嗜水气单胞菌气溶素的纯化及其溶血特性分析

Purification of aerolysin of Aeromonas hydrophila by affinity chromatography and analysis of its hemolytic activity

目的用亲和纯化法纯化嗜水气单胞菌气溶素,从单因子水平分析嗜水气单胞菌的溶血特性。方法用嗜水气单胞菌气溶素单克隆抗体McAb3C12B11为配体制备亲和层析柱,甘氨酸洗脱以获得纯化的嗜水气单胞菌ZN1气溶素。获得的纯化气溶素分别与1%欧洲鳗鲡血红细胞、1%兔血红细胞、1%羊血红细胞进行溶血特性分析,并利用单克隆抗体McAb3C12B11进行溶血抑制试验。结果经亲和纯化获得分子量约为51.7kDa的气溶素,该气溶素能在Western-blotting反应中被嗜水气单胞菌ZN1胞外产物(ECPs)免疫血清和McAb3C12B11所识别。溶血试验证实亲和纯化的ZN1气溶素和ZN1ECPs对兔血红细胞、欧洲鳗血红细胞和绵羊血红细胞的溶血效价分别为5.12×104HU/mg、3.20×103HU/mg、6.40×103HU/mg和2.56×104HU/mg、1.28×104HU/mg、3.20×103HU/mg。溶血抑制试验结果表明:当红细胞为1%兔血红细胞时,McAb3C12B11对气溶素和ZN1ECPs的溶血抑制效价均为1∶211;当红血细胞为1%欧洲鳗血红细胞时,McAb3C12B11对气溶素的溶血抑制效价为1∶210,对ZN1ECPs的溶血抑制效价仅为1∶23。结论嗜水气单胞菌气溶素在单因子条件下与嗜水气单胞菌ECPs的溶血特性存在一定的差异,嗜水气单胞菌ZN1ECPs中存在多种溶血性毒素,这些毒素对不同宿主的溶血能力存在差异,同一嗜水气单胞菌菌株对不同的宿主起主要作用的溶血性毒素不同。

To investigate the mechanism of hemolysis induced by Aeromonas hydrophila, aerolysin of this organism was purified from the extracellular products （ECPs） of A. hydrophila ZN1 strain by affinity chromatography with MeAb 3C12B11 and eluated with glycine, and its hemolytic activity was analyzed through hemolytic reactions with 1% suspension of erythro- cytes of Anguilla anguilla, rabbits and sheep. It was demonstrated that a aerolysin with molecular mass of 51.7 kDa was obtained after affinity chromatography purification and it could be recognized by immune serum against ECPs of A. hydrophila ZN1 strain and MeAb 3C12B11 . As discovered by hemolytic reaction, the hemolytic titers of the purified ZN1 aerolysin with 1% suspension of erythrocytes ofA. anguilla, rabbits and sheep were 5.20 10^3 HU^-1 , 5.12 10^4 HU^-1 and 6.4 10^3 HU^-1 respectively. In addition, the titers of hemolytic inhibition of McAb 3C12B11 against aerolysin with 1% suspension of erythroeytes of rabbits and A. anguilla ZN1 ECPs were 1：2^11 and 1：2^3 respectively. These results indicate that definite difference in hemolytic activity exists between aerolysin and its extracellular products and the acting toxin of A. hydrophilla to different hosts is also different.