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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 recombinant human 14-3-3zeta monoclonal antibody (McAb) and to detect the distribution of 14-3-3 protein in human tissues and organs. Methods The hybridoma cells secreting anti-human 14-3-3zeta were injected into the peritoneal cavity of BALB / c mice to prepare ascitic fluid McAb and purified by caprylic acid-saturated ammonium sulfate precipitation. The titer, purity, concentration, specificity Sex and affinity with natural antigens, and then use this monoclonal antibody to detect the distribution of 14-3-3 protein in human tissues and organs. Results Ascites and purified monoclonal antibody titers were 1:107 and 1:106, respectively. Purified purity of 97%, a concentration of 5mg / ml, can recognize natural human 14-3-3 protein, and can effectively identify 14-3-3 protein in rabbit and rat brain tissue. In human brain, kidney, lung, liver, stomach, ovary, breast, colon, pancreas and other tissues and organs, were detected 14-3-3 protein. Conclusion The recombinant human 14-3-3zeta subtype monoclonal antibody has been successfully prepared and applied to the actual detection, which lays the foundation for studying the mechanism of 14-3-3 protein in physiological and pathological conditions.