重组人Ⅱ型胶原250-270多肽的表达及活性研究  

Study on the expression and immunological characterization of recombinant human type Ⅱ collagen polypeptide 250-270

作　　者：李学义[1] 朱平[1] 王彦宏[1] 樊春梅[1] 黄向阳 陈庆平 

机构地区：[1]第四军医大学西京医院临床免疫科,西安710032 [2]西安市第五人民医院风湿科

出　　处：《中华微生物学和免疫学杂志》2003年第2期152-154,共3页Chinese Journal of Microbiology and Immunology

基　　金：国家重点科技攻关项目 ( 96 - 90 1- 0 5 - 2 6 2 )

摘　　要：目的　表达、分离、纯化重组人Ⅱ型胶原 2 5 0 2 70多肽 (recombinanthumancollagentypeⅡpeptide 2 5 0 2 70 ,rhCⅡ 2 5 0 2 70 )并研究其对类风湿关节炎 (rheumatoidarthritis ,RA)患者外周血单个核细胞 (PBMC)和关节滑液单个核细胞 (SFMC)中特异性T细胞的活化作用。方法　利用DNA合成、聚合酶链式反应、重组DNA等基因工程的方法构建含有人Ⅱ型胶原功能性多肽CⅡ 2 5 0 2 70多聚基因的表达载体pGEX 4T 1,转化E .coliBL2 1,融合表达目的蛋白 ,经GlutathioneSepharose○R4B亲和层析柱分离纯化 ,12 %聚丙烯酰胺凝胶电泳 (SDS PAGE)和凝胶薄层扫描测量其相对分子质量 (Mr)和纯度。以不同浓度 (0 .1、1.0、10、10 0、10 0 0 μg/ml)的 6聚rhCⅡ 2 5 0 2 70和化学合成的CⅡ 2 5 0 2 70刺激体外培养的RA患者PBMC和SFMC ,等浓度的GST蛋白和PBS作为阴性对照 ,流式细胞仪测定刺激前后 2组培养细胞中T细胞CD6 9的表达率 ,以分析其对特异性T细胞的活化作用。结果　融合表达的目的产物经SDS PAGE分析 ,相对分子质量 (Mr)大小约为 43× 10 3,与预期相符。分离纯化的 6聚rhCⅡ 2 5 0 2 70经SDS PAGE凝胶薄层扫描 ,纯度为 89% ,浓度为 2 .1mg/ml。FACS分析 :经 10 0 μg/ml浓度的 6×rhCⅡ2 5 0 2 70刺激 3d?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(P<0.01). 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. [

关 键 词：类风湿关节炎 Ⅱ型胶原 重组人Ⅱ型胶原250-270多肽 T细胞增殖 

分 类 号：R593.22[医药卫生—内科学]