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目的体外扩增肺炎链球菌(Streptococcus pneumoniae,S.pn)的溶血素(pneumolysin,ply)基因,在大肠杆菌中高效表达,并验证其生物活性。方法分离培养D39型肺炎链球菌并提取其基因组DNA,采用PCR技术体外扩增ply基因,体外重组将ply序列克隆到原核表达载体pET28(a)内,测序鉴定后将重组子转化到E.coliRosetta(DE3)中,经IPTG诱导大量表达融合6个组氨酸标签的Ply重组蛋白,经Ni-NTA树脂纯化后,获得的重组蛋白经SDS-PAGE鉴定后检测其溶血活性和细胞毒性。结果克隆的ply序列与GenBank中的数据相符,并实现了Ply蛋白的可溶表达。所获重组蛋白纯度达到95%。溶血实验显示Ply对人红细胞具有极高的溶血活性,与对照组相比较差异有统计学意义(P<0.05),并呈剂量依赖。细胞毒实验显示Ply对脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)细胞具有明显抑制作用,与对照组比较差异有统计学意义(P<0.05),并呈剂量与时间依赖性,IC50(24h)为1.525μg/ml。结论经测序鉴定ply基因正确扩增,并成功表达、纯化出具有高溶血活性和细胞毒性的Ply蛋白。
Objective To amplify the gene pneumolysin (ply) of Streptococcus pneumoniae (S. pn) in vitro and express it in E.coli efficiently and validate its bioactivity. Methods Streptococcus pneumoniae D39 was isolated and its genomic DNA was isolated. The ply gene was amplified by PCR in vitro. The recombinant plasmid ply was cloned into prokaryotic expression vector pET28 (a) in vitro. After sequencing, the recombinant plasmid was transformed into E.coli Rosetta (DE3), Ply recombinant protein with 6 histidine tag fused in large amount was induced by IPTG. After purified by Ni-NTA resin, the recombinant protein was identified by SDS-PAGE and its hemolysis activity and cytotoxicity were tested. Results The cloned ply sequence was consistent with the data in GenBank and the soluble expression of Ply protein was achieved. The purity of recombinant protein was 95%. Hemolysis experiments showed that Ply had extremely high hemolytic activity on human erythrocytes, which was significantly different from the control group (P <0.05) in a dose-dependent manner. Cytotoxicity assay showed that Ply had a significant inhibitory effect on human umbilical vein endothelial cell (HUVEC) cells in a dose-and time-dependent manner (P <0.05) 24h) was 1.525 μg / ml. Conclusion The ply gene was correctly amplified by sequencing and successfully expressed. Ply protein with high hemolysis activity and cytotoxicity was purified.