抗PSMA7单克隆抗体的制备和初步鉴定

Preparation and identification of the monoclonal antibody against human PSMA7 antigen

目的制备抗蛋白酶体α7亚基(PSMA7)的单克隆抗体,为PSMA7的功能研究奠定基础。方法以纯化的重组蛋白PSMA7为抗原免疫BALB/c小鼠,运用杂交瘤技术制备PSMA7单克隆抗体,并用间接ELISA法和Western blot法对单克隆抗体的特性进行鉴定。结果成功建立两株稳定分泌抗PSMA7蛋白的单克隆抗体杂交瘤细胞株,分别命名为B013和B001。检测杂交瘤细胞培养上清抗体效价为1∶128和1∶256,腹水效价为1∶25600和1∶32000。两株单克隆抗体的免疫球蛋白亚类均为IgG1。Westernblot及免疫组化实验证实,两株单克隆抗体均能特异性结合真核细胞内源性PSMA7蛋白。结论成功建立了两株效价高、特异性好的抗PSMA7单克隆抗体杂交瘤细胞,制备的单克隆抗体可用于PSMA7蛋白的鉴定,为其生物学功能研究奠定了基础。

Objeetive To prepare the PSMA7 hybridoma cell lines using classical hybridoma technique and to perform purification and elementary identification of the monoclonal antibodies （McAbs） against PSMA7 antigen for further study. Methods BALB/c mice were immunized with purified recombinant PSMA7 protein. Once the antibody titer of blood taken from mouse tail reached 1 ： 6 400, a cell fusion was performed between mouse splenic cells and myeloma cells （Sp2/0）, and then the hybridorna cell lines secreting monoclonal antibodies against PSMA7 antigen were screened by indirect enzyme linked immunosorbent assay （ELISA）. The immunoglobulin subtypes and titer of the monoclonal antibodies against PSMA7 antigen were identified and measured by Western blot analysis and enzyme linked immunosorbent assay, respectively. Results After cell fusion and subcloning, two hybridoma cell lines that stably secreted monoclonal antibodies against PSMA7 antigen were successfully obtained： 13013 and 13001, which belong to the subtypes of IgG1. The antibody titers in the hybridoma culture supernatant were 1：128 and 1：256, and those in the ascites fluid were 1：25 600 and 1：32 000, respectively. Western blot analysis and immunohistochemistry showed that the two antibodies can specifically bind with PSMA7 antigen derived from human eu caryotic cells or tissue. Conclusions Two monoclonal antibodies against PSMA7 antigen with high titers and specificity have been successfully prepared. These antibodies can be used for the identification of PSMA7 protein, and may be a useful tool for studying the biological properties of PSMA7 protein.