论文部分内容阅读
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。
Objective To clone the enterotoxin gene from Staphylococcus aureus SD and 104 strains in order to obtain a novel enterotoxin. Methods The primers were designed according to the conserved sequence of Staphylococcus aureus enterotoxin gene. The genomic DNA of SD and 104 strains were used as templates. The PCR product was inserted into expression vector pET28a and transformed into Escherichia coli and Isopropyl-β-D-thio The recombinant protein was purified by Ni + -NTA affinity chromatography. The activity of recombinant protein superantigen and the anti-tumor effect in vitro were determined. Results Two novel enterotoxin P genes were successfully amplified. Recombinant Escherichia coli with plasmids pET28a-SEPSD and pET28a-SEP104 were highly expressed under the induction of 1 mmol / L IPTG. The purity of the recombinant protein was> 95%. The purified protein The rate of proliferation of human lymphocytes was> 30%. The inhibitory rate was significantly higher than that of the staphylococcal enterotoxin C2 (SEC2). The best antitumor effect was achieved at 200 ng / mL, reaching 82% and 86% respectively. Conclusion The novel recombinant enterotoxin P gene of Staphylococcus aureus was successfully cloned. The recombinant proteins of SEPSD and SEP104 have the similar activity with SEC2 and high antitumor activity.