| During the research on variations of gene expression before or after Ty21a immunization, we found several novel ESTs, which were expressed differently after Ty21a immunization. The sequence of one gene, which is corresponding to EST3 10, was acquired by RACE-PCR and was named Ty21a immunization-associated gene, TIG-310. We blasted it in the GenBank and found one gene that is similar to it. But we did not get any published information about its function. We analyzed its homogenicity to other genes, its ORF, expression profile, locus on the chromosome and so on. We also get some information about the protein coded by the gene. The gene is localized on the 11th chromosome and may be expressed in the stomach and intestine. The gene has one ORF, coding a protein composed of 267 amino acids.To identify the function of TIG-310 in mucosal immune responses, we expressed the gene by pQE30 vector. After induction by IPTG, high level of target protein was expressed in M15 host strain. It was proved that the gene was expressed in stomach and intestine of normal mouse. There was low level of expression that can be detected in the Peyer's patch. Then cells expressing the TIG-310 gene in the intestinal mucosa were analyzed by in situ hybridization. It seems that the gene is mainly expressed in the intestine epithelial cells. In addition, the TIG-310 was fused to GFP and expressed in L cells and COS-7 cells. It seems that the TIG-310 gene may be a cytoplastic protein, for the fluorescence is mainly found in the cytoplasm of the transfected cells. So it was presumed that the gene might be expressed in the cytoplasm of intestine epithelial cells of normal Ba1b/c mouse.The expression of TIG-310 varied in the intestine epithelial cells after Ty21a immunization. We detailed the expression of the gene in stomach, intestine and Peyer's patch by RT-PCR. The results show that the expression of the gene in stomach and intestine was much higher than that in Peyer's patch. The expression in stomach and intestine after first Ty21a immunization was much higher than that after second and third immunization. And the expression in stomach and intestine after second and third immunization was lower than that in normal mice. From these results, we can draw some conclusion that immunization with Ty21a can induce the expression of TIG-310 instomach and intesine. The Ty21a may interact with the intestine epithelial cells and regulate the expression of TIG-310.After the injection of pEGFP-310 plasmid in muscle, there were more inflammatary cells at the injection site. Then the expression of many cytokines associated with inflammation was analyzed by RT-PCR. These results show that the expression of IFN-y and IL-12P40 was upregulated. It implied that TIG-310 might promote the inflammation response by upregulating the expression of IFN-y and IL-12P40.From the above results, we can presume that the TIG-310 may be associated with innate defence of mucosal epithelial cells.To study the function of CD1d in mucosal antigen presentation and the role of NK T cells in mucosal immunity, we need to prepare CD1d tetramer to stain the NK T cells. CD1d tetramer is a complex consisting of four CD1d and four P2m. The baculovirus expression system was selected to express mCD1d and 2m. First, the two genes were cloned into shuffle vector pFastBac Dual one after another. The recombinant plasmid was named as pBD-CDl- 2m. After homologous recombination, the GD1 and 2m were inserted into the Bacmid genome. Then the recombinant Bacmid was transfected into Sf-9 cells by Cellfectin. The expression of target protein was analyzed by SDS-PAGE. But protein expression was not detected.To study the stimulating function of GDI on lymphocytes isolated from different tissues, we detected the proliferation of lymphocytes co-cultured with CHO cells expressing CD1 molecule by MTT method and analyzed the ratio of NK1.1 and CD3 double positive cells. We found out that mCDld expressed on CHO cells could stimulate lymphocytes proliferation isolated from liver and mes...
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