EB病毒壳抗原在大肠杆菌中的表达与应用

The expression of Epstein-Barr virus-associated viral capsid antigen(VCA) in E.coli and application in diagnosis

获得足够的ＥＢ病毒壳抗原，建立ＥＬＩＳＡ方法用于鼻咽癌的诊断。方法：将 ＢＡＬＦ４基因克隆进入 载体ｐＵＲ２９１，在大肠杆菌中表达ＥＢ病毒壳抗原与β－半乳糖苷酶的融合蛋白，并通过ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ离子 交换柱层析对融合蛋白进行纯化，将纯化的蛋白用于 ＥＬＩＳＡ方法检测人血清中的ＩｇＧ／ＶＣＡ和 ＩｇＡ／ＶＣＡ抗体，用带 有ｐＵＲ２９１质粒的宿主菌裂解液吸收待检血清中的抗β－半乳糖苷酶抗体。结果：ＥＬＩＳＡ方法与免疫荧光方法检测的 结果呈正相关（ｒ＝０．６２３６，Ｐ＜０．００１；ｒ＝０．９２２５，Ｐ＜０．００１）。免疫荧光方法检测为阳性的血清，ＥＬＩＳＡ检测均为 阳性；ＩｇＧ／ＶＣＡ和ＩｇＡ／ＶＣＡ抗体的几何平均滴度（ＧＭＴ）分别是免疫荧光方法的１３倍和１５倍。在４０份免疫荧光检 测ＩｇＡ／ＶＣＡ阴性的血清中，ＥＬＩＳＡ检测有３例阳性，表明ＥＬＩＳＡ方法比免疫荧光方法更敏感。结论：在原核系统中 表达抗原，为ＥＬＩＳＡ方法在鼻咽癌的诊断和大规模血清学普查中的应用提供了一个有效手段。

Aim:To obtain sufficient Epstein-Barr viral capsid antigen and apply it to the diagnosis of Nasopharyngeal Carcinoma based on ELISA.Methods:Using the recombinant DNA technique,a fragment of gene BALF_4 was cloned into vector plasmid pUR291 and a fusion protein consisting of β－galactosidase and the major polypeptide of Epstein-Barr viral capsid antigen (VCA)was expressed in E.coli.After determining the specificity of the fusion protein,the product was purified by DEAESepharose Fast Flow ion-exhange column chromatography.ELISAswere established with the purified product,using rabbit antiserum to block β-galactosidase in human sera.Results:There was a close correlation between results of the immunofluorescence and ELISA(r=0.6236,P<0.001;R=0.9225,p<0.001).The IgG and IgA positive sera determined by immunofluoresence were also positive when determined by ELISAs,and GMT were respectively 13 times and 15 times as immunofluorescence.Three positive sera were detected by ELISA in 40 IgA negative sera determined by immunofluorescence,suggesting that ELISA is more sensitiv than immunofluorescence assay.Conclusion:The Epstein-Barr viral capsid antigen expressed in prokaryotic is useful in the application of the diagnosis of Nasopharyngeal Carcinoma based on ELISA.