Construction Of Human B7-H4 Transfected Cells And Preparation Of Anti-human B7-H4 Monoclonal Antibody

B7-H4 is a new member of costimulatory molecules,Its gene is located on chromosome 1p11.1 of Homo sapiens, encoding 282 amino acid. It belongs to the Ig superfamily. Human B7-H4 mRNA is widely distributed in lymphoid and nonlymphoid tissues. Freshly isolated human T cells, B cells, monocytes and DC do not express B7-H4 on cell surface. In contrast, B7-H4 expression can be induced on T cells, B cells, monocytes and DC after in vitro stimulation. Some research showed that B7-H4 is a negative molecule and regulates T cell response by inhibiting T cell proliferation, cytokine secretion, and cell cycle progression. Although B7-H4 protein can not be detected in most normal tissues, it is highly expressed on some tumor lines, suggesting that B7-H4 can help tumor cells to escape immunological surveillance. B7-H4 also play an important role in immunological tolerance. Therefore, B7-H4 has drawn much more attention recently.This project aims to clone human B7-H4 gene, establish B7-H4 transfected cells and prepare anti-B7-H4 monoclonal antibodies and characterize their biological functions primarily.PartⅠEstablishment of human B7-H4 transfected cells and preliminary study of its biological functionsObjective: To clone human B7-H4 gene and then inserted into the eukaryotic expression vector pIRES2-EGFP to construct human B7-H4 transfectant cell and characterize its biological functions.Methods: Human B7-H4 gene was amplified by RT-PCR.Target gene was digested with restriction endonucleases EcoR I and BamH I,and then was inserted into eukaryotic expression vector pIRES2-EGFP to construct recombinant eukaryotic expression vector pIRES2-EGFP/B7-H4. The recombinant plasmid was transfected into L929 cell line with LipofectAMINETM 2000 and the transfected cells were further selected with G418.Whether the expression vector was inserted into L929 cell line was assayed by flow cytometry analysis and RT-PCR. The biological functions of transfected cells were researched by CD69, CD25 expression detection on activated T cells and T cell proliferation test.Results: A stable cell line expressing human B7-H4 was established. The result of biological functions showed that B7-H4 could down-regulation the expression of CD25, CD69 on actived T cells and inhibited T cell proliferation.Conclusion: The transgenic cell lines stably expressing human B7-H4 molecule was obtained and afford effective immunogen for preparing anti B7-H4 monoclonal antibody.PartⅡPreparation and characterization of anti-human B7-H4 monoclonal antibodiesObjective: To prepare anti-B7-H4 monoclonal antibodies and to characterize their biological functions.Methods: A human B7-H4 transfectant cell line L929/B7-H4 was used as an immunogen to immunize BALB/c mice.By means of the B lymphoma hybridoma technique, immunofluorescent cytometry, repeated screening and multiple subcloning, the hybridoma cell lines specifically secreting anti-B7-H4 mAbs were screened.The fast-strip analysis was used to investigate murine Ig subclass.The specificity of mAbs was determined by Dot-blot and Western blot. Competitive inhibition test was employed to identify mAb binding sites on B7-H4. T cell proliferation inhibition blocking test was used to examine mAb biological function.Results: Two hybridoma cell lines were obtained and named 1F10 and 2B2,respectively.They could secret continuously and stably specific anti-B7-H4 mAbs.The result of Dot-blot indicated that two mAbs could recognize specifically B7-H4, but only mAb 2B2 recognized B7-H4 when using Western blot .The competitive inhibition test showed that two mAbs bound different epitopes on B7-H4.The two mAbs could partially block the inhibitory effects of B7-H4 on T cell proliferation in vitro.Conclusion: Two hybridoma cell lines secreting anti-B7-H4 mAbs were obtained.Obtained two mAbs provided useful tool for further studying B7-H4's biological functions.In summary, the transgenic cell lines L929/B7-H4 and two anti-hunan B7-H4 monoclonal antibodies have been obtained. T cells proliferation test in vitro showed L929/B7-H4 could inhibit T cells proliferation. B7-H4 mAb could partially block the inhibitory effects when mixed with L929/B7-H4, thus providing useful tools for further studying B7-H4'biological functions.