Abstract:

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 recombinant TIPE3 vector and it showed no cross-interaction with TIPE2 protein. Both lung cancer-derived cells (H1975) and hepatocellular carcinoma cells (HepG2) expressed TIPE3 proteins while cervical cancer cells(Hela) did not. Immunohistochemical 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 confirmed that the antibody can be used in both Western blotting and immunochemical staining procedures with high specificity.

Key words: Tumor necrosis factor-&alpha, Lung neoplasm, induced protein 8 like 3； Antibody； Specificity, Vector construction