乙型肝炎病毒主S蛋白相互作用蛋白—醛缩酶B验证及功能研究
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摘要
目的:证实醛缩酶B作为乙型肝炎病毒主S蛋白结合蛋白一种候选蛋白,探讨醛缩酶B生物学功能。
方法:(1)以人HEPG2细胞RNA为模板,用RT-PCR方法扩增出ALDOB基因。将PCR产物克隆进真核载体PCMV5内,构建含ALDOB基因的重组真核表达质粒。以实验室重组全S质粒为模板,利用PCR扩增主S蛋白基因,构建PCMV4-Flag-主S基因表达质粒,分别将重组质粒转染293FT细胞,用免疫荧光和Western blot等方法检测ALDOB及主S在293FT细胞中的表达。(2)将两种质粒共转293T细胞,激光共聚焦试验明确主S蛋白和醛缩酶B空间定位。将主S基因表达质粒稳转HepG2细胞,构建主S稳转株,通过免疫共沉淀方法,验证主S蛋白与醛缩酶B相互结合。(3)构建靶向醛缩酶B重组干扰质粒siALDOB-Pu6,导入HEPG2细胞中,筛选出干扰质粒的稳转株。通过细胞增殖实验、Transwell实验、细胞凋亡实验,探讨ALDOB在肝癌细胞HepG2生长、侵袭、凋亡等方面的作用。
结果:(1)核酸序列分析的结果表明,克隆的ALDOB基因与主S基因在GenBank中分别与已登记基因序列100%同源。免疫荧光结果显示:ALDOB蛋白在细胞质表达;主S蛋白主要存在于细胞质中。Western blot结果显示:在约40KD位置有目的条带,与预期的重组ALDOB蛋白大小一致;在约26KD位置有预期的主S蛋白表达。(2)通过免疫共沉淀方法,成功确认醛缩酶B是乙型肝炎病毒主S蛋白结合蛋白一种候选蛋白。(3)特异性抑制ALDOB表达的HepG2细胞株较没有抑制ALDOB的HepG2细胞株,细胞增殖加快、侵袭能力增强、凋亡率增加。稳定干扰ALDOB的HepG2细胞稳转株较稳转空载Pu6的HepG2细胞株生长
结论:(1)成功构建了含ALDOB基因以及主S基因的真核表达质粒。(2)醛缩酶B是乙型肝炎病毒主S蛋白的结合蛋白。(3)ALDOB能够抑制HepG2细胞增殖,使HepG2细胞侵袭能力下降,抑制HepG2细胞凋亡。
Objective: TO confirm the candidate hepatocyte binding protein ALDOB interacting with the surface antigen protein of hepatitis B virus and study on the function of Aldolase B.
Methods:(1) ALDOB gene was amplified by RT-PCR with the total RNA of human HepG2 cell as template.The PCR fragments were cloned into pCMV5 vector to construct recombinant eukaryotic expression plasmids.Whole S gene recombinant plasmid as a template by PCR amplification of the major S gene ,building PCMV4-Flag-SHBs expression vector. 293T cells were transfected with the recombinant plasmids. Immunofluorescence staining and Western blot were used to certificate the expressions of ALDOB-myc and major S in 293FT cells.(2)The two plasmids were co-transfected into 293FT, followed by confocal laser microscopy analysis to identify the co-localization of major S and ALDOB proteins. SHBs-flag gene expression plasmid was transfected into HepG2 cells to establish stable transfected major S protein cell line, using the co-immunoprecipitation method to verify binding of the major S protein and ALDOB protein. (3) Construction of recombinant targeting aldolase B interference plasmid siALDOB-Pu6, transfecting into HEPG2 cells to select the stability of interference plasmid transfer lines. By cell proliferation assays, Transwell experiments, apoptosis experiment, explore the role of ALDOB in HepG2 hepatoma cells by the growth, invasion and apoptosis.
