杆状病毒表面展示丙型肝炎病毒结构蛋白及其对丙型肝炎病毒的检测  

Detection of HCV core protein,envelope glycoprotein and core-envelope fusion protein by baculovirus surface display

作　　者：李祥[1] 金爱慧[1] 邹立林[1] 高基民[1] 吕建新[1] 

机构地区：[1]温州医学院浙江省医学遗传学重点实验室,浙江省模式生物技术与应用重点实验室,浙江温州325035

出　　处：《中国病理生理杂志》2012年第10期1910-1916,共7页Chinese Journal of Pathophysiology

基　　金：国家青年自然科学基金资助项目(No.30900344);浙江省自然科学基金资助项目(No.Y2080676);温州市科技计划项目(No.Y20080011)

摘　　要：目的:利用杆状病毒表面展示系统对丙型肝炎病毒(HCV)进行早期检测。方法:利用杆状病毒表面展示(baculovirus surface display)技术,构建展示HCV结构蛋白的重组杆状病毒vAc-Flag-HCV C-GP64、vAc-Flag-HCV E-GP64和vAc-Flag-CE-GP64。结果:(1)Western blotting分析结果表明重组杆状病毒表达了C(HCV核心蛋白)、E(HCV包膜蛋白)和CE蛋白。(2)激光共聚焦显微镜检测结果表明,重组杆状病毒感染昆虫细胞Sf9后在昆虫细胞膜上表达了C、E和CE蛋白。(3)免疫金电子显微镜观察表明重组蛋白展示在杆状病毒囊膜上。(4)重组病毒进行浓缩纯化后,利用时间分辨荧光免疫分析(TRFIA)技术对HCV样本进行检测,vAc-Flag-CE-GP64的检测阳性率可达80.2%。结论:vAc-Flag-CE-GP64可用于对HCV进行早期检测。AIM： To detect the hepatitis C virus （HCV） at early stages by the technique of baculovirus surface display. METHODS ： We constructed the recombinant baculoviruses vAc - Flag - HCV C - GP64, vAc - Flag - HCV E - GP64 and vAc - Flag - CE - GP64, which displayed HCV - related structural proteins by the technique of baculovirus surface display. RESULTS： Western blotting results showed that the core （ C）, envelope （E） and core - envelope （CE） proteins of HCV were expressed in St9 cells after the infection of recombinant baculoviruses. We also observed by confocal microscopy that the C, E and CE proteins of HCV presented and were anchored on the plasma membrane of St9 cells after infection. In addition, the results of immunogold electron microscopy demonstrated that the 3 recombinant baculoviruses displayed the C, E and CE proteins of HCV on the viral surface, respectively. To further define the role of these recombinant baculoviruses in detecting HCV at early stages, we applied time - resolved fluoroimmunoassay （TRFIA） to detect HCV samples after purification and concentration of the recombinant baculoviruses. The results showed that the positive rate of vAc - Flag - CE - GP64 was up to 80.2%. CONCLUSION： vAc - Flag - CE - GP64 is useful for detecting the hepatitis C virus at early stages.

关 键 词：肝炎病毒 丙型 杆状病毒表面展示 时间分辨荧光免疫分析 

分 类 号：R446.5[医药卫生—诊断学]