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
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。
Objective To establish a HCV Subgenomic Replicon cell line;To study the effect of ribozyme-CTE on intracellular HCV mRNA。
Methods
1.The plasmid pHCVBM4-5with HCV Subgenomic Replicon was translated into HCV Subgenomic Replicon RNA Using RiboMAX Express T7。
2.The HCV Subgenomic Replicon RNA were transfected into QSG7701 cells Using Lipofectmine~(TM)2000,and then selected the cell clone with G418。The non-transfected QSG7701 cells as control。The stable cell line was verified by RT-PCR and Western Blotting。
3.To observe the effect of pPHCV5-R1,pPHCVS-R2,pPHCV5-CR1 as well as pPHCV5-CR2 on HCV Subgenomic Replicon RNA replication。
Result
1.HCV Subgenomic Replicon QSG7701 stable cell line was stablished,which was verified by RT-PCR and Western Blotting。
2.The plasmids that contain these classic ribozyme genes and new ribozyme genes were transfected into QSG7701 cells using Lipofectmine~(TM)2000。The following experiments were carried out after 48 hours of transfection.RT-PCR and WesternBlotting were used to detect HCV RNA and corresponding protein respectively o The results of RT-PCR suggested that those new rebozymes with CTE could cut target RNAs more effective than classic ribozymes without CTE。Those new ribozymes with CTE could cut target RNAs that classic ribozymes could not cut。
Conclusion
1.successful establishment of HCV Subgenomic Replicon QSG7701 cell line。
2.CTE-ribozymes with was more effective on HCV RNA inhibition than rebozymes。
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。
Objective To establish a HCV Subgenomic Replicon cell line;To study the effect of ribozyme-CTE on intracellular HCV mRNA。
Methods
1.The plasmid pHCVBM4-5with HCV Subgenomic Replicon was translated into HCV Subgenomic Replicon RNA Using RiboMAX Express T7。
2.The HCV Subgenomic Replicon RNA were transfected into QSG7701 cells Using Lipofectmine~(TM)2000,and then selected the cell clone with G418。The non-transfected QSG7701 cells as control。The stable cell line was verified by RT-PCR and Western Blotting。
3.To observe the effect of pPHCV5-R1,pPHCVS-R2,pPHCV5-CR1 as well as pPHCV5-CR2 on HCV Subgenomic Replicon RNA replication。
Result
1.HCV Subgenomic Replicon QSG7701 stable cell line was stablished,which was verified by RT-PCR and Western Blotting。
2.The plasmids that contain these classic ribozyme genes and new ribozyme genes were transfected into QSG7701 cells using Lipofectmine~(TM)2000。The following experiments were carried out after 48 hours of transfection.RT-PCR and WesternBlotting were used to detect HCV RNA and corresponding protein respectively o The results of RT-PCR suggested that those new rebozymes with CTE could cut target RNAs more effective than classic ribozymes without CTE。Those new ribozymes with CTE could cut target RNAs that classic ribozymes could not cut。
Conclusion
1.successful establishment of HCV Subgenomic Replicon QSG7701 cell line。
2.CTE-ribozymes with was more effective on HCV RNA inhibition than rebozymes。
引文
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[2] Wada I,Hara T Kajihar S,et al. Population-based study of hepatitis c virus Infection and heptocellular carcinoma in western Japan. Hepatol Res, 2002, 23:18-24.
[3] FAMMSL.Chronic hepatitis C virus infec tion [J] .2003, 289(18): 2413- 2417.
[4] Choo QL, KH Richman, JH Han, et al. Genetic organization and diversity of the hepatitis C virus. Proc. Natl. Acad. Sci. USA 1991,88:2451-2455.
[5] Kato NM, Hijikata, Y Ootsuyama, et al. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc. Natl. Acad. Sci. USA 1990,87:9524-9528.
[6] Takamizawa AC, Mori I, Fuke S,et al. Structure and organization of the hepatitis C virus genome isolated from human carriers. J. Virol. 1991,65:1105-1113 .
[7] Hijikata MH, Mizushima T, Akagi, et al. Two distinct Proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J. Virol. 1993,67:4665-4675.
[8] Okamoto HK, Kurai S,Okada, et al. Full-length sequence of a hepatitis C virus genome having poor homology to reported isolates: comparative study of four distinct genotypes. Virology 1992,188:331-341 .
