Effect Of Porcine Indoleamine 2,3-dioxygenase In Triggering Xenogeneic Cytolytic Activity Of Human Serum Against Porcine Endothelial Cells

Partâ… . Bioinformatics prediction, cloning and characterization of porcine IDOObjective To confirm the existence of porcine IDO gene, and to clone the novel gene followed by analysis for the molecule structure properties and expression pattern.Methods The porcine expressed sequence tags(EST) homologous to human IDO were obtained from GenBank database using bioinformatics method. By the way of RT-PCR, the gene was cloned from porcine endothelial cell line(SV-40-PED) and checked for the accuracy of the nucleic acid sequence, then the expression and distribution pattern of the gene was analyzed. Chromosomal localization was performed by means of somatic cell hybrid panel(SCHP). The three-dimension structure model of porcine IDO was built referring to the tertiary structure of human IDO ectodomain using biological sequence analysis softs and database.Results Porcine IDO was identified by sequencing analysis. The nucleotide sequences were submitted to NCBI database and confirmed as a novel gene. Porcine IDO was expressed in lung,thymus,epididymis and anterior chamber with basic expression level, however in PBMC with high expression level. Chromosomal localization and three-dimension structure model of porcine IDO is yet to be done. Conclusion Simultaneous utilization of bioinformatics method and molecular biology techniques will conduce the prediction and probation of novel porcine genes, and the presumption about porcine IDO is confirmed by the very way. Identification of the structure information of porcine IDO is essential to further investigate the immunologic function of the gene.Partâ…¡. Construction, expression and purification of porcine pcDNA 3.1(-)-poIDO and pCMV-Tag2B-poIDO fusion proteinObjective To construct the expression vector (pcDNA 3.1(-)-poIDO) and fusion protein of porcine IDO (pCMV-Tag2B-poIDO) which can simulate the natural IDO molecular.Methods The open reading frame of porcine IDO was amplified from SV-40-PED using RT-PCR and cloned into T vector, then subcloned into eukaryotic expression vector pcDNA 3.1(-) and p CMV-Tag2B after sequencing. The recombinant plasmid pcDNA 3.1(-)-poIDO and the blank vector pcDNA 3.1(-) were transfected into PED cells respectively followed by G418 selection. The recombinant plasmid pCMV-Tag2B-poIDO and the blank vector pCMV-Tag2B were transfected into CHO cells respectively followed by G418 selection. PED cells which can stablely secrete pcDNA 3.1(-)-poIDO protein were identified by the way of Western Blot, immunocytochemistry, immunofluorescence and flow cytometry. And CHO cells which can stablely secrete pCMV-Tag2B-poIDO fusion protein and Tag2B were identified by the way of Western Blot, immunocytochemistry, immunofluorescence and flow cytometry. The fusion protein was purified from the supernatants of the CHO cells culture by protein affinity chromatography.Results The recombinated plasmid pcDNA 3.1(-)-poIDO,poIDO-pCMV-Tag2B was constructed and verified successfully by double enzyme digestion and sequencing.Conclusion The vector poIDO-pCMV-Tag2B was obtained successfully and can be used to investigate the interaction between porcine IDO and human serum.Partâ…¢. Study about the effect of poIDO in mediating Xenogeneic cytolytic activity of human serum against porcine endothelial cellsObjective To establish a suitable empirical method to estimate the cytotoxic effect of human serum against porcine endothelial cells objectively.Methods The effect of 1-MT triggering human serum against porcine endothelial cells was detected by flow cytometry techniques. PI labeling method was established in flow-cytometric human serum cytotoxicity assay.Results The xenogeneic cytolytic activity of human serum against pocine endothelial cells was inhibited by 1-MT.Conclusion The xenogeneic cytolytic activity of human serum against pocine endothelial cells was up-regulated by IDO. PI labeling method can test the kill ability of human serum conveniently and objectively.