摘要
目的:制备抗树鼩IgE高亲和力受体α亚基(FcεRIα)的单克隆抗体,并对其免疫学特性进行检测。方法:采用大肠杆菌原核表达树鼩IgE FcεRIα蛋白,经过纯化后免疫BALB/c小鼠,取免疫鼠脾细胞与Sp2/0细胞融合;通过ELISA法和有限稀释法筛选杂交瘤细胞;利用Protein A纯化单克隆抗体,Western印迹检测其特异性,间接免疫荧光及免疫组化对单克隆抗体进行分析。结果:用纯化的树鼩IgE FCεRIα蛋白免疫小鼠,选取效价高的小鼠脾细胞与骨髓瘤细胞Sp2/0融合,经过3轮亚克隆筛选,筛选出5株抗树鼩IgE FCεRIα单克隆抗体,分别命名为HC020、HC021、HC022、HC023和HC024,细胞间接免疫荧光表明5株单克隆抗体均能检测到293T细胞中表达的IgE FCεRIα,其中HC020、HC022、HC024能够通过免疫组化检测组织切片中的IgE FCεRIα。结论:制备了5株IgE FCεRIα单克隆抗体,为今后研究树鼩IgE FCεRIα奠定了基础。
Objective:To prepare a specific monoclonal antibody against high-affinity receptor alpha subunit(FcεRIα)of IgE of tree shrew,and characterize its immunological feature.Methods:The IgE FcεRIαprotein was expressed in E.coli and subsequently used as an antigen to immunize BALB/c mice,and immunized mice spleen cells were fused with Sp2/0cells.The hybrid cells were screened through ELISA and finite dilution method.The monoclonal antibody was purified using Protein A and analyzed by Western blot,indirect immunofluorescence and immunohistochemistry.Results:The purified IgE FCεRIαprotein was used to immunize mice and the spleen cells of high titer were fused with myeloma cell Sp2/0.After3rounds of subcloning,5strains of anti-tree shrew IgE FCεRIαwere screened,which were named as HC020,HC021,HC022,HC023and HC024.Indirect immunofluorescence showed that5monoclonal antibodies could detect IgE FCεRIαexpressed in the cells.HC020,748HC022and HC024could detect IgE FCεRIαin tissue slices by immunohistochemistry.Conclusion:Five monoclonal antibodies have been prepared by cell fusion and subcloning,which laid the basis for studying IgE FCεRIα.
作者
张晶晶
肖红剑
龙琼
浦滔
李智华
寸韡
ZHANG Jing-Jing;XIAO Hong-Jian;LONG Qiong;PU Tao;LI Zhi-Hua;CUN Wei(Institute of Medical Biology,Chinese Academy of Medical Sciences & Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Disease,Kunming 650118,China)
出处
《生物技术通讯》
CAS
2018年第6期748-751,共4页
Letters in Biotechnology
基金
云南省卫生和计划生育委员会医学学科带头人培养计划(D-2017003)
重大新药创制(2018ZX09738003-007)
中国医学科学院医学与健康科技创新工程(重大协同创新项目)(2016-I2M-1-013)
关键词
树鼩
IgE高亲和力受体α亚基
单克隆抗体
tree shrew
IgE high-affinity receptor alpha subunit(FcεRIα)
monoclonal antibody
