Gene Cloning, Expression And Exploration Of Function Of B Cell-Activating Factor Receptor

B lymphocyte stimulator (BLyS) can regulate B cell survival, proliferation, differentiation and maturation. Excessive expression of BLyS can stimulate B cell to proliferate malignantly which results in the occurrence of autoimmunity diseases. BAFF-R is the specific receptor of BLyS which plays an important role in the survival and maturation of B cell. Firstly, the extracellular domain of BAFF-R interacts with BLyS, then the complex would be associated with intracellular TRAF3, and finally activate non-canonical NF-kB signal transduction pathways. External BLyS receptors can remove the excessive BLyS in vivo also. Now the function and signal transduction pathway of BAFF-R become a hot spot for study. The function of BAFF-R was studied from two aspects as follows:1. cDNA encoding the extracellular domain of BAFF-R was gained, then expressed by fusion gene with GST. And the engineered strain and fermentation conditions were optimized, subsequently Western Blotting was used to identify the expected protein. BAFF-R was purified by GST affinity chromatography, the purity of BAFF-R was over 85%.Lastly,BAFF-R can restrain the proliferation of RA-MOS cells, which can be stimulated by BLyS and measured by MTT.2. cDNA encoding the intercellular domain of BAFF-R and TRAF3-C were gained, then the molecular binding domain of their interaction was studied by the yeast two-hybrid. Firstly, the intercellular domain of BAFF-R was inserted in BD plasmid, TRAF3-C in AD plasmid, and then these two plasmids were cotransformed to Y190.The result indicates that they strongly interact. Secondly, eleven deletion mutants of TRAF3 were constructed by deleting the aminoacids residues in N or C terminus of TRAF3 respectively. Thirdly, the mutants and intercellular domain of BAFF-R were cotransformed to yeast. The result indicates that the 382-400aa coiled-coil, 428-463aa in middle, 543-560aa of TRAF3 are critical molecular binding domains for the interaction between intercellular BAFF-R and TRAF3.The extracellular region of BAFF-R protein probably can be used for clinic treatment, and the interaction domain of the intercellular BAFF-R and TRAF3 is the basis for the investigation of the intercellular signal transduction.