EB病毒病毒壳抗原、核心抗原重组蛋白的制备及在鼻咽癌血清学诊断中的应用

目的 探讨EB病毒(EBV)病毒壳抗原(VcA)-BFRF3和核心抗原(NA)1-BKRF1重组蛋白在鼻咽癌(NPC)血清学诊断中的应用.方法 以EBV DNA为模版,用PCR扩增目的基因BFRF3和BKRF1,捅入大肠杆菌表达载体pET.51b后转化大肠杆菌JM109,诱导重组蛋白表达.表达产物经SDS-PAGE、免疫印迹分析鉴定,金属螯合亲和层析纯化后,分别制备用BFRF3、BKRF1及BFRF3,BKRF1作为包被抗原的ELISA试剂,检测NPC患者和正常人群EBV-IgA抗体.结果 大肠杆菌高效表达BFRF3和BKRF1重组蛋白,表达产物相对分子质量分别为20000和29000,均有良好免疫反应性;BKRF1的NPc诊断灵敏度(71.20%,178,250)明显高于BFRF3(64.80%,162,250)(P<0.01),但两者NPc诊断特异性差异无统计学意义(P>0.05);相对于单独应用一种抗原,联合两种重组抗原的方法诊断NPC具有高灵敏度(84.80%,212,250)和相对低的特异性(82.00%,328,400)(P<0.01).结论 NA1-BKRF1诊断NPC的灵敏度高于VCA-BFRF3,两种抗原联合使用,可提高诊断灵敏度,但特异性明显降低.

Preparation of the recombinant VCA and NA proteins of Epstein-Barr virus and application of serological diagnosis of nasopharyngeal carcinoma

Objective To study the recombinant viral eapsid antigen (VCA)- BFRF3 and NA-BKRF1 proteins of Epstein-barr (EB) virus for serological diagnosis of nasopharyngeal eareinoma (NPC).Methods EB virus DNA was used as the templates in a polymerase chain reaction (PCR). VCA-BFRF3 and NA1-BKRF1 were amplified and inserted into E.eoli expression veetor pET-51b. The reeombinant plasmids were transformed into JM 109 E.coli, and induced expression of IPTG reeombinant proteins. After analyzed by SDS- PAGE and immunoblot, expressed products were purified by immobilized metal ion affinity chromatography. The preparation of BFRF3, BKRF1 and the eombination of BFRF3/BKRF1 were served respeetively as solid phase antigens of ELISA to detect EBV-IgA antibody in NPC patients and healthy people. Results The BFRF3 and BKRF1 proteins had been expressed in E.colis. The relative molecular weight of products were approximately 20 000 and 29 000 respectively. The reeombinant proteins showed good immunoreaetivity. NPC diagnostic sensitivity of BKRF1 (71.20% , 178/250)was signifieancent higher than that of BFRF3 (64.80% , 162/250) (P<0.01) , but there was no difference in diagnostic specificity between them. Combined with BKRF1 and BKRF3, higher diagnostic sensitivity (84.80%, 212/250) and lower diagnostic specificity (82.00%, 328/400)eould be obtained. Conclusion NPC diagnostic sensitivity of BKRF1 was higher than that of BFRF3.Combined with BKRF1 and BKRF3, higher diagnostie sensitivity and lower diagnostic speeifieity could be obtained.