抗幽门螺杆菌中性粒细胞激活蛋白的单克隆抗体的制备和鉴定

Preparation and identification of monoclonal antibodies against Helicobacter pylori neutrophils activating protein

目的:从抗幽门螺杆菌(Hpylori)全菌蛋白的单克隆抗体(mAb)中,应用重组中性粒细胞激活蛋白(NAP),筛选出抗NAP单抗并进行鉴定.方法:临床分离H pylori DY01,DY04株.免疫BALB/c小鼠后,应用杂交瘤技术制备mAb.再用ELISA方法以重组表达的NAP蛋白筛选相应的单抗,对NAP单抗进行亚类鉴定和效价检测,并用Western blot和免疫组化方法鉴定其特异性.结果:获得3株针对H pylori-NAP蛋白的特异性mAb,抗体亚类为IgG1,轻链为k型.单抗细胞培养液的抗体效价为1/16-1/32,腹水的抗体效价是1/32000-1/64000.Western blot鉴定表明,抗NAPmAb针对NAP蛋白产生特异性条带,具有高度的特异性;免疫组化分析显示:3株NAP mAb能与H pylori临床菌株发生特异性结合反应,菌体染成深棕色.结论:获得抗Hpylori-NAP蛋白的特异性mAb,为幽门螺杆菌感染的诊断、预后判断及表位疫苗的研究提供基础.

AIM： To screen and identify the monoclonal antibodies （mAbs） against H pylori neutrophils activating protein （NAP）. METHODS： H pylori DY01 and DY04 strains were clinically isolated. After BALB/c mice were immunized, the mAbs against H pylori were obtained by hybridoma technique. Then, the mAbs against H pylori-NAP were screened using recombinant H pylori-NAP. The obtained mAbs were evaluated for subtype and titer, and their specificities were identified by Western blot and immunohistochemical techniques. RESULTS： Three strains of mAbs against H pylori-NAP were obtained. All of the mAbs against H pylori-NAP were identified as immunoglobulin G1 （IgG1） with κ： light chain. The titer of mAbs in the culture supernatant was 1/16 tol/32, and that in the ascites were 1/32000 to 1/64000. Western blotting showed that the mAbs had specific reaction with the recombinant H pylori-NAP. Clinical H pylori strains were stained brown with the mAbs against H pylori- NAP by immunohistochemical techniques. CONCLUSION： Three strains of mAbs against H pylori-NAP are obtained, which are helpful in the diagnosis and prognosis of H pylori infection as well as vaccine research.