摘要
为建立一种早期诊断动物细粒棘球蚴病的方法,诱导表达含PET-EgAgB8/2载体的原核表达菌,以表达纯化蛋白为抗原,制备单克隆抗体,并以制备的抗体建立检测细粒棘球蚴病夹心ELISA方法。结果显示,原核表达菌在37℃诱导培养,菌体上清中大量表达,经验证为目的蛋白,用纯化蛋白免疫小鼠,取其脾细胞与骨髓瘤细胞融合,筛选得到9株具有良好特异性、稳定分泌单克隆抗体的细胞株,经两两配对,选择75C86/15作为包被抗体,27E57/32为酶标二抗,当包被抗体浓度为1.42μg/mL,4℃包被过夜、用含50 g/L脱脂奶粉的PBST封闭、抗原作1∶100稀释、酶标二抗1∶1000稀释、反应底物作用15 min时效果最佳,检测方法的特异性为98.8%,敏感性为95.3%。成功制备细粒棘球蚴抗原B8/2蛋白单克隆抗体并建立了ELISA检测方法。
To establish a method for early diagnosis of animal echinococcosis,this study induced the expression of prokaryotic expression bacteria containing PET-EgAgB8/2 prokaryotic expression vector,purified the protein,prepared the monoclonal antibody with the expressed purified protein as the antigen,and established the sandwich ELISA method for detecting Echinococcus granulosus with the prepared antibody.The results showed that the prokaryotic expression bacteria were induced and cultured at 37℃,and the bacterial supernatants were in large amounts expressed.It was verified to be the target protein.The purified protein was used to immunize mice,and the spleen cells were fused with myeloma,and 9 strains were obtained with good specificity and stability.After pairing,75C86/15 was selected as the coating antibody,and 27E57/32 was the enzyme-labeled secondary antibody.When the concentration of antibody was 1.42μg/mL,coated overnight at 4℃,blocked with PBST containing 5%skimmed milk powder,1∶100 dilution of antigen,1∶1000 dilution of enzyme-labeled secondary antibody,and the best effect was obtained when the reaction substrate acts on for 15 minutes.The specificity and sensitivity of the method were 98.8%and 95.3%,respectively.In this study,monoclonal antibody against B8/2 protein of Echinococcus granulosus antigen was successfully prepared and a sandwich ELISA method was established.
作者
朵红
付永
沈秀英
郭志宏
张学勇
马怡隽
DUO Hong;FU Yong;SHEN Xiu-ying;GUO Zhi-hong;ZHANG Xue-yong;MA Yi-jun(Academy of Animal Science and Veterinary Medicine of Qinghai University,Xining,Qinghai,810016,China)
出处
《动物医学进展》
北大核心
2021年第10期31-36,共6页
Progress In Veterinary Medicine
基金
青海省科技厅项目(2018-NK-104)。