Results: (1) The sequence of ALDOB and major S were 100% homology with difference human gene previously registered in GenBank. The result of Immunofluorescence staining manifest: recombinate protein of ALDOB-myc protein expression in 293FT cells;Major S protein express in the cytoplasm.An interesting band about 40kD was visible in the resuult of Western blot, which was consistent with expected with expected size of recombinate protein of ALDOB-myc expressed in 293FT cells; About 26KD in the expected location of the Major S protein. (2)Co-immunoprecipitation method is successfully used for verifying of ALDOB protein as an interacting protein of surface antigen protein for hepatitis B virus.(3) Specific inhibition ALDOB expression of the HepG2 cell line, cell proliferation accelerated、invasion capacity enhanced and apoptosis rate increased than not inhibited ALDOB in HepG2 cell line.
Conclusion:(1)pCMV5-ALDOB-myc and pCMV4-majorS-flag have been constructed successfully.(2)ALDOB protein is novel binding protein of the major S protein of hepatitis B virus.(3) ALDOB can inhibit HepG2 cells proliferation、invasion ability and inhibit HepG2 cell apoptosis.
方法:(1)以人HEPG2细胞RNA为模板,用RT-PCR方法扩增出ALDOB基因。将PCR产物克隆进真核载体PCMV5内,构建含ALDOB基因的重组真核表达质粒。以实验室重组全S质粒为模板,利用PCR扩增主S蛋白基因,构建PCMV4-Flag-主S基因表达质粒,分别将重组质粒转染293FT细胞,用免疫荧光和Western blot等方法检测ALDOB及主S在293FT细胞中的表达。(2)将两种质粒共转293T细胞,激光共聚焦试验明确主S蛋白和醛缩酶B空间定位。将主S基因表达质粒稳转HepG2细胞,构建主S稳转株,通过免疫共沉淀方法,验证主S蛋白与醛缩酶B相互结合。(3)构建靶向醛缩酶B重组干扰质粒siALDOB-Pu6,导入HEPG2细胞中,筛选出干扰质粒的稳转株。通过细胞增殖实验、Transwell实验、细胞凋亡实验,探讨ALDOB在肝癌细胞HepG2生长、侵袭、凋亡等方面的作用。
结果:(1)核酸序列分析的结果表明,克隆的ALDOB基因与主S基因在GenBank中分别与已登记基因序列100%同源。免疫荧光结果显示:ALDOB蛋白在细胞质表达;主S蛋白主要存在于细胞质中。Western blot结果显示:在约40KD位置有目的条带,与预期的重组ALDOB蛋白大小一致;在约26KD位置有预期的主S蛋白表达。(2)通过免疫共沉淀方法,成功确认醛缩酶B是乙型肝炎病毒主S蛋白结合蛋白一种候选蛋白。(3)特异性抑制ALDOB表达的HepG2细胞株较没有抑制ALDOB的HepG2细胞株,细胞增殖加快、侵袭能力增强、凋亡率增加。稳定干扰ALDOB的HepG2细胞稳转株较稳转空载Pu6的HepG2细胞株生长
结论:(1)成功构建了含ALDOB基因以及主S基因的真核表达质粒。(2)醛缩酶B是乙型肝炎病毒主S蛋白的结合蛋白。(3)ALDOB能够抑制HepG2细胞增殖,使HepG2细胞侵袭能力下降,抑制HepG2细胞凋亡。
Objective: TO confirm the candidate hepatocyte binding protein ALDOB interacting with the surface antigen protein of hepatitis B virus and study on the function of Aldolase B.
Methods:(1) ALDOB gene was amplified by RT-PCR with the total RNA of human HepG2 cell as template.The PCR fragments were cloned into pCMV5 vector to construct recombinant eukaryotic expression plasmids.Whole S gene recombinant plasmid as a template by PCR amplification of the major S gene ,building PCMV4-Flag-SHBs expression vector. 293T cells were transfected with the recombinant plasmids. Immunofluorescence staining and Western blot were used to certificate the expressions of ALDOB-myc and major S in 293FT cells.(2)The two plasmids were co-transfected into 293FT, followed by confocal laser microscopy analysis to identify the co-localization of major S and ALDOB proteins. SHBs-flag gene expression plasmid was transfected into HepG2 cells to establish stable transfected major S protein cell line, using the co-immunoprecipitation method to verify binding of the major S protein and ALDOB protein. (3) Construction of recombinant targeting aldolase B interference plasmid siALDOB-Pu6, transfecting into HEPG2 cells to select the stability of interference plasmid transfer lines. By cell proliferation assays, Transwell experiments, apoptosis experiment, explore the role of ALDOB in HepG2 hepatoma cells by the growth, invasion and apoptosis.
