人内质网分子伴侣BiP的克隆、表达及其在RA患者血清抗体检测中的作用

以类风湿关节炎(rheumatoid arthritis,RA)患者滑膜组织文库cDNA为模板,用PCR方法扩增得到1965 bp人内质网分子伴侣BiP的全长cDNA,将其克隆入OmicsLinkTM表达载体pReceiver-B01a,构建重组表达质粒pReceiver-B01a-BiP。重组表达质粒导入大肠杆菌BL-21(DE3)菌株中诱导表达目的蛋白,表达产物以Ni+-NTA agarose层析柱纯化。SDS-PAGE和免疫印迹法(Western blot)分析发现,表达产物以可溶性蛋白和包涵体形式共同存在,表达量约占菌体蛋白总量的20%~30%。相对分子质量约为80000,纯度达到电泳级。经免疫印迹法鉴定,获得的BiP重组蛋白可以与RA患者血清反应,检测的抗BiP抗体在RA患者血清中的阳性率为75.4%(49/65),明显高于系统性红斑狼疮(systemic lupus erythe-matosus,SLE)患者14.6%(7/48)、原发性干燥综合征(primary Sj gren’s syndrome,pSS)患者7.3%(4/55)及正常人0%(0/71)(P<0.01)。表明,经原核表达获得的BiP全长蛋白,RA患者血清抗体可以很好结合,有望应用于临床血清学诊断。

Cloning and expression of the human endoplasmic reticulum molecular chaperone BiP and its application in the detection of serum antibodies in patients with rheumatoid arthritis

The 1 965 bp full length cDNA fragment encoding human endoplasmic reticulum molecular chaperone,BiP,was PCR amplified from a cDNA library constructed from synovium of patients with rheumatoid arthritis(RA).The DNA fragment was cloned into the plasmid vector pReceiver-B01a,and the cloned BiP gene was expressed in E.coli BL21(DE3) strain.The expressed protein products were purified by using Ni -NTA agarose affinity chromatography.As demonstrated by using SDS-PAGE and Western blotting analysis,the expressed products existed in the dissoluble proteins and inclusion body sections with a relative molecular mass of 80 000 Mr.The expression level of human BiP protein was about 20 percent of total celluar proteins after IPTG induction,and the purified BiP protein reactived with serum of RA patients as shown by Western blotting.It was found that IgG anti-BiP antibodies were detected in 49 out 65(75.4%) RA patients,but only in 7 out 48(14.6%) patients with systemic lupus erythematosus(SLE),in 4 out of 55(7.3%) patients with primary Sjgren's syndrome(pSS),and in 0 out of 71(0%) normal blood donors.The much higher positive rate of anti-BiP antibodies in the sera of RA patients than in patients with other autoimmune diseases,and the absence of the antibodies in normal persons suggest that the expressed BiP proteins can react with RA patients sera and be used as serum marker for the diagnosis of rheumatoid arthritis.