The Empirical Study Of Human Perforin And Sperm Protein 17 Radiation-inducible Expression

Spatial and temporal control of gene expression is important in biology empirical study and gene therapy. Early growth response-1(Egr-1) promoter has 6 highly conservative motifs, inducing the downstream gene expression by radiation. Follow the feature, the Egr-1 promoter is ligated upstream of cDNA encoding human perforin and Sperm protein 17, to construct radiation-inducible expression cell model.Perforin is thought to be a major mediator resposible for the cytolytic of CTL and NK cells. Perforin plays an important role in the tumor immunity. Perforin is glycosylated protein, prokaryotic expression system failing glycosylation may affect its activity. We apply eukaryotic expression system to study cytotoxic activity of perforin. Full-length cDNA of perforin was obtained by PCR, then inserted into pMD18T Vector and subcloned to pcDNA3.1(+) vector to construct recombinant eukaryotic expression vector pcDNA3.1(+)/pfn. The recombinant plasmid was transfected into COS7 cells. Expression of pfn gene in the transfected cells was detected by RT-PCR and immunocytochemistry. Perforin full-length cDNA was correctly cloned and sucessfully inserted into pcDNA3.1(+) vector. Expression of pfn mRNA in the transfected COS7 cells was confirmed with RT-PCR, but PFN protein can't be detected. To chang the CMV promoter with Egr-1 promoter, construct radiation-inducible expression vector pEgr-pfn. In the model, this pEgr-pfn construct was transfected into COS7 cells. Expression of pfn mRNA in the transfected COS7 ce11s was confirmed with RT-PCR, but PFN protein can't be detected.Sperm protein 17 (Sp17) is a protein recently identified as a novel CT antigen. It is a highly conserved mammalian protein expressed in developing spermatozoa and mature spermatids, and reported to play a role in the interaction of sperm with the zona pellucida. Sp17 is abundantly expressed in human testis,but not in other normal tissue. The restricted normal tissue expression and aberrant expression in carcinomas of Sp17 makes it a promising suitable target for diagnosis and immunotherapy of ovarian carcinoma. We aim to establish ovarian cancer cell model of Sp17 radiation-inducible expression. To construct a recombinant plasmid pEgr-Sp17, to investigate the expression of Sp17 induced by radiation in HO8910, and to explore whether Egr-1 induced expression of gene downstream and analysis the influence of Sp17 expression to ovarian cancer cell. pEgr-Sp17 vector that included Egr-1 and Sp17 gene was constructed by restriction enzyme digestion, ligation and transfected into HO8910 cells by lipofectamine-mediated transfection. The expression of Sp17 after X-ray irradiation was detected by immunocytochemistry and western blot. Sequencing and restriction enzyme digestion showed pEgr-Sp17 was correctly constructed. The HO8910 cells transfected recombinant plasmid showed positive Sp17 expression. The stably hSp17 gene transfected HO8910 cells were obtained through G418 screening,immunocytochemistry assay and western blot were further performed to confirm the expression of hSp17 in the HO8910 cells. These results provide a good hSp17 protein expression system for the study of the hSp17 in OC and lay foundation for further study of hSp17 for the diagnosis and immunotherapy target of ovarian carcinoma.In conclusion, this tentative successfully construct two radiation-inducible expression vector and construct radiation-inducible expression ovarian cancer cell model.