Results: (1) The sequence of ALDOB and major S were 100% homology with difference human gene previously registered in GenBank. The result of Immunofluorescence staining manifest: recombinate protein of ALDOB-myc protein expression in 293FT cells;Major S protein express in the cytoplasm.An interesting band about 40kD was visible in the resuult of Western blot, which was consistent with expected with expected size of recombinate protein of ALDOB-myc expressed in 293FT cells; About 26KD in the expected location of the Major S protein. (2)Co-immunoprecipitation method is successfully used for verifying of ALDOB protein as an interacting protein of surface antigen protein for hepatitis B virus.(3) Specific inhibition ALDOB expression of the HepG2 cell line, cell proliferation accelerated、invasion capacity enhanced and apoptosis rate increased than not inhibited ALDOB in HepG2 cell line.
Conclusion:(1)pCMV5-ALDOB-myc and pCMV4-majorS-flag have been constructed successfully.(2)ALDOB protein is novel binding protein of the major S protein of hepatitis B virus.(3) ALDOB can inhibit HepG2 cells proliferation、invasion ability and inhibit HepG2 cell apoptosis.
引文
[1]Liu, J. and Fan, D. Hepatitis B in China. Lancet, 2007;369(9573): 1582-3.
[2]Lok, A.S. and McMahon, B.J. Chronic hepatitis B. Hepatology, 2007; 45(2): 507-39.
[3]Galibert F, Mandart E, Fitoussi F, Tiollais P, Charnay P. Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E.coli.Nature, 1979;281:646-650.
[4]Lupberger, J. and Hildt, E. Hepatitis B virus-induced oncogenesis. World J Gastroenterol, 2007;13(1): 74-81.
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[7]Hildt, E., Saher, G., Bruss, V. and Hofschneider, P.H.The hepatitis B virus large surfaceprotein (LHBs) is a transcriptional activator. Virology, 1996;225(1): 235-9.
[8]Hildt, E., Urban, S. and Hofschneider, P.H.Characterization of essential domains for thefunctionality of the MHBst transcriptional activator and identification of a minimal MHBstactivator. Oncogene, 1995;11(10):2055-66.
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[12]Bai GQ, Cheng J, Zhang SL, Huang YP, Wang L, Liu Y, Lin SM. Screening of hepatocyte proteins binding to complete S protein of hepatitis B virus by yeast-two hybrid system. World J Gastroenterol, 2005;11:3899-3900.
[13]Zhou F, Ren JL, Lu YP, Chen MY, Chen JM, Liu M, Zhang B and Dong. Screening of hepatocyte proteins binding to whole S protein of hepatitis B virus by yeasttwo-hybrid system. Zhonghua Gan Zang Bing Za Zhi, 2008;16:304-305.
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[2]Lok, A.S. and McMahon, B.J. Chronic hepatitis B. Hepatology, 2007; 45(2): 507-39.
[3]Galibert F, Mandart E, Fitoussi F, Tiollais P, Charnay P. Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E.coli.Nature, 1979;281:646-650.
[4]Lupberger, J. and Hildt, E. Hepatitis B virus-induced oncogenesis. World J Gastroenterol, 2007;13(1): 74-81.
[5]Hildt, E. and Hofschneider, P.H. The PreS2 activators of the hepatitis B virus: activators oftumour promoter pathways. Recent Results Cancer Res, 1998;(15)4:315-29.
[6]Hildt, E., Munz, B., Saher, G., Reifenberg, K. and Hofschneider, P.H. The PreS2 activator MHBs(t) of hepatitis B virus activates c-raf-1/Erk2 signaling in transgenic mice. EMBO Journal,2002;21(4): 525-35.
