Characterization And Function Analysis Of Porcine Tim1 And Tim3 Genes

Activation of naive CD4~+ T helper (Th) cells results in the development of at least two functionally distinct effector populations, Th1 and Th2 cells. Th1 cells produce the marker cytokines interferon-γ(IFN-γ) and mediated immune responses against intracellular pathogens, inappropriate Th1 response include delayed-type hypersensitivity reactions and induction of autoimmune diseases such as experimental autoimmune encephalomyelitis and type-I diabetes. Th2 cells produce marker cytokines IL-4 and mediate immune responses to extracellular pathogens, inappropriate Th2 cell activation promotes the onset of atopic and allergic diseases. The two T helper subsets also cross-regulate each other's expansion and functions. Predominant induction of Th1 cells can regulate asthma and allergies, whereas preferential induction of Th2 cells inhibits autoimmune diseases. TIM1 and TIM3 were thought to have significant influence on Th2 cells and Th1 cells respectively. In addition to this, some polymorphisms sites that have association with asthma and allergy were also found in TIM1 and TIM3. Thus, the expression regulation and function of TIM1 and TIM3 genes have improtant impact on relative balance between Th1 and Th2 cells and body's immunity. In this study, we cloned the complete CDS of the porcine TIM1 and TIM3 using bioinformatics and comparable genome, several methods including molecular biology and cytobiology were taken to characterize the features of porcine TIM1and TIM3 in both nucleotide sequence and cell levels. The main results were listed as follows:(1) We cloned the complete CDS of the porcine TIM1 and TIM3 using bioinformatics and comparable genome. TIM1 and TIM3 were mapped to porcine chromosome 16 and closely linked to each other. It is presumed that TIM1 include a 1050bp ORF, 8 exons and 7 introns, encoding 349 amino acid residues; TIM3 include a 852bp ORF, 7 exons and 6 introns, encoding 284 amino acid residues. Each of the 5'-donor and 3'-acceptor splice sites conformed to the expected consensus sequences for eukaryotic splice junctions, namely, GT-AG rule.(2)Tissue expression patterns revealed that the porcine TIM1 mRNA expression was low in all tested tissues, whereas TIM3 mRNA expression was markedly different in diverse tissues. Both TIM1 and TIM3 mRNA expression were highest in the spleen and had nearly no expression in muscle.(3) Sub-cellular localization suggested that TIM3 gene was distributed in cellular membrane.(4) Many SNPs were identified within porcine TIM3 gene exon2, exon3, exon5, exon7 and promoter. The SNP by AccI-RFLP in exon2 were selected and association study in our purebred Landrace resource population. The results showed that the SNP by AccI-RFLP in exon2 was significantly associated with red blood cell count (RBC) of neonate piglets at 0 day and 17 days (P=0.0409 and P=0.0089 ); mean corpuscular hemoglobin (MCH) of pigs at 0 days (P=0.0294); packed cell volume (HCT) at 17 days (P=0.0168); Lymphocyte modulus of pigs at 32 days (P=0.033) and Lymphocyte percentage of pigs at 32 days (P=0.048).(5) About 2000bp sizes of the 5'-flanking region of pig TIM3 gene were cloned and constructed to PGL3-Basic vector.