JC病毒VP2蛋白的抗原抗体制备及核定位信号与入核转运受体的鉴定
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摘要
目的:
JC病毒属于人类多瘤病毒属,是一种重要的机会感染性疾病病原体。JC病毒广泛分布于人群中,有80﹪的成人血清学检测为阳性[1]。当免疫力低下时,JC病毒会引发多灶性脑白质病(PML)[2]。然而,JC病毒的临床检测,以及它的包膜蛋白VP2入核的机制和入核转运受体并不清楚。本研究拟通过原核表达、抗体制备、核定位信号鉴定和入核转运受体的鉴定几个方面初步阐释JC病毒的小包膜蛋白VP2基因在PML发生发展过程中的生物学功能。
方法:
(1)构建原核表达载体pET-32a(+)-VP2,转化BL21宿主菌后经IPTG诱导,获得了带有组氨酸标签的重组融合蛋白的优化表达。
(2)利用Ni+亲和柱纯化表达蛋白并免疫BALB /c小白鼠,获得抗VP2多克隆抗体。以纯化VP2为抗原,利用Western blot和ELISA法对多克隆抗体进行特异性和效价检测。
(3)构建重组绿色荧光表达载体pEGFP-C1-VP2,转染7402细胞,24hr后观察各截短体的亚细胞定位情况。
(4)表达纯化pGEX-2T-importin(importinα1、3、5、7和imporinβ)的GST融合蛋白。
(5)通过GST-pull down方法,寻找并确定VP2的入核转运受体。
结果:
(1)通过PCR方法从病人的脑脊液中获得VP2的目的片段,并成功构建了pET-32a(+)-VP2原核表达载体。pET-32a(+)-VP2原核表达载体在IPTG的诱导作用下,成功表达融合蛋白通过Ni+亲和层析法获得了融合蛋白纯品。
(2)用纯化的VP2融合蛋白免疫BALB/c小鼠,成功制备了高效价,高特异性的鼠抗VP2多克隆抗体。通过ELISA法表明多克隆抗体效价>1:160,000, Western blot检测证明多克隆抗体的特异性良好。
(3)通过PCR方法获得了VP2截短体的目的片段,成功构建了VP2各截短体的绿色荧光表达载体;将构建好的VP2各截短体绿色荧光表达载体转染7402人肝癌细胞,通过各截短体的亚细胞定位分析出了VP2蛋白的核定位信号为946-972段。
(4)成功表达纯化了五个pGEX-2Timportin融合蛋白。
(5)通过GST-pull down实验,分析发现VP2能够与入核转运受体蛋白importinα1、importinα3结合,从而被转运入细胞核,发挥它的生理和生化作用。
结论:
1.我们成功构建了pET-32a(+)- Vp2原核表达载体,在IPTG的诱导下,成功表达纯化了VP2融合蛋白。这就为抗体的制备以及研究VP2蛋白与其它蛋白的相互作用做了物质上的准备。
2.将VP2蛋白免疫BALB/c小鼠,制备了高效价、高特异性的鼠抗VP2多克隆抗体。这对于VP2抗原的检测,以及免疫组化奠定了基础。
3.成功的鉴定出VP2的核定位信号,通过构建四个pEGFP-C1-Vp2绿色荧光载体,脂质体转染7402细胞株,在荧光显微镜下观察pEGFP-C1-Vp2各截短体的亚细胞定位。这是第一次鉴定出VP2的核定位信号,为进一步的研究提供了信息。
4.通过GST-pull down方法,我们成功的鉴定出VP2的入核转运受体为importinα1、importinα3。初步阐明了VP2入核过程中参与的入核转运受体。
Background and Objective:
JC virus (JCV) belongs to the polyomavirus family. JCV is widespread throughout the human population with at least 80% of adults exhibiting antibodies specific for the virus. When the immune system was suppressed, JCV will replicate in oligodendrocytes cell resulting in PML(progressive multifocal leukoencephalopathy). the purpose of the study is to clarify biological function of VP2 in the course of PML through the following aspects, such as subcellular orientation, prokaryotic expression, antibody preparation and NLS and importin protein.
Methods:
(1) The prokaryotic expressive vector pET-32a(+)-VP2 was constructed and transformed into the competent BL21 E. coli. The pET-32a(+)-VP2 was induced with IPTG ( Isopropylβ- D -1-thiogalactopyranoside ) , analyzed using SDS-PAGE ( sodium dodecyl sulfate polyacrylamide gel electrophoresis) and identified using Western blot. The expressed product was purified by using Ni+ affinity column chromatography.
