抗人肝癌相关基因LAPTM4β胞内肽段单克隆抗体的制备与鉴定及其表达的检测

Preparation and identification of monoclonal antibodies against lysosome-associated protein transmembrane 4 beta intracellular 1-99(LAPTM4β-IC-N_(1-99)) and its expression

目的制备和鉴定鼠抗人肝癌相关基因溶酶体相关四次跨膜蛋白β胞内99个氨基酸残基(LAPTM4β-IC-N1-99)的单克隆抗体,检测LAPTM4β在肝癌中的表达,为深入研究其结构和功能奠定基础。方法利用本实验室构建原核表达质粒pGEX-KG-LAPTM4β-IC-N1-99,在大肠杆菌中表达融合蛋白GST-LAPTM4β-IC-N1-99。用纯化的蛋白LAPTM4β-IC-N1-99免疫雌性BALB/C小鼠,以常规杂交瘤技术制备鼠抗人LAPTM4β-IC-N1-99单克隆抗体(LAPTM4β-IC-N1-99McAb)。进行抗体亚类测定和免疫印迹法(WB)分析,然后用免疫化学技术检测LAPTM4β-IC-N1-99在肿瘤中的表达。结果诱导并纯化了重组GST-LAPTM4β-IC-N1-99蛋白,其相对分子质量约为32000;纯化得到LAPTM4β-IC-N1-99蛋白,其相对分子量约为10000。筛选出6株能稳定分泌抗LAPTM4β-IC-N1-99单克隆抗体的杂交瘤细胞株(D7,B12,H2,H8,A9和A10),D7、H8、A10分泌抗体亚类为IgG1,B12、A9、H2为IgG2b。Western blotting鉴定显示,抗LAPTM4β-IC-N1-99单抗可与融合蛋白及细胞内LAPTM4β蛋白特异性结合,具有高度特异性。免疫细胞化学证明,所得抗体能检测到肿瘤细胞中LAPTM4β基因的表达。结论以纯化的LAPTM4β-IC-N1-99蛋白免疫,获得了效价高、特异性好的LAPTM4β-IC-N1-99蛋白的单克隆抗体,用其可检测到LAPTM4β肿瘤细胞中的表达,这为LAPTM4β蛋白的生物学功能研究奠定了基础。

Objective To obtain monoclonal antibodies against lysosome-associated protein transmembrane 4 beta intracellular 1- 99（LAPTM4β-IC-N1-99） for further study of the structure and function of LAPTM4β protein.Methods Balb/c mice were immunized with purified LAPTM4β-IC-N1-99 protein, hybridoma cell lines secreting monoclonal antibodies against LAPTM4β-IC-N1-99 were screened by regular cell fusion and subcloning approach. Th~ specificities of these monoclonal antibodies were determined by ELISA, identified the McAbs and expression of LAPTM4β-IC-N1-99 in tumor by Western blotting, Immunocytochemistry. Results Six hybridoma cell lines stable in secreting specific monoclonal antibodies were successfully obtained. Subtype of IgG belonged to IgG1, IgG2b respectively. The ascite titers of these monoclonal antibodies reached 1：10（4）. They could specifically bind to recombinant LAPTM4β-IC-N1-99 and endogenous and overexpressed LAPTM4β proteins proved by ELISA and Western blotting. Both endogenous and overexpressed LAPTM4β-IC-N1-99 protein mainly located in the nucleus and cytoplasm. Conclusion Monoclonal antibodies against LAPTM4β-IC-N1-99 with high titers and specificity have been successfully prepared, which has laid the foundation for further study of LAPTM4βprotein.