Construction of HCV minigenome containning IFN-β and investigation of its inhibitory effect on viral replication
目的构建丙型肝炎病毒（HCV）反应性干扰素（IFN）-β的微型基因组及其抑制效应的初步评价。方法提取poly I:C刺激的淋巴细胞总RNA,RT-PCR扩增IFN-β基因,克隆入前期构建的HCV微型基因组pT7-5U△131-315rI3Urz;PCR并鉴定插入方向,将反向插入的质粒命名为pT7-5U△131-315rIFNI3Urz。将微型基因组5U△131-315rIFNI3URz克隆入pcDNA3.1载体,获得pCMV-5U△131-315rIFNI3URz。将体外转录的5U△131-315rIFNI3URz RNA转染Huh7.5BB7细胞,48 h后检测IFN-β的表达。将pCMV-5U△131-315rIFNI3URz转染Huh7.5 JFH-1细胞系,荧光实时定量PCR法测定细胞中HCV RNA。结果成功建立了Huh7.5JFH-1细胞系;含有IFN-β的微型基因组在复制子细胞和HCV感染细胞中可以特异表达IFN-β;携带IFN-β的HCV微型基因组可以降低细胞中病毒的RNA拷贝水平,具有一定的剂量依赖效应。结论反向插入IFN-β基因的HCV微型基因组进入HCV感染细胞内...Abstract:
Objective To construct minigenome containning IFN-β responsive to HCV and to study its inhibitory effect on viral replication. Methods Total RNA was extracted from lymphocytes stimulated by poly I:C and was reversely transcripted into cDNA. IFN-β gene was amplif ied by PCR and was cloned into pT7-5U△131-315rI3Urz in antisense direction to obtain pT7-5U△131-315rIFNI3Urz. The minigenome 5U△131-315rIFNI3URz was amplified and inserted into the plasmid pcDNA3.1 by positioning the minigenome immediately adjacent to the transcription start site of the cytomegalovirus (CMV) promoter, resulting in pCMV-5U△131-315rIFNI3URz. The pT7-5U△131-315rIFNI3Urz was transcripted into RNA in vitro, then transfected into Huh7.5BB7. After 48h, cell was harvested and total proteins were extracted for Western Blot, IFN-β antibody as the fi rst antibody. When the pCMV-5UrIFNI3URz was transfected into Huh7.5JFH-1 cell the real-time PCR assay was used to detect HCV RNA replication level. Results The Huh7.5-JFH-1 cell line which was developed by transfecting infectious RNA into Huh7.5 cell which can replicate autonomously and virus particle can be packed successfully. IFN-β could be specifi cally expressed after 5UrIFNI3Urz RNA and pCMV-5UrIFNI3URz was trasnfected into Huh7.5BB7 cell lines. The level of HCV RNA replication was distinctly decreased when Huh7.5-JFH-1 cell line was transfected with pCMV-5UrIFNI3URz. Conclusion The minigenome containning inverted IFN-β could specifi cally be expressed in HCV infected cell lines, the viral replication level was decreased to a certain extent. This study provides the new idea of anti-HCV infection.
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