重组猪溶素的生物学活性分析

Biological profiles of recombinant Suilysin

摘要:

摘要：【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0.125 nmol/L，但1 nmol/L rSLY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1～2 mmol/L）仅能阻断1 nmol/L rSLY的溶血作用。在人血清中添加15 mg/ml兔源抗猪溶素IgG可使10 nmol/L rSLY 的裂解强度从70%降到5%, 将猪溶素浓度提高到100 nmol/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。

Abstract:

Abstract: [Objective] To provide insights into the role of Suilysin in the pathogenesis of Streptococcus suis by analysing the biological profiles of recombinant Suilysin (rSLY). [Methods] sly gene of S. suis strain SC22 was cloned into pET30a(+) vector and overexpressed in E. coli Rosseta, and rSLY was purified with Ni2+ affinity chromatography, anion exchange chromatography and gel filtration. The hemolytic unit of rSLY was measured with human red blood cells. The cellular toxicity of rSLY to human peripheral white blood cell and cells derived from fetal heart (HEH), liver (HEL), kidney (HEK), lung (HPL) was detected by lactate dehydrogenase (LDH) release assay. The blocking effects of water-soluble cholesterol, serum and specific antibody on the hemolytic activity of rSLY were detected. The amount of proinflammatory cytokines in the serum of mice stimulated by rSLY was measured by Luminex-100. [Results] The hemolytic unit of rSLY was 0.125 nmol/L, but the cellular toxicity of 1 nmol/L rSLY to human peripheral white blood cell, HEH, HEL, HEK and HPF was 20%～25%. The hemolytic activity of rSLY was blocked completely by equal molar of water-soluble cholesterol. Human serum containing 2.8 mmol/L～5.7 mmol/L of cholesterol in physical condition, could merely blocked 1 nmol/L of rSLY to lyse erythrocytes. The human serum with the addition of rabbit anti-rSLY IgG to a final concentration of 15mg/ml decreased the hemolysis by 10nmol/L rSLY from 77% down to 5% successfully, but hemolysis remained 60% when tested with 100nmol/L rSLY. Although IL-1β and TNF-α didn’t display any observable change, the level of interleukin -6 (IL-6) and KC were increased continually in C57BL/6 mice injected intraperitoneumly with rSLY whereas the IL-6 and KC level of mice in the control group only increased slightly and quickly decreased. [Conclusion] The data suggest that rSLY could not only lyse cells but also induce strong inflammatory response and damage immune cells recruited to the sites of inflammation. This finding implies that serum could provide light protection against rSLY, and specific antibody show a concentration-dependent protection.