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目的 构建含异质性胞核核糖核蛋白A2 (hnRNPA2 )cDNA片段的基因克隆 ,制备并纯化重组蛋白hnRNPA2 ,用以检测抗hnRNPA2 RA33的抗体。方法 从人外周血单个核细胞提取细胞总RNA ,应用RT PCR方法扩增hnRNPA2cDNA ,将其克隆于pUC T1质粒中 ,测定其序列。构建高效表达载体pET2 8a hnRNPA2 ,转化大肠杆菌BL2 1(DE3)plysS并进行诱导表达 ,将含目的蛋白的组分经金属螯合树脂亲和层析柱进行蛋白纯化。以该纯化蛋白为包被抗原 ,ELISA方法检测类风湿关节炎 (RA) 97例、系统性红斑狼疮 (SLE) 5 0例、混合性结缔组织病 (MCTD) 8例、其他关节炎 2 9例、其他弥漫性结缔组织病 (CTD) 99例。结果 构建hnRNPA2重组表达载体并在大肠杆菌中获得hnRNPA2高效表达 ;在RA、SLE、MCTD、其他关节炎、其他CTD患者中抗hnRNPA2 RA33抗体阳性率分别为36 .8%、2 4%、75 %、3.75 %、10 .10 % ;在RA中特异性为 86 .6 7%。结论 以纯化的基因重组蛋白hnRNPA2为抗原检测hnRNPA2 RA33抗体是早期诊断RA的可靠方法
Objective To construct a cDNA clone containing hnRNPA2 cDNA and prepare and purify the recombinant protein hnRNPA2 to detect the anti-hnRNPA2 RA33 antibody. Methods Total RNA was extracted from human peripheral blood mononuclear cells. The hnRNPA2 cDNA was amplified by RT-PCR and cloned into pUC1 plasmid. The sequence of hnRNPA2 was determined. The recombinant expression vector pET2 8a hnRNPA2 was constructed and transformed into Escherichia coli BL21 (DE3) plysS. The recombinant protein was purified by metal chelate resin affinity chromatography. 97 cases of rheumatoid arthritis (RA), 50 cases of systemic lupus erythematosus (SLE), 8 cases of mixed connective tissue disease (MCTD), 29 cases of other arthritis , 99 cases of other diffuse connective tissue disease (CTD). Results The hnRNPA2 recombinant expression vector was constructed and expressed efficiently in E. coli. The positive rates of anti-hnRNPA2 RA33 antibodies in RA, SLE, MCTD, other arthritis and other CTD patients were 36.8%, 24%, 75% , 3.75%, 10. 10% respectively; the specificity in RA was 86.67%. Conclusion The detection of hnRNPA2 RA33 using purified recombinant protein hnRNPA2 as antigen is a reliable method for the early diagnosis of RA