Study Of Human B7-H4 Receptor And Anti-B7-H4 Receptor Monoclonal Antibody

As a negative costimulatory molecule, B7-H4 is a member of B7 family that regulates the adaptive immune response by inhibiting T cell proliferation, activation and cytokine production. Human B7-H4 belongs to the type Ⅰtrans membrane protein. Comparing with other B7 family members, it has a 20–30% amino acid homology in the extracellular portion. The genomic DNA of human B7-H4 is mapped on chromosome 1p11.1, comprised of six exons and five introns. Its m RNA shows an extensive expression pattern in lymphoid and non-lymphoid tissues. However, the protein of B7-H4 only presents weak expression in normal tissues because of the stringent regulation at the post-transcriptional level. Recent studies have shown that freshly isolated human T cells, B cells, monocytes and DC do not express B7-H4 on cell surface, nevertheless,it can be activated by stimulation in vitro. The studies also have found that B7-H4 molecules is over-expressed on the tumor cells and tumor-associated macrophages in tumor tissues such as ovarian cancer, breast cancer, lung cancer and other malignant cancers. All of the evidence indicates that B7-H4 over-expression play an important role in tumor cells escaping from immune surveillance in tumor microenvironment. Although B7-H4 receptor has not been identified, studies of B7-H4 receptor has been one of the most important area in immunology. The discovery of B7-H4 receptor will help us cast new light on the action mechanism of B7-H4 molecule. Using the flow cytometry, we found that there is a subset of lymphocytes from tonsil constitutively expressing B7-H4 receptor. Furthermore, T lymphocyte cells from lung cancer tissues and its metastatic lymphonodus also express B7-H4 receptor.In this study, utilizing immune precipitation, we purified B7-H4 receptor by B7-H4-h Fc fusion protein. In addition, a primary characterization test was carried and based on this receptor, two suspect anti- B7-H4 receptors founded recently by us were determined.Objective:Obtain B7-H4 receptor, and use the B7-H4 receptor to test anti-B7-H4 receptor antibodies.Methods:An affinity column was prepared with B7-H4-h Fc fusion protein then B7-H4 receptor protein was separated from tonsil cells. After electrophoresis, observe the band and the interaction with goat Anti-human HRP and goat anti-mouse HRP. ELISA detect the B7-H4 receptor after labeling the suspect anti-B7-H4 receptor and B7-H4-h Fc fusion protein with biotin.Results:After SDS-PAGE, a electrophoretic band about 170 k D was observed. ELISA also shows that the B7-H4 recepor could react with goat Anti-human-HRP and goat anti-mouse-HRP. In addition, with the receptor protein was covered with plates, identified by ELISA, monoclonal antibody 10A10 and B7-H4 receptor showed positive reaction.Conclusion: We have successfully got the B7-H4 receptor and utilizing this receptor a preliminary appraisal for two suspect anti-B7-H4 receptors was carried. Finally, we got a well anti-B7-H4 receptor monoclonal antibody. No Doubt, this research has a lot advantage for further study of B7-H4 receptor.