HCV亚基因复制子QSG7701细胞株的建立及CTE-核酶对HCVRNA复制的影响

发布时间：2018-06-05 11:16

本文选题：亚基因复制子 + 丙型肝炎　；参考：《中南大学》2008年硕士论文

【摘要】： 目的建立RNA转染的HCV Subgenomic Replicon(HCV亚基因复制子)稳定转染和表达的细胞株;探讨CTE-核酶对丙型肝炎病毒亚基因组RNA复制的影响。研究方法 1.在T7体外转录RNA试剂盒介导下,将含有HCVSubgenomic Replicon的质粒pHCV BM4-5转化为RNA。 2.建立HCV Subgenomic Replicon稳定转染和表达的细胞株在脂质体lipofectamine ~(TM)2000介导下,将含有HCV SubgenomicReplicon的RNA导入人肝癌细胞系QSG7701中,经G418筛选抗性克隆,扩大培养,建立转染克隆细胞系。用反转录聚合酶链反应(RT-PCR)和蛋白印迹法证实HCV Subgenomic Replicon RNA复制及其蛋白表达。 3.观察普通核酶pPHCV5-R1、pPHCV5-R2和CTE-核酶pPHCV5-CR1、pPHCV5-CR2瞬时转染含HCV亚基因复制子QSG7701。48小时后收集细胞,提取细胞总RNA,采用RT-PCR方法,观察核酶对HCV5'-NCR切割效果。结果 1.经RT-PCR和Western Blot证实,成功建立HCV SubgenomicReplicon RNA稳定转染QSG7701细胞株。 2.经RT-PCR证实:pPHCV5-CR1转染组未见明显HCV5'-NCR扩增目的条带,pPHCV5-R1转染组可见扩增目的条带,亮度低于未转染组;pPHCV5-CR2转染组可见扩增目的条带,亮度低于未转染组,pPHCV5-R2转染组可见扩增目的条带,亮度与未转染组接近。这些结果提示:CTE-核酶在细胞内的切割靶RNA的效率明显高于普通核酶。普通核酶难以切割的位点,CTE-核酶能进行有效的切割;普通核酶能进行切割的位点,带CTE-核酶的切割效率明显提高。结论 1.成功建立HCV Subgenomic Replicon QSG7701细胞株。 2.CTE-核酶在细胞内切割HCVRNA的效率明显高于普通核酶。普通核酶难以切割的位点,CTE-核酶也能进行有效的切割;普通核酶能进行切割的位点,带CTE-核酶的切割效率明显提高。
[Abstract]:Objective to establish a stable transfection and expression cell line of RNA transfected HCV Subgenomic Replicon(HCV subgene replicas and to investigate the effect of CTE- ribozyme on the replication of hepatitis C virus subgenomic RNA. Research method 1. The plasmid pHCV BM4-5 containing HCVSubgenomic Replicon was transformed into HCVSubgenomic Replicon by T7 in vitro transcription RNA kit. 2. Establishment of a cell line stably transfected and expressed with HCV Subgenomic Replicon The RNA containing HCV SubgenomicReplicon was introduced into human hepatoma cell line QSG7701 by liposome lipofectamine / TMN 2000. The resistant clones were screened by G418, and the transfected clone cell line was established. HCV Subgenomic Replicon RNA replication and protein expression were confirmed by reverse transcriptase polymerase chain reaction (RT PCR) and Western blot. 3. After transient transfection of pPHCV5-CR1, pPHCV5-CR2, pPHCV5-CR2 and pPHCV5-R1pPHCV5-CR2 into QSG7701.48 containing HCV subgene, cells were collected and total RNAs were extracted. The effect of ribozyme on HCV5'-NCR cleavage was observed by RT-PCR method. Result 1. RT-PCR and Western Blot confirmed that the stable transfection of QSG7701 cells by HCV SubgenomicReplicon RNA was successfully established. 2. RT-PCR confirmed that there was no significant HCV5'-NCR amplification target band in the pPHCV5-CR1 transfection group. The brightness was lower than that in the untransfected pPHCV5-CR2 transfection group, and the brightness was lower than that in the pPHCV5-R2 transfection group, and the amplification target band was lower in the pPHCV5-R2 transfection group than in the non-transfected pPHCV5-R2 transfection group. The brightness was close to that of the untransfected group. These results suggest that the efficiency of cleavage target RNA of 1: CTE- ribozyme is significantly higher than that of normal ribozyme. The cleavage efficiency of CTE- ribozyme was improved when the common ribozyme was difficult to cleavage and the common ribozyme cleavage site could be effectively dissected by CTE- ribozyme. Conclusion 1. HCV Subgenomic Replicon QSG7701 cell line was successfully established. 2. The efficiency of HCVRNA cleavage by CTE- ribozyme was significantly higher than that of normal ribozyme. CTE- ribozyme which is difficult to cleavage by ordinary ribozyme can also be effectively dissected, and the cleavage efficiency of CTE- ribozyme with common ribozyme can be improved obviously because of the cleavage of common ribozyme.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373.21

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