(2) The purified pET-32a(+)-VP2 fusion protein was used to immunize BALB/c mouse to produce polyclonal antibody. The specificity and potency of polyclonal antibody were evaluated by using Western blot and ELISA.
(3) Construction of recombined green fluorescent protein expressive vector pEGFP-C1- VP2(complete,1-972,1-945,946-972). 7402 cells were transfected, and observed by fluorescent inverted microscope after 24hr.
(4) The expressive vector pGEX-2T-importin was transformed into the competent BL21 E. coli. The importin protein was induced with IPTG and analyzed using SDS-PAGE. The expressed product was purified by using Ni+ affinity column chromatography.
(5) We found the importin binding to VP2 by using the GST-pull down assay.
Results:
(1) The purified pET-32a(+)-VP2 fusion protein was used to immunize BALB/c mouse to produce polyclonal antibody. The ELISA manifested the titer of polyclonal antibody >1:160,000. The high specificity was testified using Western blot.
(2) The eukaryotic expressive vector pEGFP-C1-VP2(complete,1-972,1-945 , 946-972) was successfully constructed. The eukaryotic expressive vector pEGFP-C1-VP2 was transfected into the 7402 cell strain, pEGFP-C1-VP2(complete) and pEGFP-C1-VP2(1-972) and (946-972)can be subcellularly located in nucleus. pEGFP- C1-VP2(1-945) can be subcellularly located in cytoplasm by its visible green fluorescent signal. These evidence indicated that its nuclear localization signal (NLS)located in 946-972bp.
(3) The importin fusion protein was expressed successfully in the prokaryote expressing system through SDS-PAGE analysis.
(4) We found VP2 binding to imprtinα1、α3 by GST-pull down assay, the binding complex could pass through the NPC and come into nucleus.
Conclusions:
1. The VP2 fusion protein was induced and expressed successfully by E. coli prokaryote expressing system. The protein provided materials for Polyclone antibody and interaction between two protein.
2. Highly specific and potent polyclonal antibody of VP2 was prepared. The polyclonal antibody provided valuable material for the further study on the meaning of VP2 in the course of PML by other methods such as immune histochemistry
3. Our work proved that VP2 subcellularly located in nucleus and NLS located in 946-972bp. This is the first time that we identified the VP2 NLS, and provided information for the further study.
4. We found VP2 binding to imprtinα1andα3, and the binding complex could pass through NPC and come into nucleus. We found that the importin was binding to VP2.
JC病毒属于人类多瘤病毒属,是一种重要的机会感染性疾病病原体。JC病毒广泛分布于人群中,有80﹪的成人血清学检测为阳性[1]。当免疫力低下时,JC病毒会引发多灶性脑白质病(PML)[2]。然而,JC病毒的临床检测,以及它的包膜蛋白VP2入核的机制和入核转运受体并不清楚。本研究拟通过原核表达、抗体制备、核定位信号鉴定和入核转运受体的鉴定几个方面初步阐释JC病毒的小包膜蛋白VP2基因在PML发生发展过程中的生物学功能。
方法:
(1)构建原核表达载体pET-32a(+)-VP2,转化BL21宿主菌后经IPTG诱导,获得了带有组氨酸标签的重组融合蛋白的优化表达。
(2)利用Ni+亲和柱纯化表达蛋白并免疫BALB /c小白鼠,获得抗VP2多克隆抗体。以纯化VP2为抗原,利用Western blot和ELISA法对多克隆抗体进行特异性和效价检测。
(3)构建重组绿色荧光表达载体pEGFP-C1-VP2,转染7402细胞,24hr后观察各截短体的亚细胞定位情况。
(4)表达纯化pGEX-2T-importin(importinα1、3、5、7和imporinβ)的GST融合蛋白。
(5)通过GST-pull down方法,寻找并确定VP2的入核转运受体。
结果:
(1)通过PCR方法从病人的脑脊液中获得VP2的目的片段,并成功构建了pET-32a(+)-VP2原核表达载体。pET-32a(+)-VP2原核表达载体在IPTG的诱导作用下,成功表达融合蛋白通过Ni+亲和层析法获得了融合蛋白纯品。
(2)用纯化的VP2融合蛋白免疫BALB/c小鼠,成功制备了高效价,高特异性的鼠抗VP2多克隆抗体。通过ELISA法表明多克隆抗体效价>1:160,000, Western blot检测证明多克隆抗体的特异性良好。
