Studies Of BAFF Antagonist Peptides

BAFF(B-cell-activating factor) is B lymphocyte stimulator, belonging to thetumor necrosis factor (TNF) family. BAFF is essential for B cell survival,maturation， antibody secretion, and is critical for normal immune. It’s reported thatthere are some relations between BAFF deregulation and diseases, such asimmunodeficiency diseases, systemic lupus erythematosus, infection and therioma.There exist different BAFF antagonists: anti-BAFF antibody, BAFF receptorinhibitor, immunotoxin of BAFF. BAFF monoclonal antibody-Belimumab hasreceived FDA’s approval for treating SLE, which is a ratification of the feasibility ofBAFF-targeting therapies. However, there are still many problems to be solved forclinical applications of BAFF antagonist, such as, stability and side effects. The mainpurpose of this subject is to study the inhibition activity of three peptides on theinteraction of BAFF with its receptors, to explore the feasibility of using lowmolecular peptide as BAFF antagonist, and to provide a reference for BAFFantagonist peptide research and clinical trial. Three peptides used in our study,TA,TC and BC, were designed by Insight II2000software and molecular dockingmethod, according to the key amino acid residues related to the interaction ofTACI-BAFF, BCMA-BAFF bindingIn this study, PCR of bacterial colonies, enzyme digestion and gene sequencingwere used to verity the correctness of recombinant TACI-Fc and BCMA-Fc. Aftertransferred to the prokaryotic expression host cells, TACI-Fc and BCMA-Fc wereexpressed and purified by protein A column, fusion proteins were gained. Thebinding activity of fusion proteins and BAFF was investigated by indirect ELISA.And, competitive ELISA was used to study the inhibition activity of peptides on thebinding of BAFF-TACI and BAFF-BCMA. The results are as follows:(1)Verify the correctness of recombinants pET28a-TACI-Fc andpET30a-BCMA-Fc, and successfully express the two fusion proteins in prokaryoticsystem.(2)Investigate the binding activity of fusion proteins and BAFF, and determinethe best saturation concentration of binding of BAFF-TACI-Fc andBAFF-BCMA-Fc,5ng/μL and30ng/μL, respectively.(3)Competitive ELISA test proved that the three peptides, TA, TC and BC, hadinhibition activity on the binding of BAFF-TACI and BAFF-BCMA, in certainextent. And，the mix of two peptides had increased the inhibition activity, inhibition rates were increased by the increase of the concentration of peptides. When theconcentration was100ng/μL, the inhibition rate of TA, TC, BC, TA+BC, TA+TCand BC+TC on the binding of BAFF-BCMA was42.2%,44.8%,48.6%,46.4%,46.6%and56.3%; and on the binding of BAFF-TACI was30.8%,40.9%,40.3%,34.6%,43.9%and44.6%, respectively. A good foundation was made for the furtherstudy of this three antagonist peptides.