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

B7-H4, also known as B7 x or B7S1, is a new member of the B7 family recently discovered, which plays an important role in the negative regulation of T cell mediated immune response. It mRNA is widely expressed in human tissues, including placenta,liver, skeletal muscle, kidney and so on, while the expression of B7-H4 protein on the cell surface is limited. The study found that B7-H4 is expressed in many kinds of tumor tissues, including lung cancer, ovarian cancer, gastric cancer and so on. It is considered as a prognostic marker of cancer, which is closely related to the occurrence and development of tumor. At present, B7-H4 receptor has not yet been cloned. Sica constructed B7-H4.Ig fusion protein and separated health fresh human T lymphocyte,found B7-H4.Ig could not bind to it, but after activated B7-H4.Ig could bind to20%-30% of T lymphocytes, confirming the expression of B7-H4 receptors on activated T cells. At present, though it’s unclear how B7-H4 and B7-H4 receptor interact and how signal pathway changes down-regulate T cell activation, proliferation and cytokine production, but the literature reported that B7-H4 molecules binding to its receptor could inhibit the activity of ERK, JNK, p38 and AKT molecules, thereby affecting the activation of T cells.B7-H4 receptor in lung cancer tissue infiltration of T cells, lung cancer cells metastasis lymph node derived T lymphocytes and inflammatory infiltration of the tonsil cells was detected with B7-H4-Fc fusion protein and flow cytometry in our laboratory before. On this study, we used Magrose-NHS beads and hydrogen bromide activated sepharose 4B affinity column chromatography to purify B7-H4 receptor protein from the tonsil and Jurkat cells. After SDS-PAGE, Coomassie brilliant blue staining and decolorization, target protein band was observed. The expression and localization of B7-H4 receptor in Jurkat cells and inflammatory infiltrate tonsil byimmunohistochemistry and immunofluorescence provided basis for future exploring the expression of B7-H4 receptor in tumor infiltrated T cells.Part Ⅰ: Isolation and identification of human B7-H4 receptorObjective: To purify the B7-H4 receptor and test it. The purified B7-H4 receptor is used for future study.Methods: Through Magrose-NHS beads and hydrogen bromide activated sepharose 4B affinity chromatography column, we separated B7-H4 receptor protein from the tonsil cells and Jurkat cells and observed the target protein bands by SDS-PAGE, Coomassie brilliant blue staining, decolorization.Results: We successfully isolated and purified B7-H4 receptor from tonsil and Jurkat cells lysates, respectively, by magnetic Magrose-NHS and hydrogen bromide activated sepharose 4B-10A10 column chromatography. After SDS-PAGE, Coomassie brilliant blue staining and decolorization, target protein band above 170 KD was detected. The purified B7-H4 receptor protein coated on plate could bind to B7-H4-Fc-Bio and 10A10-Bio to produce positive results, which showed the statistical significance(P < 0.05) by graph pad prism analysis.Conclusion: We separated and purified B7-H4 receptor protein through Magrose-NHS beads and hydrogen bromide activated sepharose 4B-10A10 affinity column chromatography and lay the foundation for the further study of biological function of B7-H4 receptor.Part Ⅱ: Detection of the expression of B7-H4 receptor in cells and tissues by B7-H4 receptor antibody 10A10Objective: To study B7-H4 receptor in tonsil tissue and Jurkat cells for providing the basis of exploring the expression and localization of B7-H4 receptor cells in tumor infiltrated T cells in the future.Method: Jurkat cells and the frozen sections of the tonsil were prepared to detect the expression and localization of B7-H4 receptor by immunohistochemistry and immunofluorescence assay.Result: The immunohistochemistry staining results showed that the brown areawere scattered in the surface membrane of Jurkat cells and the amygdala, and that brown granules were diffuse distribution in the tonsil frozen sections. The immunofluorescent staining results showed that red fluorescence was detected on the membrane of the Jurkat cells, separated tonsil and tonsil frozen section.Conclusion: B7-H4 receptor were distributed in Jurkat cells, amygdala cells and frozen tonsil. It located on the cell membrane.