Polyclonal Antibody Preparation And Application Of Human CIDE-3 Protein

Apoptosis is a conserved suicide process controlled by many genes that plays critical roles in embryonic development and in the homeostasis. One of the biochemical hallmarks of apoptosis is genomic DNA fragmentation. DNA fragmentation factor (DFF) 40 is the enzymatically active form of the nuclease which plays an important role in DNA fragments. Cell death-inducing DFF45-like effector (CIDE), a novel family of cell death activators, encodes highly related proteins with homology to the N-terminal region of DFF. Among CIDEs, three from human (CIDE-A, CIDE-B and CIDE-3) and three from mouse (CIDE-A, CIDE-B and FSP27) have been reported. CIDE-3 was first reported in 2003. The corresponding sequence of CIDE-3 was present in the genomic clone AC007783, which is about 169 kb long. CIDE-3 has been mapped to human chromosome 3p25. The cDNA is consisted of 1305 bp with alternative splicing of the CIDE-3. The CIDE-3 could induce apoptosis. But, the distribution, physiological and pathological function and the mechanism of inducing apoptosis are not clear. Therfore, in this research, CIDE-3 gene fragment was amplified by RT-PCR and was cloned into the pET28a(+) vector. After identified by restriction endonuclease digestion analysis and DNA sequencing, the vector was transformed into E.coli BL 21(DE3) to express the target protein. CIDE-3 protein was purified and used as antigen to immunize rabbit. The polyclonal antibody was identified and then used to show the distribution of CIDE-3 in various tissues by immuhistochemistry staining.The results of these studies include:1. The prokaryotic expression vector pET28a(+)-CIDE-3 was constructed. The target gene was identical to the published sequence in Genbank through restriction endonuclease digestion analysis and DNA sequencing.2. The 172 amino acids of Human CIDE-3 protein was expressed in E.coli BL 21(DE3) and purified by Ni-NTA Agarose, SDS-PAGE analysis and Western blot showed that purified human CIDE-3 protein was obtained.3. Polyclonal antibody of human CIDE-3 protein was prepared. ELISA analysis showed the antibody with high titer of 1:30000. Western Blot analysis showed this antibody could specifically recognize CIDE-3 protein. Immuhistochemistry staining estimates that human CIDE-3 protein may be located in parietal cells of stomach tissue.4. CIDE-3 immunoactivity was shown in renal tubules (5/5) (2 cortex, 3 medulla immuhistochemistry staining), squamous epithelium of esophaguses (5/5), hearts (4/5), colons (7/10), pacreases (3/5) (1 pancreatic islet, 2 pancreatic gland immuhistochemistry staining), livers (2/5). Brains (0/5) and lungs (0/5) were both negative.5. CIDE-3 immunoreactivity was also demonstrated in hepatocellular carcinomas (HCC) (10/37), gastric cancers (4/19), breast cancers (1/17). Positive rates of human CIDE-3 protein in HCC decreased significantly in comparison with hepatic cells around HCC(P<0.05). And positive expression rates of well differentiated, moderately differentiated and poorly differentiated HCC also decreased significantly in comparison with hepatic cells around HCC(P<0.05).In conclusion, in the present research prokaryotic expression vector pET28a(+)-CIDE-3 was constructed, expressed. The human CIDE-3 protein was purified and a polyclonal antibody was prepared. The distribution of normal tissues and tumor tissues was determined primarily. All of those will become useful tools for further studies and draw a light on the therapy, diagnosis and preventation of tumor.