摘要
目的研究核包膜蛋白gp210基因在大肠杆菌中的稳定表达、纯化和免疫学活性鉴定。方法从人外周血单个核细胞中提取细胞总RNA,应用RT-PCR方法扩增gp210蛋白中含优势表位的基因片段。将该段基因克隆入PET-30a表达载体并转化到大肠杆菌BL21中表达。融合蛋白经Ni-NAT树脂柱进行亲和层析纯化,并通过SDS-PAGE电泳及ELISA方法进行鉴定。结果在原核表达载体中成功构建了PET30a-gp210重组体。重组体诱导培养后,SDS-PAGE电泳分析可见在相对分子量大约69 kD处有gp210融合蛋白的高效表达,经纯化获得纯度达90%的表达蛋白。ELISA检测结果显示该重组蛋白具有较好的抗原性和特异性。结论应用基因工程方法,获得了gp210重组蛋白,为检测抗gp210抗体提供了特异性抗原,也为临床将抗gp210抗体作为PBC的特异性诊断指标奠定了基础。
Objective To express, purify nuclear envelope protein gp210 gene in E. coil, and identify its immunoactivity. Methods Gene fragment of nuclear envelope protein gp210 containing the predominant autoepitope was amplified by extract/ng total RNA from human peripheral blood mononuclear cell and RT-PCR. The target gene was cloned into the expressive vector PET-30a and tronsformated into E coli BL21. The recombinant was induced by IPTG to express target protein. This fusion protein was purified by NAT chromatography and identified by SDS-PAGE and ELISA. Results The plasmid PET30a-gp210 was reconstructed successfully and expressed a Mr 69kD gp210 fusion protein in E. toll by seeing a single special strip on SDS-PAGE gel. It reached a purity of 90% by purification. The result of ELISA showed its good antigenicity and specificity. Conclusion Using the method of gene engineering, we obtain recombinant polypeptide. It can be used as diagnostic material to develop a simple assay to detect gp210 autoantibodies for its good antigenicity. It also provides test reference for clinical diagnosis of PBC.
出处
《中国实验诊断学》
2007年第10期1343-1346,共4页
Chinese Journal of Laboratory Diagnosis
