嵌合中国河北株包膜蛋白基因的HCV细胞培养模型的传代分析  

Characterization of Serial Passage of 1b/2a Chimera Hepatitis C Virus Cell Culture System Carrying Envelope E1E2 Coding Gene from Hebei Strain of China

作　　者：卢莎[1,2] 张玲[2] 陶格斯[1,2] 蔡敏[1] 包丽丽[1] 李恋[1] 邓瑶[2] 沈晓玲[1] 谭文杰[2] 

机构地区：[1]内蒙古医科大学微生物教研室,呼和浩特010059 [2]中国疾病预防控制中心病毒病预防控制所,北京102206

出　　处：《病毒学报》2015年第6期647-652,共6页Chinese Journal of Virology

基　　金：国家自然科学基金(81373229);传染病科技重大专项(2013ZX10004601;2013ZX10004609);内蒙古医科大学科技百万项目(NY2011BW003)

摘　　要：建立稳定的嵌合中国河北株包膜蛋白基因的丙型肝炎病毒（HCV）细胞培养体系,进行传代特性分析。本研究经体外转录获得嵌合中国河北株包膜蛋白基因的丙型肝炎病毒（HCV）全长RNA,脂质体法转染Huh7.5-CD81细胞,连续传代培养,进行Real Time RT-PCR、间接免疫荧光、Western blot、再感染试验与感染滴度检测及序列分析。结果表明：嵌合重组HCV RNA转染Huh7.5-CD81细胞后可产生感染性的病毒颗粒（HCVcc）;传代过程中,Western blot可检测细胞内HCV蛋白的表达;IFA检测阳性细胞数逐渐增多,41d升至高峰,达80%~90%;Real Time RT-PCR检测传代细胞上清中HCV RNA拷贝数在104~107拷贝/mL;再感染实验嵌合HCVcc最高感染滴度为104ffu/mL。序列分析显示在传代后期嵌合的HCV包膜基因发生了适应性突变,导致6处氨基酸改变。结论嵌合中国河北株1b亚型包膜蛋白基因的HCV细胞培养体系可以产生具有感染性的嵌合HCV病毒颗粒,传代后感染性增强并且嵌合的HCV包膜基因发生了适应性突变。To character a novel chimera（1b/2a）hepatitis C virus cell culture（HCVcc）system carrying envelope E1E2 coding gene from Hebei strain of China,chimera HCVcc（cHCVcc）was developed from Huh7.5-CD81 cells after transfection with in vitro transcribed full-length 1b/2achimera RNA,which carrying envelope E1E2 coding gene from Hebei strain of China.Then the replication,expression and infectious titer of serial passage HCVcc were assessed by Real Time RT-PCR,indirect immunofluorescence assay（IFA）and Western blotting（WB）.In addition,chimeric envelope gene from HCVcc was sequenced after serial passage.We found that the number of HCV positive focus increased gradually in cell post-transfection with chimera HCVcc（1b/2a）RNA and reach a peak platform（80%to 90%）at 41 days post-transfection;the expression of HCV protein was also confirmed by WB during serial passage.At meantime,HCV RNA copy number in the supernatant peaked at 104~ 107copies/mL and the highest infectious titer of this 1b/2acHCVcc reinfection were tested as 104 ffu/mL.Sequence analysis indicated 6of adaptive amino acid substitutes occur among chimeric envelope E1E2 during serial passages.We concluded that a novel 1b/2achimera HCVcc carrying envelope E1E2 coding gene from Hebei strain of China was developed and its infectious titer increased after serial passage of HCVcc.This novel cHCVcc will be an effective tool for further evaluation of anti-virus drugs and immune effects against the major genotype from Chinese.

关 键 词：丙型肝炎病毒 嵌合 包膜蛋白 细胞培养 

分 类 号：R373.9[医药卫生—病原生物学]