人TIPE家族新成员TIPE3特异性抗体的鉴定及应用

Identification and application of specific antibody against human TIPE3,a new member of TIPE family

目的制备人肿瘤坏死因子-α诱导蛋白8样蛋白3(TIPE3)的特异性抗体,分析将其用于Western blotting和免疫组化检测TIPE3蛋白表达的可行性和特异性。方法利用人TIPE3抗原制备兔抗人TIPE3多克隆抗体;构建人TIPE3真核表达载体;采用Western blotting方法检测抗体的特异性、TIPE3在肿瘤细胞系中的表达并用免疫组化方法检测人TIPE3蛋白在组织中的表达。结果制备的兔抗人TIPE3多克隆抗体的滴度为1∶400,该抗体可特异性地检测细胞中的TIPE3蛋白,与TIPE2无交叉反应;发现人肺腺癌细胞系H1975、肝癌细胞系HepG2高表达TIPE3,但其在宫颈癌细胞系Hela低表达;用于免疫组织化学检测,可检测到TIPE3在肺癌组织中高表达于细胞浆和细胞膜,在癌旁组织和正常肺组织未见表达。结论成功制备了人TIPE3多克隆抗体,特异性高,可用于TIPE3的Western blotting和免疫组化检测。

Objective To prepare the antibody specifically against human tumor necrosis factor-α induced protein 8 like 3 （TIPE3） and investigate the feasibility and specificity of the antibody in detecting TIPE3 protein in human tissues. Methods The specific polyclonal antibody was prepared by immunization of rabbits with TIPE3 antigen. Human TIPE3 eukaryotic expression vector was constructed. The specificity of TIPE3 antibody and the expression of TIPE3 protein in tumor cell lines were analyzed by Western blotting. TIPE3 expression in tumor tissues was detected by immunohistochemistry. Results The polyclonal antibody specifically against human TIPE3 was successfully prepared and the full-length human TIPE3 gene was constructed into pflag-cmv2 eukaryotic expressional vector. The prepared antibody was able to detect TIPE3 protein specifically by Western blotting in 293 cells which were transfected with human recom- binant TIPE3 vector and it showed no cross-interaction with TIPE2 protein. Both lung cancer-derived cells （H1975） and hepatocellular carcinoma cells （HepG2） expressed T1PE3 proteins while cervical cancer cells （Hela） did not. Immuno- histochemical reactions showed that strong positive staining of TIPE3 appeared in lung cancer cells, mainly in cytoplasm and on cell membrane, but not in the normal or adjacent tissues. Conclusion The polyclonal antibody against human TIPE3 is successfully prepared and it is conf＇Lrmed that the antibody can be used in both Western blotting and immunochemical staining procedures with high specificity.