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

摘要

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.The 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.%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-N1-99 McAb).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.