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Study on the Expression and Immunological Characterization of Recombinant Human Type Ⅱ Collagen Polypeptide 250-270

Author: LiXueYi
Tutor: ZhuPing
School: Fourth Military Medical University
Course: Internal Medicine
Keywords: Rheumatoid arthritis(RA) Type Ⅱ collagen(C Ⅱ) peptides rh CⅡ250-270 T lymphocyte proliferation oral tolerance Flow cytometry Cytokine
CLC: R593.22
Type: Master's thesis
Year: 2003
Downloads: 82
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Abstract

Rheumatoid arthritis (RA) is a common human autoimmune disease with a prevalence of about 0.3 % among adults in our country. It is characterized by a chronic inflammation of the synovial membrane which is associated with destruction of cartilage and bone. Several distinct patterns of cartilage loss and bone destroy have been observed in individuals with RA.These include juxtaarticular osteopenia adjacent to inflamed joints and the presence of focal erosions of subchondral bone and at joint margins in areas of direct pannus invasion, leading to a progressive destruction of articular cartilage inflammation in multiple joints. Conventional therapies suppress the immune system nonspecifically and are associated with significant side effects, including infections.Although the pathogenesis is not completely understood, an antigen-driven autoimmune process is proposed to mediated the joint pathology.Type II collagen (CII ) ,which is one of the major components of articular cartilage and provides the tensile strength of this tissue,is a signification autoantigen associated with the development and therapy of RA. Autoantibodies to CII have been detected in the serum of RA patients. Oral tolerance induced by CII and it’s synthesis polypeptide 250-270 was demonstrated to be a nondangerous,effective way for preventing harmful inflammatory in animal model of RA. However, it is more difficult to extract CII from animal Cartilage tissue. Furthermore, the source of material is much limited to supply the demand of basal and clinical application for research and treatment of RA and the other diseases.The objective of this paper is to high-performance express the recombinant polymerized gene encoding human collagen type II polypeptide 250-270 (rh C11250-270) .Then, to explore its ability to stimulate the T cells of PBMC and SFMC in patients with RA. Chemical synthesis and PCR techniques were used to recompose human C11250-270 gene, and the preference codon was adopted in the E.coli. Fusion protein was expressed in pGEX-4T-l and isolated and purified by Glutathione Sepharose affinity chromatography. The relative molecular mass (Mr) of expressed product was 43 KD which is in accord with predicted. The purity of purified fusion protein reached 89 percent assessed by SDS-PAGE gel thin-layer scanning.Assessment of T cell activation has traditionally been performed by measuring proliferation as a function of 3H-thymidine incorporation, or secretion of cytokines from activated PBMC in culture. However, the diagnostic value is often influenced by a diminished signal to noise ratio because of increased background proliferation. Moreover, the significant disadvantage of this type of method is that it does not measure proliferative events in single cells. Thus, a new method for measuring lymphocytic proliferative effect and intracellular cytokines producing by new Flow cytometry assays was establish at the single cell level in this paper. BrdU, an analog of thymidine, was used and incurpo-rated into cellular DNA as a marker of proliferation event in individual cell. After appropriate fixation and permeabilization of the cells, a monoclonalantibody (mAb) against BrdU conjugated with a fluorescent dye is employed to measure the incorporated BrdU by FCM. Cell surface activated phenotype (CD69 or CD25), cell cycle phase and intracellular cytokines producing were detected by 3-color flow- cytometry .The optimal experimental conditions were selected to improve the sensitivity and specifity of assays. This method make it passible to Simultaneous detect of lymphocytic proliferation, phenotype analysis and cytokine production of the individual cells at the single cell level. It may be well used to analyse the proliferative events of mononuclear cell in varies field.To investigate the ability of rhC II 250-270 peptide overlaps, with immunodominant epitopes to stimulate the antigen specific T cell proliferation in the PBMC and SFMC of RA patients.The cellular events that characterize lymphocyte responses to specific stimul

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CLC: > Medicine, health > Internal Medicine > Systemic disease > Autoimmune diseases > Autoimmune diseases, connective tissue disease > Rheumatoid arthritis
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