| Ankylosing spondylitis (AS) is an articulatory disease with high disability rate and its incidence is 0.2%-6% in Chinese population, so it is a common disease in our country. The disease begins from articulation sacroiliac to neck, and arthrentasis happens in the late phase and leads patients to lose their working capacity and decrease their life quality. For the treatment of AS, no specific and effective treatment has been reported so far. A variety of therapeutic as well as preventional measures have been studied. Therefore, the strategies for AS therapy have been a problem remained to be solved urgently.In 1973, Brewerton and Schlosstein found that there was strong association between HLA-B27 and AS. And later lots of findings give the support for the correlation between HLA-B27 and AS genetically. Experiments also indicate that reduce or block the expression of B27 heaven chain (H-chain) can lessen symptom and cut down the incidence of AS. The mechanism of this treatment is to intercept the presentation function of HLA-B27 molecules by blocking the peptide-binding sites of them, or block the immunopathologic reaction induced by B27 H-chain through interfere the recognition of lymphocyte to B27. In this paper, we used bacteriophage display technology to biospanning for short peptides which can highly specific combind to HLA-B*2704 and B*2705 H-chains, which we call it antagonistic peptide by its function. This method can be better therapic effect and less side-effect compared with non-specific therapies, such as general immune suppressive therapy andantiinflammatory therapy.HLA-B27 antigen is expressed on all the nucleated cells and belongs to major histocompatibility complex class I molecular, which is composed ofα-chain (H-chain) encoded by MHC in the sixth chromosome andβ2 microglobuhn (light chain) encoding by non-MHC encoding region in another chromosome. Its subgroups are from HLA-B*2701 to HLA-B*2725 and B*2704 and B*2705 are the dominant subtypes with the frequencies of 50.85% and 40.68%, respectively, in Chinese Han population. As to Chinese Han AS patients, B*2704 and B*2705 is also the predominant subtypes with the rates of 54.8% and 41.1%, respectively. Therefore, HLA-B*2704 and B*2705 were selected as the research targets.In the first section, we established HLA-B*2704 and B*2705 immortalized cell lines and then cloned and verified their full-length cDNA. Firstly, we obtained the B lymphocytes from HLA-B*2704/B*2705 positive individual peripheral blood and then established HLA-B*2704/B*2705 immortalized B cell lines by transforming Epstein-barr virus (EBV) into these cells. And then a serias biological characteristic study was carried out for the EBV transformed cells. The results demonstrated that immortality was verified in the EBV transformed HLA-B*2704/B*2705 cell lines and malignant transformation features were not deteced with normal karyotypes and no oncogenicity to nude mouse. It maintains the original biological characters of B lymphocytes. Therefore, we established HLA-B*2704/B*2705 specific immortalized cells lines and provided a stable sample bank for the consequency study and international cooperation. The total RNA was extracted from both the normal HLA-B*2704/B*2705 positive B cells and the immortalized cells to obtain the full-length cDNA which was then cloned into pGEM-T vector and verified by DNA sequencing. By sequence similarity blasting with the published HLA-B*2704/B*2705 cDNA sequences in Genbank database, we confirmed our full-length cDNA sequences, which will enable the consequency HLA-B27 H-chain soluble expression in prokaryotic system. The second part is the soluble protein expression of HLA-B*2704 and B*2705 extracellular region of their H-chains in E.coli. The HLA-B*2704/B*2705 extracellular region DNA were amplified by PCR and then inserted into fusion expression vector pET32a-c ( + ) and transformed into Origami host bacterium, and then we got positive clones. After induced by IPTG, the recombinated fusion protein was obtained, and the expression throughput reached to more than 50% of total protein in the positive bacterium. The further studies showed that the ratio between soluble protein and inclusive bodies correlated to induction temperature. In 30°C, the production exits most in soluble pattern, and in 37°C, the most expression form is inclusive bodies. In different temperature, the throughput of soluble protein was in 30°C>25°C>37°C pattern, which may be due to the different dissolvability caused by different bacteria microenvironment changing under different tempreture enviroments. However, the concentration of IPTG made no sense to the soluble protein production. The fusion expression pET32a vector used in this study overcomes lots of defects in procaryon expression system and can gain soluble protein expression with high efficiency. By using this expression system, we gained purified protein by affinity purification, and then cut the target protein from fusion protein by zymoplasm to remove the carrier protein. After purification of the target protein, its purity>80% was procured, and the lymphocytotoxicity reaction mediated by anti-HLA-B27 antibody to B27 positive cells were blocked by the soluble expression products, which provided a relative better target for the next screen for its antagonistic peptide by biospanning phage random peptide library.The third part aims to screen and identify the HLA-B*2704/B*2705 antagonistic peptides. Proteins were coated onto ELISA plate to screen phage displayed random 12 -peptide library, with a 3-round biopanning to enrich the bacteriophagus clone. 20-40 bacteriophagus clones were chosen randomly for ELISA detection. 12 clones for B*2704 and 10 clones for HLA-B*2705 were selected for ssDNA sequencing. 5 binding-peptide sequences for HLA-B*2704 are HTSFCSTHLCLI (×4), QHCSPTLCQIHR (×5), ARCTTTLCYLSN (×1), YGLCTDWYCHIT (×1) and YPLCDAILCRLP (×1). 10 B*2705 binding peptides share 4 sequences: SHCSPHWCALPF(x6), HLCSNSLCLL PW (×2), EPMCSWFWCTLP (×1), WTCSPLLCTWGA (×1). B*2704 and B*2705 binding peptides have a consensus sequence, CS(T)TXXL(W)CXL, which indicates that the peptides have common binding epitope between them. By immunofluorescence analysis, we found that the screened bacteriophage clone could bind HLA-B*2704 and B*2705 cell lines while no binding effect was observed in normal B cell. By flow cytometry analysis, bacteriophage clones of B*2704 binding peptide can bind HLA-B*2704 cell line with positive rate of 43.55%, while bacteriophage clone of B*2705 binding peptide can recognize HLA-B*2705 cell line with a 45.69% positive rate. These results indicate that the B27 binding peptides show some affinity and specificity to B*2704 and B*2705 molecules expressed in cell surface but do not bind to normal B cell.In summary, HLA-B*2704 and B*2705 immortalized EBV-transformed B cell lines were established and their full-length cDNA were cloned and verified by sequencing. The soluble proteins were expressed using prokaryotic expression system and purified. Phage displayed random peptide library was screened and the possible HLA-B*2704 and B*2705 antagonistic peptides were obtained. The expression of HLA-B*2704/B*2705 antagonistic peptides and their possible biological functions were investigated. The role of HLA-B27 antagonistic peptide in reducing AS occurrence may be elucidated further based on these results and it may also provide a new method for AS prevention.
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