重组猪链球菌2型溶血素的表达及其溶血活性和致炎作用  

Expression of recombinant Streptococcus suis type 2 hemolysin and its hemolytic activity and inflammatory effect

作　　者：王俊捷 王洋 柳增善 WANG Junjie;WANG Yang;LIU Zengshan(Ministry of Education Key Laboratory for Zoonosis Research,College of Veterinary Medicine,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学动物医学学院人兽共患病研究教育部重点实验室,吉林长春130062

出　　处：《中国兽医学报》2022年第7期1393-1398,共6页Chinese Journal of Veterinary Science

摘　　要：PCR扩增溶血素(suilysin,SLY)基因,克隆后测序,利用原核表达载体pET-28a及表达宿主菌E.coli BL21(DE3)构建SLY原核表达系统,采用SDS-PAGE检测蛋白表达情况,借助Ni-NTA亲和层析法纯化蛋白,获得具有序列保守且可溶性的SLY蛋白。显微镜形态学观察显示,质量浓度为0.4 mg/L的SLY在37℃条件下作用2 h可使贴壁hCMEC/D3(人脑微血管内皮细胞)全部死亡。通过分光光度法测定SLY对人红细胞的溶血活性,结果显示1μg SLY可使2.25×10^(10)个人红细胞完全溶解。Real-time RT-PCR结果显示,SLY处理所致破碎的红细胞上清可使hCMEC/D3细胞炎性因子IL-6和IL-8转录水平分别升高80倍和100倍以上,且该作用效果显著高于超声破碎红细胞的方式。结果表明,SLY具有显著的细胞毒性,其导致红细胞溶血可能是猪链球菌2型(Streptococcus suis type 2,SS2)引起机体炎症反应的原动力。Streptococcus suis type 2(SS2)is an important zoonotic pathogen,and Streptococcus suis hemolysin(suilysin,SLY)is its main virulence factor,which has cytotoxic and inflammatory effects.Our previous research found that the internal environment disorder caused by the interaction of SLY and red blood cells(erythrocyte,RBC)is an important cause of meningitis induced by bacteremia caused by SS2.This finding is worthy of further investigation.In this experiment,the SLY gene was amplified by PCR,cloned and sequenced,and the prokaryotic expression vector pET-28 a and the expression host strain E.coli BL21(DE3)were used to construct the SLY prokaryotic expression system.SDS-PAGE was used to detect the protein expression,with the help of Ni-NTA,the protein was purified by affinity chromatography to obtain sequence-conserved and soluble SLY proteins.Microscopic morphological observations showed that the concentration of 0.4 mg/L SLY can cause death to adhered hCMEC/D3(human brain microvascular endothelial cells)after 2 h at 37℃.The hemolytic activity of SLY on human red blood cells was measured by spectrophotometry,and it was found that 1μg SLY could completely dissolve 2.25×10^(10)human red blood cells.Real-time RT-PCR experiments showed that the supernatant derived from broken red blood cells caused by SLY treatment could increase the transcription levels of the inflammatory factors IL-6 and IL-8 of hCMEC/D3 cells by more than 80-fold and 100-fold,respectively,and the effect was significantly higher than that of ultrasound disrupting red blood cells.In conclusion,this study confirms that SLY has significant cytotoxicity.It is found that the hemolysis of erythrocytes caused by SLY may be the driving force of SS2to cause the inflammatory response.This discovery is of great significance for the study of the mechanism of SLY-mediated SS2infection and the development of anti-SS2drugs,provides a material basis for subsequent in-depth research on the interaction of SLY and red blood cells in vivo and in vitro.

关 键 词：猪链球菌2型 SLY 红细胞 IL-6 IL-8 

分 类 号：S852.61[农业科学—基础兽医学] R535[农业科学—兽医学]