Effect Of DNAM-1-nectin-2 Signal Pathway In Triggering Xenogeneic Cytolytic Activity Of Human NK Cells Against Porcine Endothelial Cells

PartⅠ.Bioinformatics prediction,cloning and characterizationof pig nectin-2Objective To confirm the existence of porcine nectin-2 gene,and to clone the novelgene followed by analysis for the molecule structure properties and expression pattern.Methods The pig expressed sequence tags(EST) homologous to human nectin-2 wereobtained from GenBank database using bioinformatics method.By the way of RT-PCR or3'RACE techniques,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 anddistribution pattern of the gene was analyzed.Chromosomal localization was performed bymeans of somatic cell hybrid panel(SCHP).According to the inverse PCR principle,theunknown sequence of 5' control region of the gene was amplified.The three-dimensionstructure model of porcine nectin-2 ectodomain was built referring to the tertiary structureof human PVR ectodomain using biological sequence analysis softs and database.Results Two different isoforms,pig nectin-2αand -2δ,with identical extracellularregion and unique transmembrane region and cytoplasmic tail region were identified by sequencing and homology analysis.The nucleotide sequences of the two splicing variantswere submitted to NCBI database and confirmed as novel genes and the GenBankaccession numbers were EF 195266 and EU069826.Nectin-2 was ubiquitously expressed invarious cells and tissues including SV-40-PED with high expression level。Pig nectin-2 wasmapping to chromosome 6q21.A 371bp long unknown sequence was definited in 5'uncoding region of nectin-2α.The three-dimension structure model showed that the pignectin-2 protein contained three Ig-like loops V-C-C in the extracellular region and the Vlike domain was folded into a secondary structure with nineβ-strands.Conclusion Simultaneous utilization of bioinformatics method and molecular biologytechniques will conduce the prediction and probation of novel porcine genes,and thepresumption about pig nectin-2 is confirmed by the very way.Identification of the structureinformation of pig nectin-2 is essential to further investigat the immunologic function ofthe gene.PartⅡ.Construction,expression and purification of pignectin-2ED-IgGFc fusion proteinObjective To construct extracellular region fusion protein of pig nectin-2(nectin-2ED-IgGFc) which can simulate the natural nectin-2 molecular to interact withDNAM- 1.Methods The extracellular region of pig nectin-2 was amplified from SV-40-PEDusing RT-PCR and cloned into T vector,then subcloned into eukaryotic expression vectorCD51negl after sequencing.The recombinant plasmid nectin-2ED-CD51negl and the blankvector CD51negl were transfected into CHO cells respectively followed by puromycinselection.CHO cells which can stablely secrete nectin-2ED-IgGFc fusion protein andIgGFc 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 A+G affinity chromatography.Results The recombinated plasmid nectin-2ED-CD51negl was constructed andverified successfully by double enzyme digestion and sequencing.After puromycinselection,CHO cells which can stably secrete nectin-2ED-IgGFc fusion protein and IgGFcwere obtained,and highly purified ectogenic proteins were isolated from the CHO cellsculture supernatants.Conclusion The nectin-2ED-IgGFc fusion protein was obtained successfully and canbe used to investigate the interaction between pig nectin-2 and human DNAM-1.PartⅢ.Study about the effect of DNAM-1- nectin-2 pathway inmediating Xenogeneic cytolytic activity of human NKcells against porcine endothelial cellsObjective To identify the heterogeneic binding ability between pig nectin-2 andhuman DNAM-1.To establish a suitable empirical method to estimate the cytotoxic effectof human NK cells against porcine endothelial cells objectively,and contrast theheterogeneic kill efficiency between different human NK cell lines including NK92 andYT.Methods The affinity ability between nectin-2ED-IgGFc fusion protein and DNAM-1was detected by flow cytometry and co-immunoprecipitation techniques.The expressionlevel of DNAM-1 of NK92 and YT cell lines was compared by semiquantitative analysisusing Western Blot.The binding efficiency of fusion protein and anti-DNAM-1 neutralizingantibody to YT cells was tested by flow cytometry.CFSE/PI double labeling method wasestablished in flow-cytometric NK cells cytotoxicity assay to compare heterogeneic killingability against SV-40-PED between NK92 and YT cell lines.Results The fusion protein could bind specifically to human DNAM-1 expressed onYT cell surface.There was no significant difference of expression level of DNAM-1 between NK92 and YT cells.After 10μg/ml anti-DNAM-1 neutralizing antibody incubatingwith YT cells at room temperature for 1 hour,the binding rate of the antibody to YT cellswas 48.53%.Adopting CFSE/PI double labeling method,when the effctor/target ratio was10:1,coincubating time was 5h,6h and 7h,the detected cytotoxicity of NK92 againstSV-40-PED was 44.68%,53.99% and 57.44% respectively and that of YT was 20.44%,20.60% and 26.71% respectively.Conclusion Identify the specifical conjunction between pig nectin-2 and humanDNAM-1 which is the important precondition in studying the effect ofDNAM-l-nectin-2 passway.CFSE/PI double labeling method can test the kill ability of NKcells conveniently and objectively.Under the same condition,NK92 shows higherheterogeneic cytolytic activity comparied with YT.All the data establish a favourablefoundation for further investigation of the potency of DNAM-l-nectin-2 pathway inmediating human NK cells heterogeneic cytotoxic effect against pig endothelial cells.