MICA-α3和NKG2D蛋白的表达及其抗体结合功能检测  

MICA-α3 and NKG2D expression and antibody binding functional analysis

作　　者：陆瑾 梁红远 吴丽娜 瞿爱东 Lu Jin;Liang Hongyuan;Wu Lina;Qu Aidong(No.1 Reaseach Laboratory,Shanghai Institute of Biological Products Co.,Ltd.,Shanghai 200051,China)

机构地区：[1]上海生物制品研究所有限责任公司第一研究室,上海200051

出　　处：《国际生物制品学杂志》2022年第1期47-51,共5页International Journal of Biologicals

基　　金：上海市企业技术中心能力建设项目(沪创新-J-08-25)。

摘　　要：目的重组表达人MHCⅠ类链相关蛋白Aα3结构域(MHCⅠchain related protein Aα3 domain,MICA-α3)蛋白及自然杀伤细胞受体2D(natural killer group 2D,NKG2D)蛋白,制备抗MICA-α3单克隆抗体,并检测其结合功能。方法构建MICA-α3及NKG2D蛋白表达载体,经转化大肠埃希菌BL21菌株表达重组蛋白,通过分子筛、组氨酸标签蛋白层析纯化。用重组MICA-α3蛋白免疫小鼠,采用杂交瘤技术筛选单克隆抗体,用细胞ELISA检测单克隆抗体功能。结果重组蛋白在大肠埃希菌BL21菌株中以包涵体形式表达。筛选得到5株能和表达MICA-α3的MDA-MB-453细胞结合的单克隆抗体,且不影响MDA-MB-453细胞与NKG2D结合。结论制备的抗MICA-α3单克隆抗体能与肿瘤细胞结合,同时不影响肿瘤细胞与NKG2D结合。Objective To express recombinant human MHCⅠchain related protein Aα3 domain(MICA-α3)and natural killer group 2D(NKG2D),prepare anti-MICA-α3 monoclonal antibodies and test antibody binding function.Methods MICA-α3 and NKG2D expression vectors MICA-α3-pET11c and NKG2D-pET16b were constructed and transformed into BL21 for expression.Recombinant proteins were purified by polyhistidine-tag chromatography and gel chromatography.After immunizing mice with MICA-α3 protein and screening monoclonal antibody by hybridoma technology,the monoclonal antibody function was tested by cell-ELISA.Results Recombinant proteins were expressed in the form of inclusion body in E.coli BL21.Five monoclonal antibodys were screened,which could bind to MDA-MB-453 cells expressing MICA-α3,and this binding did not affect the binding of MDA-MB-453 cells to NKG2D.Conclusion Anti MICA-α3 monoclonal antibody prepared can bind to tumor cells without affecting the binding of tumor cells to NKG2D.

关 键 词：受体 自然杀伤细胞 抗体 单克隆 主要组织相容性复合体Ⅰ类链相关蛋白A 细胞杂交瘤技术 

分 类 号：Q78[生物学—分子生物学]