多瘤病毒样颗粒的制备及血清学检测方法的建立  

Preparation of John Cunningham virus-like particles and establishment of serological detection assay for John Cunningham virus

作　　者：赵洪兰[1] 刘熙君[1,2] 徐承富[3] 王岳[1] 江永珍[1] 宋敬东[1] 毕胜利[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052 [2]粤北人民医院 [3]浙江大学医学院附属第一医院

出　　处：《疾病监测》2013年第11期936-939,共4页Disease Surveillance

基　　金：国家重大传染病防治专项课题(No.2009ZX10004-711)~~

摘　　要：目的制备多瘤病毒(John Cunningham virus,JCV)病毒样颗粒并建立JCV血清学检测方法,了解我国一般人群JCV流行情况。方法通过基因合成技术合成JCV衣壳蛋白基因VP1,将其克隆至PET-21a质粒,转化BL21(DE3)大肠埃希菌,IPTG诱导表达,用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定目的蛋白表达。采用2次超速离心法对目的蛋白进行纯化,通过SDS-PAGE、透射电子显微镜及血凝试验鉴定纯化产物。以纯化产物为抗原建立间接酶联免疫吸附试验(ELISA)法,筛查一般人群血清标本,分析该人群JCV感染情况。结果通过透射电子显微镜观察及血凝试验鉴定,显示纯化产物具有和天然JCV颗粒类似的形态结构和血凝活性。以纯化的JCV病毒样颗粒为抗原建立了间接ELISA法,筛查了一般人群抗JCV IgG抗体,结果显示一般人群抗JCV IgG抗体阳性率为54.2%。结论本研究制备了JCV病毒样颗粒,所建立的ELISA法可应用于JCV人群血清流行病学调查。Objective To prepare John Cunningham virus （JVC）-like particles and establish a serological detection assay for JCV,and understand the seroepidemiology of JCV in general population in China. Methods The synthesized sequence of VP1 was inserted into the prokaryotic expression plasmid PET -21a, the resulting plasmid was transferred to the Escherichia coli BL21 （DE3）, and then the protein expression was induced with IPTG, the expression products were analyzed with SDS-PAGE. Two steps of ultracentrifugation were utilized to purify the VP1 protein, the JCV-like particles were analyzed with transmission electron microscopy and hemagglutination assay. Then the purified JCV-like particles were used as antigen to establish the indirect enzyme-linked immunosorbent assay （ELISA） to detect the anti- JCV antibody in serum from general population. Results The results of transmission electron microscopy and hemagglutination assay demonstrated the shape and hemagglutination activity of JCV-like particles were similar to the natural JCV particles. ELISA was established by using JCV-like particles as antigen to screen the antibody to JCV. The seropositive rate of JCV in general population was 54. 2%. Conclusion JCV-like particles were successfully prepared, the established ELISA could be used to analyze the seroepidemiology of JCV.

关 键 词：多瘤病毒 病毒样颗粒 酶联免疫吸附试验 血清学检测 

分 类 号：R440[医药卫生—诊断学]