombinant plasmid was transformed into E.coli BL21.Transformants Recombinant plasmid was identified by double enzyme digestion and sequence analysis.The sequence results were compared with the gene sequence in the GenBank.The expression of recombinant fusion protein was induced by IPTG and identified by west-blotting probed with HRP conjugated anti-His-Tag mouse monoclonal antibody.Results SEA gene fragment of 774 bp was successfully amplified from Staphylococcus aureus,and identified by double enzyme digestion.DNA sequence showed that cloned SEA gene was in the correct open reading frame as fused with the tag.Sequence analysis showed its nucleotide sequence was identical with SEA sequence of Staphylococcus aureus in GenBank.Recombinant fusion protein was 30 000 Mr,and identified by west-blotting.Conclusions SEA gene of Staphylococcus aureus was successfully cloned,and was expressed in E.coli as a fusion proein,which made a good foundation for Staphylococcus aureus SEA application research.