融合抗原ELISA检测EB病毒抗体的临床价值  

Detection of antibodies to Epstein-Barr virus with ELISA based on fusion capsid protein

作　　者：邱清芳[1] 王云[1] 罗兵[1] 

机构地区：[1]青岛大学医学院微生物教研室,266021

出　　处：《中华检验医学杂志》2009年第9期1002-1005,共4页Chinese Journal of Laboratory Medicine

摘　　要：目的探讨以EB病毒（Eptein-Barr virus，EBV）衣壳抗原（VCA）小分子量蛋白p23和p18融合蛋白为抗原，建立检测EBV特异性VCAIgG和IgM抗体的ELISA方法的临床应用价值。方法采用重叠延伸PCR技术，将p23编码基因BLRF2（氨基酸1～162）和p18编码基因BFRF3的C端（氨基酸105～176）基因序列通过多肽接头（Gly4Ser）3DNA序列连接获得融合基因p23-p18。融合基因导入原核表达载体获得融合蛋白，经镍-敖合物琼脂糖树脂柱亲和层析纯化获得融合蛋白，免疫印迹（Western blot，WB）鉴定其特异性。以纯化融合蛋白作为抗原，建立了特异性VCAIgM和IgG间接ELISA检测方法。以该方法对60例传染性单核细胞增多症、56例慢性。肾功能不全透析患者和326名健康献血员的血清进行VCAIgG和IgM抗体检测，并与间接免疫荧光（IFA）检测结果进行比较，综合评价融合抗原ELISA检测方法的敏感度、特异度和符合率等指标。结果（1）融合蛋白鉴定：p23和p18融合基因成功构建并有效表达，WB显示融合抗原与10份VCA特异性IgM阳性血清和10份VCA特异性IgG阳性血清均发生反应，与8份两种抗体均阴性血清不发生反应。（2）临床检验：融合抗原ELISA和IFA同时检测上述442份标本。以tFA为标准，融合抗原检测VCAIgG的敏感度、特异度和准确性分别为97．3％（392／403）、97．4％（38／39）和97．3％（430／442）；检测VCAIgM的分别为94．2％（65／69）、99．5％（371／373）和98．6％（436／442）。结论融合抗原ELISA特异性强，灵敏度高，可用于临床EBV感染诊断及流行病学调查。Objective To develop enzyme-linked immunosorbent assay （ELISA） for detecting antibody to viral capsid antigen （VCA） of Epstein-Barr virus （EBV） by using fusion capsid protein of p23 and p18 as antigen and evaluate its diagnostic implication. Methods Full-length p23 （amino acids 1-162） and carboxy half of p18 （105-176） gene fragment were connected by a （Gly4Ser）3 linker using overlap extension PCR method. The prokaryotie expressed fusion protein of p23-p18 was purified by nickel-chelating sepharose resin affinity chromatography and used as the antigens for ELISA detection of IgG and lgM antibodies to VCA. Western blot was used to detect its antigenicity and specificity. The serum samples from 60 patients with infectious mononucleosis （IM） , fifty-six chronic renal dysfunction patients on hemodialysis and 326 serum samples from healthy blood donors were examined for the presence of VCA-specific IgG and IgM by fusion protein ELISA and indirect immunoflurorescene assay （IFA） simultaneously. Tile specificity, sensitivity and accuracy of ELISA were detected. Results （ 1 ） Identification of the fusion protein ： p23-pl 8 fusion gene was constructed correctly and expressed successfully in the prokaryotic expression vector. The fusion protein showed good reactivity with 10 cases of VCA IgM and 10 cases of IgG positive sera in Western blot, while no reactive bands were found with 8 cases of VCA IgM and IgG negative sera. （2） Clinical tests： IFA and ELISA with fusion proteins were used to detect VCA IgG and IgM in 442 serum samples listed above. As compared with IFA, the sensitivity, specificity and accuracy of the recombinant protein IgG ELISA were 97.3% （392/403）, 97.4% （38/39） and 97.3% （430/442）, respectively,and those of IgM ELISA were 94. 2% （ 65/69）, 99. 5% （ 371/373 ）, and 98. 6% （ 436/442 ）, respectively. Conclusion The ELISA based on fusion viral eapsid proteins is sensitive, specific and accurate method for determining antibodies to VCA of EBV for

关 键 词：疱疹病毒4型 人 抗原 病毒 衣壳蛋白质类 重组融合蛋白质类 酶联免疫吸附测定 

分 类 号：R686[医药卫生—骨科学]