自身抗原gp210融合蛋白的重组表达及其临床应用研究

The recombinant Expression and clinical application of Autoantigen gp210

目的重组表达人核包膜蛋白自身抗原gp210融合蛋白,以用于原发性胆汁性肝硬化(PBC)的临床诊断和病情监测。方法针对基因库中人gp210的cDNA序列设计引物,从正常人的淋巴细胞中提取RNA,通过反转录PCR方法扩增得到相应的基因片段,经测定序列验证后插入表达载体PET28a(+),构建重组表达载体PET28a(+)-gp210,转化大肠杆菌BL21(DE3)后诱导表达蛋白质,并经SDS-PAGE、Western-blot鉴定重组表达的gp210融合蛋白,进一步经Ni2+亲合层析柱纯化。应用重组gp210融合蛋白建立间接ELISA,检测PBC患者血清中的抗gp210抗体。结果经核苷酸序列测定和酶切鉴定结果表明,成功地构建了PET28a(+)-gp210重组质粒。经SDS-PAGE、Western-blot鉴定,获得了具有免疫原性的重组gp210融合蛋白。经ELISA检测,PBC患者中抗gp210抗体的阳性率为40.5%,与疾病对照组及正常对照组比较有显著性差异(P<0.05)。PBC患者临床资料分析表明,抗gp210阳性患者中IgM浓度显著高于抗gp210阴性患者;经UDCA治疗后,28位PBC患者中,3例抗gp210抗体由阳性转为阴性,9例抗gp210抗体持续阳性,1例抗gp210抗体由阴性转为阳性,16例抗gp210抗体持续阴性;其他临床症状与抗gp210抗体无显著相关。结论应用重组gp210融合蛋白建立ELISA检测自身抗体,对PBC诊断具有较好的特异性,为PBC的临床诊断和病情监测提供了有力依据。

Objective To express gp210 recombinant fusion protein in E. eoli and apply to clinical assay.Methods The eDNA encoding gp210 was obtained by RT - PCR, confirmed by DNA sequencing, subcloned into the bacterial expression plasmid pET28a （ ＋ ） and then transformed into E. coil. BL21 （DE3） to express the recombinant fusion protein induced by IPTG. ELISA was established to detected anti - gp210 antibody in PBC patients.Results The recombinant firsion protein was 69kDa as expected and exhiited the antigenicity of gp210 by Western- blot.The positive ratio of anti - gp210 antibody in PBC was 40.5% .The concentrations of IgM in patients with anti - gp210 antibody were significantly higher than patients without anti - gp210 antibody. Conclusion The recombinant gp210 fusion protein could be used for monitoring the course of Primary biliary cirrhosis.