摘要
为制备特异性的鸡源志贺菌IpaC蛋白单克隆抗体和鉴定其受体,以纯化的重组鸡源志贺菌IpaC蛋白制备多克隆抗体,Western blot和间接ELISA分析表明重组IpaC蛋白具有很好的免疫反应原性。将纯化的IpaC蛋白免疫BALB/c小鼠,通过淋巴细胞杂交瘤技术制备单克隆抗体。经过杂交瘤细胞阳性孔的筛选、单克隆抗体效价测定、单克隆抗体亚型鉴定、单克隆抗体Western blot鉴定后,获得1株杂交瘤阳性细胞5A12B5。同时,通过鸡胚肠组织提取总蛋白,利用铺覆蛋白印迹技术,初步测定IpaC蛋白受体相对分子质量约为55 000。试验为IpaC蛋白检测和该蛋白与鸡肠上皮细胞之间的相互作用研究奠定基础,同时也为评估鸡源志贺菌在公共卫生等方面的重要性提供参考依据。
To prepare a specific monoclonal antibody a dentify the receptor of IpaC protein. The recombinant gainst chicken Shigella chicken Shigella IpaC IpaC p protein rotel was n and i- purified to prepare polyclonal antibody. The immune activity of recombinant chicken Shigella IpaC protein was verified by Western blot and indirect ELISA. The recombinant chicken Shigella IpaC protein was used to immunize BALB/c mice and the chicken Shigella IpaC protein monoclonal antibody was obtained by hybridoma technique. One hybridoma cell line SA12B5 secreting monoclonal anti- body suitable for chicken Shigella IpaC protein were established by screening hybridoma-positive wells,titer determination, identification of subtype and Western blot of monoclonal antibody. At the same time, total protein was extracted by chicken embryos intestinal tissue, the receptors mo- lecular weight of chicken Shigella IpaC protein was identified about 55 000 by Western blot and o- verlay protein blot assay （OPBA）. It lays a foundation for detecting the interaction between the protein and intestinal epithelial cells, but also provides a reference for assesing the importance of chicken Shigella in public health.
作者
蒋大伟
张彬
马金玉
韩志霞
王亚宾
许兰菊
苗银萍
JIANG Da-wei ZHANG Bin MA Jin-yu HAN Zhi-xia WANG Ya-bin XU Lan-ju MIAO Yin-ping(College of Animal Husbandry and Veterinary Medicine, Zhengzhou 450002,China Henan Agricultural University, Zhengzhou 450002,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2016年第12期2061-2066,共6页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(31272568)
