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【目的】通过研究重组猪溶素(Recombinant Suilysin,rSLY)的生物学活性来分析猪溶素(Suilysin,SLY)在猪链球菌致病过程中的作用。【方法】利用原核表达载体pET30a(+)表达SC22株猪链球菌的猪溶素基因(sly),经Ni2+亲和柱、阴离子交换柱和凝胶过滤纯化获得rSLY制品。用微量法测定rSLY对人红细胞的溶血效价,用LDH法测定rSLY对人外周血白细胞、人胚胎肺源细胞、人胚胎肝细胞和人胚胎心肌细胞的毒性强度,并用水溶性胆固醇、人血清和特异性抗体阻断rSLY的溶血活性。用Luminex测定rSLY刺激小鼠后血清中前炎性细胞因子的浓度。【结果】我们测得重组猪溶素的溶血效价仅为0.125nmol/L,但1nmol/Lr SLY对人外周血白细胞等的细胞毒性强度为20%～25%。水溶性胆固醇可阻断等摩尔浓度的rSLY,人血清样品(游离胆固醇的平均浓度为1～2mmol/L)仅能阻断1nmol/Lr SLY的溶血作用。在人血清中添加15mg/ml兔源抗猪溶素IgG可使10nmol/Lr SLY的裂解强度从77%降到5%,将猪溶素浓度提高到100nmol/L时,红细胞裂解强度为60%。C57BL/6小鼠经腹腔注射rSLY后血清中白介素6、趋化因子KC持续上升,这与注射牛血清白蛋白的对照组小鼠血清中相应细胞因子浓度有显著性差异。【结论】重组猪溶素不仅能直接损伤宿主细胞还能导致机体产生强烈的炎症反应,并进而损伤招募到炎症部位的免疫细胞;血清阻断rSLY溶血作用的能力很有限,特异性抗猪溶素抗体的保护作用表现为剂量依赖性。
【Objective】 The purpose of this study was to investigate the role of Suilysin (SLY) in the pathogenesis of Streptococcus suis by studying the biological activity of recombinant lysine (rSLY). 【Method】 The sly of SC22 Streptococcus suis was expressed by using prokaryotic expression vector pET30a (+). The purified rSLY was purified by Ni2 + affinity chromatography, anion exchange chromatography and gel filtration. The titer of rSLY against human erythrocytes was determined by the microtiter method. The toxicity intensity of rSLY to human peripheral blood leucocytes, human embryonic lung derived cells, human embryonic lung cells and human embryonic cardiomyocytes was measured by LDH method. The cytotoxicity of rSLY with water-soluble cholesterol and human serum And specific antibodies block the hemolytic activity of rSLY. Luminex was used to determine the concentration of pro-inflammatory cytokines in the serum following rSLY-challenged mice. 【Result】 We measured the hemolysis titer of recombinant porcine lysine was only 0.125nmol / L, but the cytotoxicity of 1nmol / L SLY on human peripheral blood leucocytes was 20% ~ 25%. Water-soluble cholesterol blocked equimolar concentrations of rSLY, human serum samples (free cholesterol average concentration of 1 ~ 2mmol / L) can only block 1nmol / Lr SLY hemolysis. The addition of 15mg / ml rabbit anti-porcine lysozyme IgG to human serum reduced the cleavage intensity of 10nmol / L SLY from 77% to 5%, and when the concentration of porcine lysostain increased to 100nmol / L, the cleavage intensity of erythrocytes was 60% . Serum interleukin-6 and chemokine KC were continuously increased in C57BL / 6 mice after intraperitoneal injection of rSLY, which was significantly different from that of the control mice injected with bovine serum albumin. 【Conclusion】 Recombinant porcine lysine can not only directly damage host cells, but also cause the body to produce a strong inflammatory reaction, which in turn damages the immune cells recruited to the inflammatory site. The ability of serum to block rSLY hemolysis is limited, The protective effect of the antibody is dose-dependent.