[7]Hildt, E., Saher, G., Bruss, V. and Hofschneider, P.H.The hepatitis B virus large surfaceprotein (LHBs) is a transcriptional activator. Virology, 1996;225(1): 235-9.
[8]Hildt, E., Urban, S. and Hofschneider, P.H.Characterization of essential domains for thefunctionality of the MHBst transcriptional activator and identification of a minimal MHBstactivator. Oncogene, 1995;11(10):2055-66.
[9]Blanchet M, Sureau C. Analysis of the cytosolic domains of the hepatitis B virus envelope proteins for their function in viral particle assembly and infectivity. J Virol, 2006;80:11935-11945.
[10]Lwa SH, Chen WN.Hepatitis B virus X protein interacts with beta5 subunit of heterotrimeric guanine nucleotide binding protein. J Virol,2005;2:1-8.
[11]Nijhara R, Jana SS, Goswami SK, Rana A, Majumdar SS, Kumar V, Sarkar DP. Sustained activation of mitogen-activated protein kinases and activator protein 1 by the hepatitis B virus X protein in mouse hepatocytes in vivo. J virol, 2001;75:10348-10358.
[12]Bai GQ, Cheng J, Zhang SL, Huang YP, Wang L, Liu Y, Lin SM. Screening of hepatocyte proteins binding to complete S protein of hepatitis B virus by yeast-two hybrid system. World J Gastroenterol, 2005;11:3899-3900.
[13]Zhou F, Ren JL, Lu YP, Chen MY, Chen JM, Liu M, Zhang B and Dong. Screening of hepatocyte proteins binding to whole S protein of hepatitis B virus by yeasttwo-hybrid system. Zhonghua Gan Zang Bing Za Zhi, 2008;16:304-305.
[14]A. Baldini, D. I. Smith, M. Rocchi, O. J. Miller, D. A. Miller .A human alphoid DNA clone from the EcoRI dimeric family: Genomic and internal organization and chromosomal assignment. Genomics,1989;5(4): 822-828.
[15]周玉球,卢义钦,刘俊凡.醛缩酶及其同工酶研究进展.国外医学分子生物学分册,1993;15:237-238.
[16]Hai Song, Shuang-Luo Xia, Cheng Liao, Yi-Liang Li, Yi-Fei Wang, Tsai-Ping Li, Mu-Jun Zhao.Genes encoding Pir51,Beclin1,RbAp48,and aldolas b are up or down-regulated in human primary hepatocellular carcinoma, World J Gastroenterol 2004;10(4):509-513.
[17]Liu J and Fan D. Hepatitis B in China. Lancet,2007;369:1582-1583.
[18]Lok AS,and McMahon BJ. Chronic hepatitis B. Hepatology, 2007;45:507-508.
[19]Oishi N,Shilagardi K,Nakamoto Y,Honda M,Kaneko S,Murakami S.Hepatitis B virus X protein overcomes oncogenic RAS-induced senescence in human immortalized cells.CancerSci,2007;98:1540-1548.
[20]Ou DP, Tao YM, Tang FQ, Yang LY. The hepatitis B virus X protein promotes hepatocellular carcinoma metastasis by upregulation of matrix metalloproteinases. Int J Cancer,2007;120:1208-1214.
[21]De Falco S, Ruvo M, Verdoliva A, Scarallo A, Raimondo D, Raucci A, Fassina G. N-terminal myristylation of HBV preS1 domain enhances receptor recognition. J Pept Res, 2001;57:390-400.
[22]蔺淑梅,张树林,成军,刘敏,王琳,王建军,杨倩,黄燕萍,白桂芹.肝细胞cDNA文库中乙型肝炎病毒前-前-S蛋白结合蛋白基因筛选.世界华人消化杂志,2004;12:2907-2910.
[23]Ryu CJ, Cho DY, Gripon P, Kim HS, Guguen-Guillouzo C, Hong HJ. An 80-kilodalton protein that binds to the pre-S1 domain of hepatitis B virus. J Virol, 2000;74:110-116.
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