| Human platelet and T cell activation antigen 1 (PTA1), mainly expresses on platelet and activated T cells, was discovered in 1985. The cDNA of human PTA1 was cloned in 1997, and the molecule was designated as CD226 in the 7th Workshop and Conference on Human Leukocyte Differentiation Antigen (HLDA7). Many researches have done on its structure, distribution, function as well as the relationship with clinic diseases since its discovery. It was shown that human PTA 1/CD226 plays a key role in the T cell differentiation, as well as platelet activation and aggregation. It was also found that human PTA1/CD226 is the member of IgSF, and is a signalling molecule. Recently, human PTA1 was confirmed in the formation of immune logical synapse. Although its ligand has not been cloned, it was demonstrated that many cell lines express the ligand of human PTA1, such as Colo205 and HUVEC. Furthermore, some molecules may associate with human PTA1, including LFA-1,4.1G, hDlg and Rapl. Finally, human PTA1 has close relationship with such diseases as platelet function abnormality, autoimmune disease, graft versus host reaction, virus infection and tumour. In short, human PTA1 is found to be a very important molecule with essential biological function and potential clinic application.In the present study, based on the findings of human PTA 1 we cloned mouse PTA 1 cDNA, and explored its expression, distribution, molecule characteristics, as well as its function. The major results are as follows:By searching the EST database of GenBank, we found a sequence which had 51% of homology with human PTA1 at the amino acid level. And then, specific primers were designed and synthesized according to this sequence, and the full length of mouse PTA1 was cloned from the thymus of BALB/c mouse by the method of RACE. Unexpectedly, three isoforms were also obtained. It was found that the ORF of mouse PTA1 has 1002bp, encoding 333 amino acids, which is 3aa shorter than that of human PTA1. Compared with human PTA 1, mouse PTA 1 has 67% identity at nucleotide level, and 53% identity at amino acid level. Mouse PTA1 is also a transmembrane molecule, with four Cystines in the extracellular region forming two V-like Ig fold. The phosphorylation sites and signalling molecules binding sites can be found at its cytoplasmic region. Finally, mouse PTA1 gene is located at chromosome 18, composed of 7 exons. Besides, from thegenomic sequence it was confirmed that the three isoforms comply the rules of RNA alternative splicing.In order to obtain the antigen for antibody preparation, the extracellular region of mouse PTA1 was cloned into eukaryotic expression vector pSecTag2B, and the construct was transfected into COS7 cells. mPTAl-Myc fusion protein was purified from the expression supernatant by anti-Myc affinity column. Rabbit anti-mPTA 1 serum was obtained via immunizing New Zealand Rabbit with mPTAl-Myc fusion protein. At the mean time, the extracellular region of mouse PTA1 was cloned into prokaryotic expression vector pGEX-4T-1, and the construct was transferred into E. coli. BL21. GST-PTA1 fusion protein was obtained from the bacteria induced by IPTG, and used to immunize BALB/c mouse. Finally, mouse anti-mPTAl monoclonal antibody hybridoma was set up by fusion of immunized splenocytes with Sp2/0 cell lines. It was demonstrated that the rabbit antiserum can recognize natural mouse PTA 1, therefore can be used in the experiments of immunofluorescent staining, immunoprecipitation, as well as functional study. Whereas monoclonal antibody 7G10 can recognize denatured mouse PTA1, therefore can be used in Western Blot.In order to explore the cell and tissue distribution of mouse PTA1, several experiments were performed. It was found from the results of RT-PCR that mRNA of mouse PTA1 is present in immunological tissues, such as thymus, spleen and lymph nodes. Whereas mRNA of mouse PTA1 was positive in EL-4 and P815 cell lines. Furthermore, it was shown from the in situ hybridization that mouse PTA1 positive cells are located at periarterial lymp...
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