Screening Of The Peptides Binding To Human Soluble B-lymphocyte Stimulator And Analysis Of Their Immunosuppressive Functions

B lymphocyte stimulator (BLyS), also known as THANK, BAFF, TALL-1 or zTNF4, is an essential survival factor for B cells. It can stimulate B cells to mature into antibody-producing plasma cells. BLyS deficiency can result in loss of mature B cells. However aberrant overexpression of BLyS is related close with several autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and Sjogren's syndrome (SS). So BLyS is considered as an attractive target for the treatment of these autoimmune diseases. Research and development of BLyS antagonists has a very potential benefit in illustrating the mechanism of BLyS action and developing novel therapy strategy for BLyS-associated autoimmune diseases.Phage display has been proved to be a powerful tool for analyzing protein-protein interactions.It is a well-established technology that provides for the screening of large peptide libraries and identifing of the peptide binding to the proteins. It has been suggested that small molecule inhibitors are likely to be effective when they are able to interact with key amino acids of the interface.Indeed, it is now possible to find peptides that bind to BlyS by phage display.Peptides binding to BLyS have the potential to be potent antagonists of BLyS and could be exploited therapeutically.Here the purified recombinant human soluble BLyS(112-285 amino acids, rhBLyS_112-285) was prepared first, and subsequently rhBLyS_112-285 binding oligo-peptides were selected and their immunosuppressive effects were analyzed. Following were the brief protocol and the major results.1. Preparation of the purified functional rhBLyS_112-285 E.coli. DH5a containing the rhBLyS_112-285 expression plasmid was harvested after induction by 1 mmol/L IPTG for 4 hours at 37℃ and then sonicated. Analyzing the fractions by SDS-PAGE showed that the desired protein(about 23kD) was successfully expressed and mainly located in inclusionbodies. By using Ni2+-NTA resin affinity chromatography and Sepharcryl S-200 chromatography rhBLySn2-285 was purified. Then the purified protein was refolded under different conditions. SDS-PAGE analysis showed that the purity of the protein was up to 90 percent. Lymphocyte proliferation assay revealed that the refolded purified rhBLySi 12-285 had a good biological activity.2. Screening of rhBLySn2-285 binding oligo-peptides The phage-displayed C7C-peptide library was bio-panned taking purified rhBLyS 112-285 coated on polystyrene plate as the bait. After 3 rounds of bio-panning, specific enrichment of the phage-displayed rhBLySn2-285 binding oligo-peptide was realized. By using ELISA and competitive ELISA 12 positive phage clones were identified.3. DNA sequencing and functional analysis of the positive clones Based the DNA sequences the 12 positive phage clone displaying C7C-peptide sequences could be deduced and classified into three subgroup amino acid sequences. Functional analysis by using MTT assay showed that two of three phage clones could inhibit the activity of BLyS.4. Synthesis and functional analysis of the oligo peptides The oligo-peptides corresponding to the amino acid sequences displayed by the above two positive phage clones were synthesized using FMOC method. And the functional analysis by using MTT assay indicated that the synthetic peptides could suppress the BLyS-stimulated lymphocyte proliferation.In summary, the C7C phage displayed peptide library was bio-panned with purified rhBLyS H2-285, and 12 positive phage clones were selected. Two out of the positive clones could inhibit BLyS activity, and the corresponding synthesized oligo-peptides could also suppress BLyS activity. The in vitro successful selection and identification of the two antagonist peptides of BLyS may pave a way for further study on the mechanism of BLyS action and development of potential novel therapeutic agents for BLyS-associated autoimmune diseases.