抗人LOC51255单克隆抗体的制备及亚细胞定位

Preparation of the monoclonal antibody against LOC51255 antigen and subcellular localization

目的:制备抗人LOC51255单克隆抗体,并构建pDsRed1-C3/LOC51255真核表达质粒,了解LOC51255在人肝癌细胞株HepG2中的亚细胞定位情况,为进一步研究其功能提供实验工具和基础。方法:以纯化的重组蛋白pET28b/LOC51255为抗原,免疫BALB/c小鼠,运用杂交瘤技术制备LOC51255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;通过构建含红色荧光蛋白(pDsRed1)的pDsRed1-C3/LOC51255真核表达质粒,转入人肝癌细胞株HepG2表达重组蛋白,并对LOC51255进行亚细胞定位。结果:成功建立2株稳定分泌抗LOC51255单克隆抗体的杂交瘤细胞株;ELISA检测抗LOC51255单克隆抗体的腹水效价分别为1∶12 800和1∶25 600。2株单克隆抗体的免疫球蛋白亚类均为IgG1。通过Western blot实验证实,2株单克隆抗体均能特异性结合真核细胞内源性LOC51255蛋白。荧光显微镜观察发现LOC51255主要定位于HepG2细胞的细胞浆。结论:成功制备了2株效价高、特异性好的抗LOC51255单克隆抗体,制备的单克隆抗体可用于LOC51255蛋白的鉴定;亚细胞定位分析证实LOC51255主要表达于HepG2细胞的胞浆,为LOC51255的生物学功能研究奠定了基础。

Objective：To prepare the monoclonal antibody （McAbs） against LOC51255 antigen and to detect the subcelhdar localization of LOC51255 in HepG2 cells line for further study. Methods： BALB/c mice were immunized with recombinant pEY28b/LOC51255 protein. Hybridoma cell lines were set up by using hybridon,a technique and McAbs were prepared. The McAbs were identified by Western blot and enzyme linked immunosorbent assay （ELISA）. Recombinant plasmid pDsRedl-C3/LOC51255 was constructed and transfected into HepGz cells by lipofectamine 2000.The expression and subcellular localization of LOC51255 was observed tmder fluorescence microscope. Results： Two hybridoma cell lines that stably secreted LOC51255 McAbs were obtained： E001 and E002 belonging to IgG1. Western blot proved that the two McAbs could specifically bond with human endogenous LOC51255 protein. Their antibody titers were 1 ： 12 800 and 1：25 600 in the ascites, respectively. Fluorescence microscope showed that LOC51255 was located in the cytoplasm of HepG2 cell lines. Conclusion： McAbs of IDC51255 with high titer and specificity have been successfully prepared and can be used to identify LOC51255 protein. Subcellular localization analysis shows that LOC51255 is mainly expressed in the cytoplasm of HepG2 cell lines. This will be benefit to further research of the functions of LOC51255 protein.