[9] Okamoto HS, Okada Y, Sugiyama K, et al. Nucleotide sequence of the genomic RNA of hepatitis C virus isolated from a human carrier: comparison with reported isolates for conserved and divergent regions. J. Gen. Virol. 1991, 72:2697-2704.
[10] Tanaka TN, Kato M, Nakagawa Y,et al. Molecular cloning of hepatitis C virus genome from a single Japanese carrier: sequence variation within the same individual and among infected individuals. Virus Res. 1992,23:39-53 .
[11] Poynard T, Yuen MF, Ratziu V et al. Viral hepatitis C. Lancet. 2003 12(20);362(9401 ):2095-2100 .
[12] Hahn JA.Sex, drugs, and hepatitis C virus.J Infect Dis. 2007;195(11): 1556 -1559
[13] Poynard T, Ratziu V, Benhamou Y et al.Natural history of HCV infection.Baillieres Best Pract Res Clin Gastroenterol. 2000 ;14(2):211-228.
[14] Poynard T. Hepatitis C: natural history, biology, treatment monitoring. Pathol Biol (Paris). 1999 ;47(9):911-916.
[15] Tanaka T, Kato N, Cho MJ ey al. Structure of the 3' terminus of the hepatitis C virus genome. J Virol. 1996 5;70(5):3307-3312,
[16] Tanji Y, Kaneko T, Satoh S, et al. Phosphorylation of hepatitis C virus encoded nonstructural protein NS5A. J Virol. 1995; 69 : 3980-3986c
[17] Van Doorn LJ. Molecular biology of the hepatitis C virus. J Med Virol. 1994; 43: 345-456.
[18] Simmonds P,Smith DB, McOmish F et al. Identification of genotypes of hepatitis C virus by sequence comparisons in the core, E1 and NS-5 regions. J Gen Virol. 1994; 75: 1053-1061.
[19] LohmannV, Korner F, Koch J,et al. Replication of subgenomi chepatitis C virus RNAs in a hepatoma cell line [J]. Science, 1999; 285(5424): 110-113 .
[20] Blight K J, Kolykhalov a A, Ricec M, Efficient in itiation of HCV RNA replication in cell culture[J]. Science, 2000,290. 1972-1974.
[21] Blight K J, Mckeating J A, Marcotrigiano J, et al Efficient replication of hepatitis C Vitus genotype la RNAs in cell culture[J], J Virol, 2003, 77(5): 3181-3190.
[22] Lohmann V, Korner F, Koch J, et al Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line[J].Science, 1999, 285:110-113.
[23] kato T, Date T, Miyamoto M, et al Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon[J]. Gastroenterology, 2003,125(6): 1808-1817 .
[24] IKEDA M, YIM , LIK, et al Selectable subgenomic and genome length dicistronic RNAs derived from an infectious molecular clone of the GCv-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cell[J], J Virol 2002,76: 2997-2907.
[25] Luo G, Xin S,Cai Z. Role of the 5'proximal stem-loop structure of the 5 untranslated regionin replication and translation of hepatitis C viru RNA[J]. J Virol, 2003; 77(5): 3312-3318.
[26] Yi M, Lemon SM.Structure-function analysis of the 3'stem-loop of heatitis C virus genomic RNA and its role in viral RNA replication[J] RNA, 2003; 9(3): 331-345.
[27]Cech TR, ZaugAJ, Grabow ski PJ. In vitro splicing of the ribosomal RNA precursor of tetrahymena: Involvement of a guano sine nucleotide in the excision of the interverning sequence. Cell 1981, 27: 487 .
[28]Guerrier-Takada C, Gardiner K, Marsh R, Pace N, Altman S. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 1983, 35: 8492857
[29] Ryu KJ, Lee SW. Identification of the most accessible sites to ribozymes on the hepatitis C virus internal ribosome entry site. J Biochem Mol Biol. 2003 11(30);36(6):538-544.
[30] Macejak DG, Jensen KL, Pavco PA,et al. Enhanced antiviral effect in cell culture of type 1 interferon and ribozymes targeting HCV RNA. J Viral Hepat. 2001 Nov;8(6):400-405.
[31] Ali N, Siddiqui A. The La antigen binds 5' noncoding region of the hepatitis C virus RNA in the context of the initiator AUG codon and stimulates internal ribosome entry site-mediated translation. Proc Natl Acad Sci U S A. 1997 3(18);94(6):2249-2254.
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