The Study Of BLyS Antagonists And Mutant

B lymphocyte stimulator(BLyS)is essential in B cell homeostasis and immunoglobulin secretion.BLyS plays a key role in B cells by binding to its receptors.BLyS regulates the expression of the gene by a series of signal transduction.BLyS overexpression is associated with autoimmune diseases such as sj?gren’s syndrome,lupus and rheumatoid arthritis.Thus,it is being a research hotspot to treat the diseases by blocking the BLyS activity and decreasing the level.We previously obtained the BLyS antagonist peptides BC and TA by computeraided drug design.To maintain the stability of the peptide,human IgG1 Fc fragment was fused to BC/TA to form BC/TA-Fc fusion protein.BP-Fc without a His tag possesses the identical sequence as BC-Fc.The 3-copy of BC(3-BC)fused with Fc was also constructed.BLyS-binding peptide 814 selected from the phage display library was also fused with Fc.In this study,the activities of the above fusion proteins were compared and the binding sites were analyzed.Moreover,the novel fusion proteins 814-TA-Fc and 814-BC-Fc were obtained and their bioactivities were investigated.The BLyS mutant gene was constructed and the activity of the mutant was analyzed.The following were results:1.The fusion proteins BC-Fc,BP-Fc,3-BC-Fc and 814-Fc were purificated.The direct ELISA result showed that 814-Fc had higher binding activity and BC-Fc had lower activity compared to other proteins.The indirect ELISA result revealed that 814-Fc had better competitive activity and BP-Fc had lower activity compared to other proteins.2.The result of peptide 814 inhibiting the BP-Fc binding to BLyS showed that peptide BC and 814 owned the same BLyS binding site,but shared the detached orientations.3.The recombined vectors pET30a-814-TA-Fc and pET30a-814-BC-Fc were constructed,the ELISA result revealed that they two had the binding activity to BLyS,but not better than 814-Fc.4.BLyS mutant with key amino acid mutation was constructed and expressed,the ELISA result revealed that it had lower activity compared with BLyS.