| Department of Immunology, Fourth Military Medical University, Xian, 710032 ABSTRACT Platelet and I cell activation antigen 1 (PTA1) is a novel molecule whose cDNA was cloned in 1997, and was designated as CD226 in the 7~ workshop and conference on human leukocyte differentiation antigen (HLDA7). A great deal of research has performed on the CD226 structure, distribution, function as well as its relationship with some diseases since its discovery in 1985, and it is shown that: (I) PTA1 is a member of IgSF with two Ig V-like domain in its extracellular region; (2) PTA1 is expressed on activated I cell, NK cell, megakaryocyte/platelet lineage, as well as activated endothelial cell; (3) PTA 1 plays roles in T cell activation and differentiation, platelet activation and aggregation as well as its signal transduction; (4) PTA1 has a close relationship with such diseases as platelet function abnormal, autoimmune disease, graft versus host reaction, virus infection and tumor. In short, PTAI is found to be a very important molecule with essential biological function and potential clinic application. It is a common way to study a molecule along the following procedure: first, exploring the modulatory function of this molecule on human physiological function, based on the knowledge of this molecule structure; second, identifying and cloning the ligand of this molecule; third, observing the signal transduction of this molecule. Department of Immunology, FMMU Since PTA1 plays a key role in the physiology and pathology procedure of human body, it is essential to identify its ligand. However, the cloning of PTA1 ligand (PTA 1L) is unsuccessfi,1, moreover, publications on PTA1L are very rare. Therefore, the purpose of the present study is to identify PTA 1 L, which can lay a foundation for the PTA 1 L cloning, as well as for the further research on physiology and pathology function of PTA1-PTA1L, and their signal transduction. Firstly, we purified monoclonal antibody LeoA 1 against human PTA 1, and then coupled it to Sepharose 4B to prepare affinity column. By using the column, we massively purified PTA 1/Ig fusion protein from the supematant of transfected stable cell line CHO. Secondly, with the help of flow cytometory scanning, we observed the distribution of PTA1L on different cell lines. It was shown that the distribution and expression level of PTA 1 L varied according to different cell lines, among which, the expression level on human endothelial cell line ECV3O4 was highest (67.8%). Finally, we surveyed the blocking activity of a panel of mAbs against PTA I on the binding of PTA I hg to PTA1L, and found that LeoAl could block this binding completely, which suggested that the epitope recognized by LeoAl on PTAI was possibly the same or adjacent one with which PTA1 could bind to its ligand. In order to determine the molecule weight of PTA IL, we Iabled ECV3O4 cell line with biotin. And then lysed the cells. After preclearation with Protein A and human IgGl, immunoprecipitation was perfonned by using PTA 1/Ig fusion protein. Finally, Western Blot was conducted by using avidin-HRP conjugate. It turned out that PTA1L was possibly a protein of 22OkDa. Besides, we also explored the modulatory function of PTAI on the mixed lymphocyte culture (MLC) after its functional epitope was blocked. The secre...
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