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.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
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)