(3)通过PCR方法获得了VP2截短体的目的片段,成功构建了VP2各截短体的绿色荧光表达载体;将构建好的VP2各截短体绿色荧光表达载体转染7402人肝癌细胞,通过各截短体的亚细胞定位分析出了VP2蛋白的核定位信号为946-972段。
(4)成功表达纯化了五个pGEX-2Timportin融合蛋白。
(5)通过GST-pull down实验,分析发现VP2能够与入核转运受体蛋白importinα1、importinα3结合,从而被转运入细胞核,发挥它的生理和生化作用。
结论:
1.我们成功构建了pET-32a(+)- Vp2原核表达载体,在IPTG的诱导下,成功表达纯化了VP2融合蛋白。这就为抗体的制备以及研究VP2蛋白与其它蛋白的相互作用做了物质上的准备。
2.将VP2蛋白免疫BALB/c小鼠,制备了高效价、高特异性的鼠抗VP2多克隆抗体。这对于VP2抗原的检测,以及免疫组化奠定了基础。
3.成功的鉴定出VP2的核定位信号,通过构建四个pEGFP-C1-Vp2绿色荧光载体,脂质体转染7402细胞株,在荧光显微镜下观察pEGFP-C1-Vp2各截短体的亚细胞定位。这是第一次鉴定出VP2的核定位信号,为进一步的研究提供了信息。
4.通过GST-pull down方法,我们成功的鉴定出VP2的入核转运受体为importinα1、importinα3。初步阐明了VP2入核过程中参与的入核转运受体。
Background and Objective:
JC virus (JCV) belongs to the polyomavirus family. JCV is widespread throughout the human population with at least 80% of adults exhibiting antibodies specific for the virus. When the immune system was suppressed, JCV will replicate in oligodendrocytes cell resulting in PML(progressive multifocal leukoencephalopathy). the purpose of the study is to clarify biological function of VP2 in the course of PML through the following aspects, such as subcellular orientation, prokaryotic expression, antibody preparation and NLS and importin protein.
Methods:
(1) The prokaryotic expressive vector pET-32a(+)-VP2 was constructed and transformed into the competent BL21 E. coli. The pET-32a(+)-VP2 was induced with IPTG ( Isopropylβ- D -1-thiogalactopyranoside ) , analyzed using SDS-PAGE ( sodium dodecyl sulfate polyacrylamide gel electrophoresis) and identified using Western blot. The expressed product was purified by using Ni+ affinity column chromatography.
(2) The purified pET-32a(+)-VP2 fusion protein was used to immunize BALB/c mouse to produce polyclonal antibody. The specificity and potency of polyclonal antibody were evaluated by using Western blot and ELISA.
(3) Construction of recombined green fluorescent protein expressive vector pEGFP-C1- VP2(complete,1-972,1-945,946-972). 7402 cells were transfected, and observed by fluorescent inverted microscope after 24hr.
(4) The expressive vector pGEX-2T-importin was transformed into the competent BL21 E. coli. The importin protein was induced with IPTG and analyzed using SDS-PAGE. The expressed product was purified by using Ni+ affinity column chromatography.
(5) We found the importin binding to VP2 by using the GST-pull down assay.
Results:
(1) The purified pET-32a(+)-VP2 fusion protein was used to immunize BALB/c mouse to produce polyclonal antibody. The ELISA manifested the titer of polyclonal antibody >1:160,000. The high specificity was testified using Western blot.
(2) The eukaryotic expressive vector pEGFP-C1-VP2(complete,1-972,1-945 , 946-972) was successfully constructed. The eukaryotic expressive vector pEGFP-C1-VP2 was transfected into the 7402 cell strain, pEGFP-C1-VP2(complete) and pEGFP-C1-VP2(1-972) and (946-972)can be subcellularly located in nucleus. pEGFP- C1-VP2(1-945) can be subcellularly located in cytoplasm by its visible green fluorescent signal. These evidence indicated that its nuclear localization signal (NLS)located in 946-972bp.
(3) The importin fusion protein was expressed successfully in the prokaryote expressing system through SDS-PAGE analysis.
