原核表达和制备EB病毒GST/BFRF3融合蛋白用于鼻咽癌的诊断筛查

摘要

目的:构建表达EB病毒衣壳抗原BFRF3基因的原核细胞表达载体并探讨其在鼻咽癌血清学诊断中的应用.方法:以EB病毒DNA为模版,采用PCR法扩增目的基因BFRF3,与原核表达载体PGEX-5X-1连接,构建PC-EX-5X-BFRF3重组质粒,转化大肠杆菌BL21 (DE3),IPTG诱导表达GST/BFRF3融合蛋白.表达产物经SDS-PAGE和免疫印迹法鉴定后,纯化目的蛋白作为包被抗原,制备ELISA试剂检测鼻咽癌患者和正常人群BFRF3-IgA抗体.结果:在大肠杆菌中成功地表达了GST/BFRF3融合蛋白,相对分子质量为44 kD,免疫印迹证实目的蛋白带有免疫原性,目的蛋白经纯化后作为包被抗原检测鼻咽癌患者的灵敏度和特异度分别为65％和87％.结论:采用原核表达系统成功构建并表达了GST/BFRF3融合蛋白,其在鼻咽癌血清筛选中具有诊断价值.%AIM:To construct a prokaryotic expression plasmid containing Epstein-Barr viral (EBV) capsidantigen BFRF3 gene and to observe the application of recombinant BFRF3 protein in the serological diagnosis of nasopharyngeal carcinoma (NPC).METHODS:DNA extracted from the B95-8 cells was used as the templates.Polymerase chain reaction (PCR) was used to generate a DNA fragment of BFRF3 gene,and a 531-bp DNA fragment was inserted into a PGEX-5X-1 vector.The recombinant plasmid was transformed into E.coli BL21 (DE3).The expression of GST/BFRF3 fusion protein was induced by IPTG,identified by both SDS-PAGE and Western blotting,and then purified by glutathionesepharose beads.The purified recombinant protein was coated to microplate for ELISA detection of EBV-IgA antibody in NPC patients.RESULTS:The GST/BFRF3 fusion protein was successfully expressed in E.coli.The molecular weight of the product was approximately 44 kD.The recombinant fusion protein GST/BFRF3 showed good immunoreactivity.A novel ELISA was established using GST/BFRF3 protein.Serum samples collected from the NPC patients and healthy controls were tested by this ELISA.The sensitivity and specificity of GST/BFRF3 tests for NPC patients were 65％ and 87％,respectively.CONCLUSION:The recombinant protein GST/BFRF3 is expressed in E.coli,and it has diagnostic value for screening of NPC patients.