EB病毒核抗原1羧基端的原核表达、纯化及其免疫学特性

为进一步研究EB病毒核抗原 1(EBNA1)的功能及提高EB病毒 (EBV)相关疾病辅助诊断的特异性 ,对EBNA1基因 3′端的部分片段进行了原核表达、纯化并初步研究其免疫学特性 .采用PCR法扩增了EBNA1基因编码区 ,经酶切鉴定、序列分析后 ,将其 3′端 5 73bp片段克隆至原核表达载体pET30a中 ,得到重组质粒pET30a SS5 80 .该重组质粒转化大肠杆菌BL2 1(DE3)感受态细胞并经异丙基 β D 硫代半乳糖苷 (IPTG)诱导表达出分子量约 2 5kD的融合蛋白 (2 5 kDEBNA1) .该蛋白以包涵体和可溶形式存在 ,均可用Ni2 + 离子亲和柱纯化 .Western印迹结果显示 ,该蛋白能与鼻咽癌 (NPC)病人血清发生特异性反应 .纯化的 2 5kDEBNA1蛋白免疫BALB c小鼠后 ,经ELISA检测获得了高效价的多克隆抗体 .免疫印迹和间接免疫荧光结果显示制备的免疫小鼠血清能够与HeLa细胞中瞬时表达的EBNA1蛋白发生特异性反应 ,且特异性优于鼻咽癌病人血清 .以上结果表明成功构建了EBNA1羧基端的原核表达质粒 ,并在大肠杆菌中高效表达了 2 5kDEBNA1蛋白 ,该蛋白具有良好的抗原性和免疫原性

Prokaryotic Expression, Purification and Immunological Analysis of Carboxyl-Terminal Domain of Epstein-Barr Virus Nuclear Antigen 1

To further study the functional characteristics of EBNA1 and to increase the specificity of EBV-associated diseases detection rate, a prokaryotic vector of EBNA1 gene 3′-terminal was constructed. The fusion protein was expressed and purified in prokaryotic system and its immunological characteristics were studied. The coding sequence of EBNA1 was amplified by PCR method. After identified by the restriction digestion and sequencing, the 3′-terminal 573 bp of EBNA1 gene was inserted into pET30a plasmid to yield an identified recombinant plasmid pET30a-SS580, which was used to transform the competent expressive cells of E.coli BL21(DE3). After induction with IPTG, samples analysis results revealed that a fusion protein approximately 25 kD was yielded(25-kD EBNA1), and it occurred both in the form of inclusion bodies and soluble protein,and both of them could be purified with Ni 2+ affinity chromatography. The fusion protein could be detected by Western blotting with nasopharyngeal carcinoma (NPC) patients' sera. Then BALB/c mice were injected with purified 25-kD EBNA1 to induce immunoreactions and a highly reactive and specific antiserum was prepared. Both the prepared antiserum against 25-kD EBNA1 and NPC patients' sera could specifically reacted with transiently expressed EBNA1 on HeLa cells by immunofluorescence assay and Western blot analysis. These results indicated that the carboxyl-terminal domain of EBNA1 was highly expressed and the fusion protein (25-kD EBNA1) was highly antigenic and immunogenic.