重组人14-3-3zeta亚型单克隆抗体的制备、鉴定及初步应用  

Preparation,Characlerization and Preliminary Application of Monoclonal Antibody Against Recombinant Human 14-3-3 Zeta Isoform

作　　者：钟政荣[1] 沈继龙[1] 汪学龙[1] 胡元生[1] 沈继录[2] 王家传[1] 李小月[1] 

机构地区：[1]安徽医科大学病原生物学教研室,安徽省基因研究重点实验室,合肥230032 [2]安徽医科大学第一附属医院检验科,合肥230022

出　　处：《中国生物制品学杂志》2005年第4期316-318,共3页Chinese Journal of Biologicals

基　　金：国家自然科学基金(30170841);安徽省自然科学基金(0044547;98436329)资助

摘　　要：目的制备重组人14-3-3zeta亚型单克隆抗体(McAb),并检测14-3-3蛋白在人体组织器官的分布。方法将筛选出分泌抗人14-3-3zeta的杂交瘤细胞注射入BALB/c小鼠腹腔,制备腹水McAb,用辛酸-饱和硫酸铵沉淀法纯化,并鉴定其效价、纯度、浓度、特异性以及与天然抗原的亲和力,再用此单抗检测14-3-3蛋白在人体组织器官的分布。结果腹水及纯化后的单克隆抗体的效价分别为1∶107和1∶106。纯化后的纯度达97%,浓度为5mg/ml,能识别天然的人14-3-3蛋白,并能有效地识别兔和鼠脑组织的14-3-3蛋白。在人脑、肾、肺、肝、胃、卵巢、乳腺、结肠、胰腺等组织器官中,均检出14-3-3蛋白。结论已成功制备出重组人14-3-3zeta亚型单克隆抗体,并应用于实际检测,为研究14-3-3蛋白在机体生理和病理情况下的作用机制奠定了基础。Objective To prepare, purify and identify the monoclonal antibody(McAb)against recombina nt human 14-3-3 zeta isoform on the basis of screening of hybridoma cells,and explore the distribution of 14-3-3 protein in human tissues and organs. Methods Inject i.p. the hybridoma cells secreting McAb against human 14-3-3 zeta isofo rm into BALB/c mice.Collect the ascites of mice to prepare McAb.Purify the McAb by caprylic acid-saturated ammonium sulfate precipitation and determine its tit er,purity,concentration,specificity and affinity to natural antigen.Detect the d istribution of 14-3-3 protein in human tissue and organ with the McAb. Results The titers of ascites and purified McAb were 1∶107 and 1∶106 respectively. The McAb after purification reached a purity of 97% and a concentration of 5 mg/ ml,and recognized natural human 14-3-3 protein as well as the protein in rabbi t and murine brain tissues.The 14-3-3 protein were detected in many human tiss ues or organs,such as brain,kidney,lung,liver,stomach,ovary,mammary gland,colon and pancreas. Conclusion The McAb against recombinant human 14-3-3 zeta isoform was successfully prepar ed and preliminarily applied to detecting the distribution of 14-3-3 protein i n human tissues or organs.It laid a foundation of study on the action mechanism of 14-3-3 protein physiological and pathological states.

关 键 词：重组人14-3-3zeta亚型单克隆抗体 制备 鉴定 分布 作用机制 

分 类 号：R392.11[医药卫生—免疫学]