[Preparation of rabbit polyclonal antibodies against human copine-8 protein]

Objective To produce human polyclonal antibodies against copine-8 (CPNE8) protein. Methods Full-length cDNA of CPNE8 gene in human bronchial epithelial cells (HBEs) was amplified by reverse transcription PCR (RT-PCR), and then cloned into prokaryotic expression vector pGEX-4T-1. The recombinant plasmid was transformed into E.coli Rosetta (DE3). The recombinant protein was induced by IPTG, purified by glutathione-sepharose affinity chromatography, and then identified by MALDI-TOF MS. New Zealand white rabbits were immunized with the purified protein to generate polyclonal antibodies. Titer and specificity of the purified polyclonal antibodies were analyzed by ELISA, Western blotting and immunohistochemistry (IHC). Results Prokaryotic expression vector of pGEX-4T-1-CPNE8 was constructed. Recombinant CPNE8 protein was efficiently expressed in E.coli Rosetta (DE3). ELISA showed that the titer was 1:64 000. Prokaryotically expressed CPNE8 protein was detected by Western blotting using the human CPNE8 polyclonal antibody. The expression and distribution of CPNE8 protein in normal human lung tissue and lung cancer tissue was detected by IHC using the human CPNE8 polyclonal antibody. Conclusion We have successfully produced human polyclonal antibodies against CPNE8 protein.