Milman G, Scott A L, Cho M S, Hartman S C, Ades D K, Hayward G S, Ki P F, August J T, Hayward S D
Proc Natl Acad Sci U S A. 1985 Sep;82(18):6300-4. doi: 10.1073/pnas.82.18.6300.
The carboxyl-terminal one-third of the Epstein-Barr virus nuclear antigen (EBNA-1) encoded by the BamHI restriction fragment K was synthesized in Escherichia coli by use of a high-expression plasmid. The resultant 28-kDa EBNA fusion polypeptide, comprising 5-10% of the total soluble bacterial protein, was purified to apparent homogeneity by phosphocellulose and hydroxylapatite column chromatography. Both rabbit monospecific antibodies and mouse monoclonal antibodies against 28-kDa EBNA gave nuclear immunofluorescence staining on Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines and recognized the appropriate intact EBNA polypeptide bands on immunoblots. An ELISA with the purified 28-kDa EBNA as antigen was used to quantitate anti-EBNA antibody in human serum samples. The ELISA method was approximately 100-fold more sensitive than the classical anticomplement immunofluorescence assay. Anti-EBNA antibody was detected in sera from 100% of normal individuals who were seropositive for the viral capsid antigen, and low anti-EBNA titers were detected in serum from most patients with acute infectious mononucleosis. The assay gave the expected pattern of titers in sera from patients with rheumatoid arthritis, Burkitt lymphoma, or nasopharyngeal carcinoma, thus confirming the validity of this purified reagent for assessing EBNA antibody status. Approximately 10% of normal individuals and rheumatoid arthritis patients had anti-EBNA titers as high as those seen in nasopharyngeal carcinoma patients. In these high-titer individuals, greater than 1% of the total IgG are antibodies that recognize 28-kDa EBNA, which indicates that the carboxyl-terminal domain of EBNA is highly immunogenic.
利用高表达质粒在大肠杆菌中合成了由BamHI限制性片段K编码的爱泼斯坦-巴尔病毒核抗原（EBNA-1）羧基末端的三分之一。所得的28 kDa EBNA融合多肽占细菌总可溶性蛋白的5%-10%，通过磷酸纤维素和羟基磷灰石柱色谱法纯化至表观均一。针对28 kDa EBNA的兔单特异性抗体和小鼠单克隆抗体在爱泼斯坦-巴尔病毒（EBV）感染的淋巴母细胞系上均产生核免疫荧光染色，并在免疫印迹上识别出相应的完整EBNA多肽条带。以纯化的28 kDa EBNA作为抗原的ELISA用于定量人血清样本中的抗EBNA抗体。ELISA方法比经典的抗补体免疫荧光测定法敏感约100倍。在病毒衣壳抗原血清阳性的100%正常个体的血清中检测到抗EBNA抗体，大多数急性传染性单核细胞增多症患者的血清中检测到低抗EBNA滴度。该测定在类风湿性关节炎、伯基特淋巴瘤或鼻咽癌患者的血清中给出了预期的滴度模式，从而证实了这种纯化试剂用于评估EBNA抗体状态的有效性。约10%的正常个体和类风湿性关节炎患者的抗EBNA滴度与鼻咽癌患者的滴度一样高。在这些高滴度个体中，总IgG的1%以上是识别28 kDa EBNA的抗体，这表明EBNA的羧基末端结构域具有高度免疫原性。
