Expression And Purification Of A Novel Recombinant Chicken C â…¡ Peptide Containing Only Two T-cell Tolerance Epitopes And Study Of Its Preventive Effect On Collagen-Induced Arthritis In Mouse

Rheumatoid arthritis(RA) is a systemic auto-immune disease drived by autoantigens and CD4+CD28+Th1 cells. As the synovitis and arthrentasis in RA are closely related to the immunological function, researchers trend to find some new drugs that can modulate immune system. Oral tolerance is the specific immunological therapy emerged in 1990s. The therapeutic strategy which induces immunological tolerance by oral administration of bovine or chicken typeⅡcollagen(CⅡ) had been proved effective.At present, most CⅡused to treat RA are purified from animal cartilage. So it is difficult to maintain the natural conformation, antigenicity and proper modification of the collagen. As these collagens may be lack of the conformation related epitopes, tolerance induced by them is different from the natural ones. Furthermor, the half-life of CⅡin vivo is so short that it must be used frequently. So all the factors restrict the widely use of nCⅡ. Further research reveal that the functional epitope of CⅡlocated on 260-270aa in CB11 fragment, called collagen tolerance epitope 2(CTE2). Administration of recombinant or synthetic peptides containing CTE2 can improve the condition of collagen-induced arthritis(CIA) and reduce the anti-CⅡantibody level in serum and synovial fluid. The structure of CⅡis similar in different species. CTE2 exist in almost all species, but a unique epitope was found in chicken CⅡ190-200aa, we call it CTE1. All these epitopes can combine with HLA-DR1/DR4 and T cell. The hydrogen bond between HLA-peptide compounds and T cells can be interrupt by remove the side chain of amino acid in TCR epitope or replace the T cell binding amino acid. And then, the antigen present reaction was suppressed. These peptides are called CTE or Altered peptide ligand(APL), which belong to immunological and micromolecular peptides. Treat RA with these CTEs or APLs is considered to be one of the most crucial advancement in recent years. Therapeutic effects of recombinant or synthetic immunological peptides have been confirmed in animal models. Notably, phaseⅠ/Ⅱclinical trials had been completed in America and Canada in 2000, and phaseⅢclinical trial is being carry on. We cloned the DNA fragment coding CTE1-2 from CCOL2A1 and successfully developed the yeasty and prokaryotic expression vector. This research is aimed to optimize the expression and purification of rcCTE1-2. Therapeutic effects of rcCTE1-2 on CIA mouse are also observed meanwhile.To obtain the highly purified rcCTE1-2 peptides, we first optimized the express conditions of rcCTE1-2 in E coli. The results display that 1% inoculate volume, culturing in LB 16h at 37℃, induced 4h at 30℃in 1mM IPTG, we got the highest production of rcCTE1-2. The peptide is expressed in form of inclusion bodies and its level is 41%. When induced at 25℃and 30℃, there are some soluble rcCTE1-2 in supernatant. To deal the E. coli/rcCTE1-2 with 30% sucrose, 3 times of treatment by sonicator, the inclusion bodies can be released totally. After the inclusion bodies were washed by 0.5% Triton X-100,2M NaCl,4M urea twice, denature by 8M urea. The western-blot confirmed that the purified rcCTE1-2 can specific binding to anti-CⅡmonoclonal antibody, which indicated that the epitopes in rcCTE1-2 were still remain the biological activities.As we know, the formation of collagen is a very complex process, which involves post-translational processing, such as glycosylation, hydroxylation and many other kinds of steps. At least eight kinds of enzymes participate in the modification of collagen. P4Hα,βare particularly important, they enable the collagen-specific tripeptide repeat Gly-X-Pro to be hydroxylated on proline residues. Proline hydroxylation under physiological conditions in the formation of stable collagen triple helix is essential. Our research is base on the successfully developing of rcCTE1-2 expression vector in E. coli which lack of post-translational modification compared to eukaryotic system, that is, non-hydroxylation and non-glycosylation. Dose this recombinant collagen peptide has biological function? Whether it can induce immune tolerance effectively in mice or not? What mechanism of the induction of tolerance?All the questions will be most concerned about in our research. Therefore, we used AMMS / 1 female mice to establish the CIA model. The swelling of the limbs, performance of Histopathology, serum anti-CⅡantibody levels in this model are fully consistent with the standards of RA model. In vivo results show that after the 28d of collagen immunization, 50μg/kg rcCTE1-2 and 50μg/kg nCCⅡcan significantly reduce the degree of joint swelling of limbs, and the differences were significant (P <0.05). Compared with the 50μg/kg nCCⅡgroup, 50μg/kg rcCTE1-2 group in regard to inhibited the joint swelling better (P <0.05). Histological analysis showed that, CIA control group marked intra-articular pannus formation, synovial hyperplasia, inflammatory cell infiltration, thinning and loss of articular cartilage, bone marrow cavity distribution of a large number of osteoclasts. 50μg/kg nCCⅡgroup and 50μg/kg rcCTE1-2 group decreased synovial hyperplasia, inflammatory cell infiltration in a significant decrease in precipitation rare fiber material. And there were little fundamental changes in cartilage and bone. Compared with the positive control group other dose group had no significant change. Test of anti-CⅡantibody level in serum showed that compared to positive control group, nCCⅡwith the rcCTE1-2 were able to reduce the level of anti-CⅡantibody, while only 50ug/kg nCCⅡgroup and 5μg/kg, 50μg/kg rcCTE1-2 group got significant difference (P <0.05). Anti-CⅡantibodie is considered to reflect the severity of arthritis of RA and the level of inflammation and the destruction of bone. It confirmed that the rcCTE1-2 without post-translational modification can effectively prevent the occurrence of CIA and the severity of arthritis symptoms.In order to further explore the mechanism of the prevention of CIA by rcCTE1-2, in the 38d after immuned by nCCⅡ, we separated the spleen cells of 50μgg/kg nCCⅡand rcCTE1-2 group and detected the Th3 factor TGF-βand Th1 factor IFN-γin the culture supernatant. The results show that, IFN-γof nCCⅡgroup and rcCTE1-2 group declined significantly compared with CIA group(P <0.01), and rcCTE1-2 group declined more significant than nCCⅡgroup ( P <0.05). In CIA control group, the IFN-γwere significantly higher than the negative control group (P <0.01). Detection of TGF-βshowed that, secretion of TGF-βin nCCⅡgroup and rcCTE1-2 group was significantly higher than that CIA group and negative control group (P <0.01), rcCTE1-2 group was significantly higher than nCCⅡgroup (P <0.05) . These results suggest that, 50μg/kg rcCTE1-2 and nCCⅡare able to regulate the secrete of cytokines, the mechanism may be through reduced Th1-type cytokines, while increase Th2-type cytokines to control and mitigate the occurrence and development of CIA.In short, this research successfully optimized the expression conditions of rcCTE1-2 which contain two tolerogenic epitopes in a prokaryotic expression system ,and got a highly purified rcCTE1-2 through metal chelate chromatography. Further research show that rcCTE1-2 is effective in preventing morbidity and incidence of CIA on mouse. And we also investigate some mechanisms that the rcCTE1-2 may be acted. More deeply research is required of rcCTE1-2 for clinical application future.