Study on the expression and immunological characterization of recombinant human type II collagen polypeptide 250-270
Objective To express the recombinant polymerized gene encoding human collagen type Ⅱ polypeptide 250-270(rhCⅡ250-270), so as to obtain the recombinant polypeptide with high-performance expression and convenient purification. Moreover, to explore its ability to stimulate T cells of PBMC and SFMC in patients with rheumatoid arthritis (RA). Methods Using chemical synthesis and PCR techniques, constructing human CⅡ250-270 gene, adopting the preference codon in E.coli. Fusion protein expressed in pGEX-4T-1 was isolated and purified. The proliferative effect of human T cells in RA patients responses to rhCⅡ250-270(0.1, 1.0, 10, 100μg/ml) was evaluated by FACS. Results The relative molecular mass of expressed product was 43kD as predicted. The purity of purified fusion protein reached 89% as assessed by SDS-PAGE gel thin-layer scanning. It was shown by FACS that 100μg/ml was the optimal activation concentration, CD69 expression arrived at top level at the third day, CD69+ T cells of the PBMC and SFMC activated by rhCⅡ250-270 were (18.05%±4.34%, 25.17%±4.89%) respectively as well as synthesized CⅡ250-270 groups, which was significantly higher than those in the GST′s groups (1.02%±0.48%, 1.25%±0.23%). The express level of CD69 in SFMC is much higher than that in PBMC(P0.01). Conclusion rhCⅡ250-270 has the ability to activate the T cells of PBMC and SFMC from RA patients as well as synthesized CⅡ250-270. The high-level expression of rhCⅡ250-270 may provide a base land for further researching about the pathogenesis and treatment of rheumatoid arthritis. [