The Preparation Of Human B7-H4-Fc Fusion Protein And The Detection Of The Expression Of B7-H4Receptor

B7-H4molecule is a member of B7family that was newly discovered in recentyears. It could inhibit the proliferation of T lymphocyte and cytokine produce bydepressing the activation of ERK, JNK, p38, AKT, and so on. The genomic DNA ofhuman B7-H4is mapped on chromosome1p11.1, comprised of six exons and fiveintrons. Its mRNA shows an extensive expression pattern in lymphoid andnon-lymphoid tissues, however, the protein of B7-H4only presents weak expression innormal tissues because of the stringent regulation at the post-transcriptional level.Recent studies have shown that freshly isolated human T cells, B cells, monocytes andDC do not express B7-H4on cell surface, but it can be induced to express by activestimulation in vitro. The studies also have found that B7-H4molecules isover-expression on the tumor cells and tumor-associated macrophages in tumor tissuessuch as ovarian cancer, breast cancer, lung cancer, and other malignant cancers. All ofthe evidence indicate that B7-H4over-expression play an important role in tumor cellsescaping from immune surveillance in tumor microenvironment. Although B7-H4receptor has not been identified, the studies of B7-H4receptor is one of hot-spots in theimmunology. The discovery of B7-H4receptor will help us cast new light on the actionmechanism of B7-H4molecule. In addition, it is important to investigate the expressionof B7-H4receptor on the lymphocyte separated from various tissues.PartⅠ The preparation of B7-H4-Fc fusion protein and theresearch of its applicationObjective: To clone the extracellular domain of human B7-H4gene and human Fcgene, construct recombinant retrovirus vectors carrying the extracellular domain ofhuman B7-H4gene and human Fc gene, which can be stably expressed in CHO cell lines, and study the B7-H4-Fc fusion protein’s biological function.Methods: The extracellular domain of human B7-H4gene and human Fc gene,were amplified by PCR. The gene fragments were cloned into pMD19-T vector that wasdigested with two restriction endonucleases, and inserted into retrovirus vectorpEGZ-Term. Constructed and authenticated recombinant vector was namedpEGZ–Term/B7-H4-Fc, which was then expressed in eucaryote. The recombinantvector together with its two helper virus vectors was co-transfected into the package cell293T with Lipofectamine2000. The supernatant of293T was used to infect CHO cells.CHO cells stably expressing B7-H4-Fc fusion protein were selected in the presence ofZeocin, and then analyzed by FCM and Western-blot. The B7-H4-Fc fusion protein waspurified and identified, and its biological function studied.Results: The recombinant expression vectors pEGZ–Term/B7-H4-Fc wasconstructed successfully. After identified by FCM and Western blot analysis, thetransfectants stably expressing human B7-H4-Fc fusion protein on the cells wereestablished successfully, then the fusion protein was purified. In vitro, B7-H4-Fc fusionprotein had an inhibitory effect on the proliferation of T cells. B7-H4receptor wasisolated with the B7-H4-Fc fusion protein by immunoprecipitation.Conclusion: The gene-transfected cell line that could stably express B7-H4-Fcfusion protein was constructed successfully. The purified B7-H4-Fc fusion protein, laysthe foundation for the further study on the biological function of B7-H4molecule.Using the method of immunoprecipitation, B7-H4receptor molecule was isolated.Part II The detection the expression of B7-H4receptor onlymphocyte membrane separated from various tissueObjective：To detect the expression of B7-H4receptor on lymphocyte membraneseparated from various tissue with fluorescein FITC-conjugated B7-H4-Fc fusionprotein.Methods：The B7-H4-Fc fusion protein was labeled with FITC, and then used toexamine the expression of B7-H4receptor on lymphocyte membrane separated fromvarious tissue by FCM.Results：There is a subset of B lymphocyte cells from tonsil constitutivelyexpressing B7-H4receptor, and the actived T lymphocyte cells, T lymphocyte cells from lung cancer tissue and its metastatic lymphonodus also express B7-H4receptor.Conclusion：The constitutive expression of B7-H4receptors on the subpopulationof tonsil B lymphocytes makes it possible to isolate and purify B7-H4receptor proteinfor further research. The expression of B7-H4receptor on T lymphocyte cells from lungcancer tissue and its metastatic lymphonodus suggested that T lymphocytes may bereeducated in the tumor microenvironment to express B7-H4receptor, and interact withB7-H4molecules on the surface of tumor cells, tumor associated macrophage andsoluble B7-H4molecules in tumor microenvironment, then leading to T cell immuneneglect and helping tumor cells to escape immune surveillance and immune response totumor.