摘要
目的:制备增强型绿色荧光蛋白(EGFP)的兔多克隆抗体。方法:用谷胱甘肽-增强型绿色荧光蛋白(GST-EGFP)融合抗原加福氏完全佐剂背部皮内注射首次免疫新西兰大白兔,第28天用GST-EGFP融合抗原加福氏不完全佐剂同样剂量加强免疫,第35天时再次免疫。第49天心脏采血30mL,4℃静置过夜,收集血清,用酶联免疫吸附试验(ELISA)检测抗血清中EGFP多克隆抗体的效价;免疫印迹法检测EGFP多克隆抗体的特异性;利用荧光免疫细胞化学检测该抗体和观察转染EGFP贴壁生长的大鼠脑皮层混合培养细胞中EGFP的表达。结果:ELISA检测EGFP多克隆抗体滴度最高为1:9549;免疫印迹检测结果显示该抗体特异性高;荧光免疫细胞化学检测显示在混合培养细胞中EGFP呈阳性表达。结论:直接使用GST-EGFP融合蛋白免疫新西兰大白兔,可在较短时间内制备大量EGFP多克隆抗体。
Aim：To prepare the polyclonal antibody of enhanced green fluorescent protein（EGFP） in rabbits.Methods：The New Zealand white rabbits were immunized with Freund＇s complete adjuvant plus GST-EGFP fusion antigen at the back skin intradermal injection for the first time.The same dose Freund＇s incomplete adjuvant plus GST-EGFP was injected to strengthen the immune on the 28th day and the 35th day separately.On the 49th day,blood sample was collected from heart and antisera was extracted after 4 ℃ settlement overnight.The titer of antisera was detected by ELISA.The specificity of EGFP polyclonal antibody was observed by Western blot.The expression of EGFP was examined by EGFP polyclonal antibody through fluorescence immunocytochemistry in the EGFP transfected rat cerebral cortex cells.Results：EGFP polyclonal antibody titer was up to 1：9 549 by ELISA.Western blot showed that the antibody had a high specificity.The expression of EGFP was positive in the mixed culture cells by fluorescence immunocytochemistry.Conclusion：EGFP polyclonal antibody can be produced in large quantities in a short time by using GST-EGFP fused protein to immunize the New Zealand white rabbit directly.
出处
《郑州大学学报（医学版）》
CAS
北大核心
2010年第1期87-89,共3页
Journal of Zhengzhou University(Medical Sciences)
关键词
增强型绿色荧光蛋白
多克隆抗体
兔
enhanced green fluorescent protein
polyclonal antibody
rabbit
