Expression of B7-H4 gene from mouse in eukaryofic system and its suppressive effect on proliferation of lymphocytes cell

Objective To provide subjects for lucubrating the effect of B7-H4 on T cell activation and graft rejective reaction through cloning and constructing the eukaryotic expression vector encoding the gene of extracellular region of B7-H4 from mouse,and investigate the effect of B7-H4 on the proliferation of lymphocytes in vitro.Method The total RNAs of mouse lung and spleen were extracted and cDNA was transcribed from RNA using RT-PCR technique.The gene of extracellular region of B7-H4 was amplified according to the template of cDNA by PCR.The amplified cDNA was imported into pGEM-T Easy vector to construct TA-mB7-H4 plasmid.The plasmid was cut by restriction enzyme of XBaI and HindIII and was identified by the agarose gel electrophoresis and sequence scanning.Then the mB7-H4 corroborated by sequencing was inserted into the fluorescence expression vector MYC-HIS-EGFP-N after cut by the restriction enzymes to construct B7-H4-EGFP and control-EGFP eukaryotic expression vectors.The recombinant plasmids were transfected into JM109 competence bacteria,and were extracted and identified by the agarose gel electrophoresis and the sequencing after cut with the restriction enzymes.They were transfected into CHO cell through lipofectamineTM 2000,and the CHO cell lines expressing stably the fusion protein were obtained through G418 selection.MTT colorimetry was used to assess the effect of B7-H4 on the proliferation of lymphocyte in the culture of lymphocyte from BALB/c or C57 mouse respectively and in co-culture of lymphocyte from both BALB/c and C57 mouse.Result The gene sequences of B7-H4 cDNA cloned from mouse and TA-mB7-H4 constructed were correct by sequencing.The transfective CHO cells stably expressed the recombinant transmembrane B7-H4 protein.The B7-H4 protein suppressed the lymphocyte proliferation either in the culture of lymphocyte respectively and in co-culture of lymphocyte from BALB/c and C57 mouse.Conclusion The B7-H4 eukaryotic expression vector was constructed successfully and can suppress recombinant B7-H4 protein with biological activity.It lay the foundation for further studies on the role of B7-H4 in transplant rejection and T cell activation.