Molecular cloning of staphylococcal enterotoxin gene from wild Staphylococcus aureus strains
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摘要: 目的 从金黄色葡萄球菌SD和104菌株中克隆肠毒素基因,以期获得新型肠毒素。方法采用已知的金葡菌肠毒素基因保守序列设计引物,以SD和104菌株基因组为模板,PCR扩增产物插入表达载体pET28a并转化大肠埃希菌,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,镍离子金属螯合(Ni+-NTA)亲和层析纯化重组蛋白,测定重组蛋白超抗原活性及体外抑瘤效果。结果 成功扩增到2个新型肠毒素P基因;带有质粒pET28a-SEPSD和pET28a-SEP104的重组大肠埃希菌,在1 mmol/L IPTG诱导下高效表达,重组蛋白纯度>95%;纯化蛋白刺激人淋巴细胞增殖率分别>30%;抑瘤率明显高于金黄色葡萄球菌肠毒素C2(SEC2),200 ng/mL时抑瘤效果最佳,分别达到82%和86%。结论 成功克隆到金葡菌新型肠毒素P基因,SEPSD和SEP104重组蛋白具有与SEC2相近的活性并有较高的抑瘤效果。
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关键词:
- 金黄色葡萄球菌肠毒素 /
- 超抗原 /
- PBMC增殖 /
- 体外抑瘤
Abstract: Objective To clone novel staphylococcal enterotoxin P(SEP)gene from Staphylococcus aureus(S.auresu)strains SD and 104 for the production of a novel staphylococcal enterotoxin. Methods Primers were designed according to the conserved sequence of SEP from published data.PCR products amplified from genomic DNA of S.aureus SD and 104 were inserted into the plasmid pET-28a.The resultant plasmids were transformed into E.coli BL21(DE3)and induced to express the gene with isopropyl-β-D-thiogalactoside(IPTG).Proteins purified through nickel-nitrilotriacetic acid(Ni+-NTA) affinity chromatography were used to analyze the superantigenic activity and anti-tumor effect in vitro. Results Two novel staphylococcal enterotoxin P genes were obtained according to the results of sequence alignment.E.coli harboring pET28aSEPSD and pET28a-SEP104 could high-efficiently express the soluble protein under 1 mmol/L IPTG induction.The proteins were purified through Ni+-NTA affinity chromatography.The purified protein was used to stimulate peripheral blood mononuclear cell(PBMC)proliferation.Compared with staphylococcal enterotoxin C2(SEC2),the proliferation rates of the proteins were higher than 30%.SEP could obviously inhibit the growth of tumor cells,even higher than SEC2. Conclusion New novel SEP genes were successfully cloned and the expressed proteins SEPSD and SEP104 of the genes exhibited anti-tumor effect stronger than SCE2. -
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