Liu M T, Lin L S, Yu Y, Chung T T, Hsu C Y, Chen J T, Jeng K C
Department of Medical Research, Taichung Veterans General Hospital, Taipei, Taiwan, R.O.C.
Zhonghua Yi Xue Za Zhi (Taipei). 1996 Jan;57(1):7-15.
Nasopharyngeal carcinoma (NPC) is one of the most common cancers in southern China and Taiwan. Serological studies revealed the close-relationship between NPC and Epstein-Barr virus (EBV). Elevated serum and saliva levels of anti-EBV antibodies are detected in patients with NPC. Therefore, Development Center for Biotechnology prepared the EBV-early antigen (EA-D) by recombinant DNA technique for screening the serum and throat washing samples from patients with head and neck cancers.
The BMRF1 gene for EBV early antigen (EA-D) was placed into the plasmid pDB18, then transformed into an Escherichia coli strain containing the lambda cI857 temperature-sensitive repressor. Heat treatment of the transformant, at exponential growth phase, inactivated the cI protein and induced an over-expression of the EA-D protein. Next, the EA-D was purified by chromatography and characterized as a protein of molecular weight 47 kDa, by sodium dodecyl sulfate-polyacry lamide gel electrophoresis (SDS-PAGE) and Western blot analysis using monoclonal anti-EA antibody and sera from patients with nasopharyngeal carcinoma (NPC). Enzyme-linked immunosorbent assay (ELISA) with the purified EA-D antigen was used to screen 129 serum and throat washing (TW) samples from patients with head and neck tumors, 24 from patients with a nonmalignant disease and 44 from normal donors.
Experimental results indicated significantly higher positive rates of EA-D IgA (69%) and EA-D IgG (91%) in NPC sera than in the sera of patients with other head and neck tumors and normal controls. TW samples from patients with NPC also showed a higher positive rate (34%) than the other groups (7-20%).
Results in this study demonstrate that the bacterially expressed EA-D antigen could be recognized by sera from patients with NPC and monoclonal anti-EA antibody. Thus, it has potential use in ELISA for screening EBV-related diseases such as NPC.
鼻咽癌（NPC）是中国南方和台湾地区最常见的癌症之一。血清学研究表明鼻咽癌与爱泼斯坦-巴尔病毒（EBV）关系密切。鼻咽癌患者血清和唾液中的抗EBV抗体水平升高。因此，生物技术开发中心采用重组DNA技术制备了EBV早期抗原（EA-D），用于筛查头颈癌患者的血清和洗喉样本。
将EBV早期抗原（EA-D）的BMRF1基因插入质粒pDB18，然后转化到含有λcI857温度敏感型阻遏物的大肠杆菌菌株中。在指数生长期对转化体进行热处理，使cI蛋白失活并诱导EA-D蛋白的过表达。接下来，通过色谱法纯化EA-D，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）以及使用单克隆抗EA抗体和鼻咽癌（NPC）患者血清进行的蛋白质印迹分析，将其鉴定为分子量为47 kDa的蛋白质。使用纯化的EA-D抗原进行酶联免疫吸附测定（ELISA），以筛查129例头颈肿瘤患者的血清和洗喉（TW）样本、24例非恶性疾病患者的样本以及44例正常供体的样本。
实验结果表明，鼻咽癌血清中EA-D IgA（69%）和EA-D IgG（91%）的阳性率显著高于其他头颈肿瘤患者血清和正常对照。鼻咽癌患者的TW样本阳性率（34%）也高于其他组（7 - 20%）。
本研究结果表明，细菌表达的EA-D抗原可被鼻咽癌患者血清和单克隆抗EA抗体识别。因此，它在ELISA中具有筛查EBV相关疾病（如鼻咽癌）的潜在用途。