(4) We found VP2 binding to imprtinα1、α3 by GST-pull down assay, the binding complex could pass through the NPC and come into nucleus.
Conclusions:
1. The VP2 fusion protein was induced and expressed successfully by E. coli prokaryote expressing system. The protein provided materials for Polyclone antibody and interaction between two protein.
2. Highly specific and potent polyclonal antibody of VP2 was prepared. The polyclonal antibody provided valuable material for the further study on the meaning of VP2 in the course of PML by other methods such as immune histochemistry
3. Our work proved that VP2 subcellularly located in nucleus and NLS located in 946-972bp. This is the first time that we identified the VP2 NLS, and provided information for the further study.
4. We found VP2 binding to imprtinα1andα3, and the binding complex could pass through NPC and come into nucleus. We found that the importin was binding to VP2.
引文
[1] Major EO, Amemiya K, Tornatore CS, Houff SA, Berger JR. Pathogenesis and molecular biology of progressive multifocal leukoencephalopathy, the JC virus-induced demyelinating disease of the human brain. Clin Microbiol Rev 1992; 5: 49-73.
[2] Koralnik IJ. Overview of the cellular immunity against JC virus in progressive multifocal leukoencephalopathy. J Neurovirol 2002; 8 Suppl 2: 59-65.
[3] Padgett BL, Walker DL, ZuRhein GM, Eckroade RJ. Cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy. Lancet1971; 1: 1257-60.
[4] Gordon J, Khalili K. The human polyomavirus, JCV, and neurological diseases (review). Int J Mol Med 1998; 1:647-55.
[5] Khalili K, Gordon J, White MK. The polyomavirus, JCV, and its involvement in human disease. In Ahsan Ned. Polyomaviruses and human diseases . Landes Bioscience,2005: 21.1-21.14.
[6] Safak M, Khalili K. An overview: human polyomavirus JC virus and its associated disorders. J Neurovirol 2003; 9 Suppl 1: 3-9.
[7] Richardson EP. Progressive multifocal leukoencephalopathy. N Engl J Med 1961; 265: 815-23.
[8] White MK, Khalili K. Polyomaviruses and human cancer: molecular mechanisms underlying patterns of tumorigenesis. Virology 2004; 324: 1-16.
[9] White MK, Khalili K. Signaling pathways and polyomavirus oncoproteins: importance in malignant transformation.Gene Ther Mol Biol 2004; 8: 19-30.
[10] Khalili K,White MK, Sawa H, Nagashima K, Safak M. The agnoprotein of polyomaviruses: a multifunctional auxiliary protein. J Cell Physiol 2005; 204: 1-7.
[11] Ishii, N., N. Minami, E. Y. Chen, A. L. Medina, M. M. Chico, and H.Kasamatsu.. Analysis of a nuclear localization signal of simian virus 40 major capsid protein Vp1. J. Virol. 1996;70:1317–1322.
[12] Shishido-Hara, Y., Y. Hara, T. Larson, K. Yasui, K. Nagashima, and G. L.Stoner. Analysis of capsid formation of human polyomavirus JC (Tokyo-1 strain) by a eukaryotic expression system: splicing of late RNAs, translation and nuclear transport of major capsid protein VP1, and capsid assembly. J. Virol. 2000;74:1840–1853.
[13]查锡良。医学分子生物学(供研究生用)。北京:人民卫生出版社,2003.274
[1] Dingwall C,Sharnick S V, Laskey RA. A poly peptide domain that Specifies migration of nucleoplasmin into the nucleus[J].Cell,1982,30(2):449-458
[2] Jain A K, Bloom D A,Jaiswal A K.Nuclear import and export signals in control of Nrf2[J].J Biol Chem,2005,280(32):29158-29168
[3] Zhang X,Wang K S,Wang Z Q, et al.Nuclear localization signal of ING4 plays a key role in its binding to p53[J].Biochem BiophysRes Commun,2005,331(4): 1032-1038
[4] Cronshaw JM,Krutchinsky AN,Zhang W,et al.Proteomic analysis of the mammalian nuclear pore complex[J].J Cell Biol,2002,158 (5):915-927
[5] Rout M P,Aitchison J D,Suprapto A,et al.The yeast nuclear pore complex: composition,architecture,and transport mechanism[J].J Cell Biol,2000,148(4): 635-651
[6] Suntharalingam, M. & Wente, S. R. Peering through the pore: nuclear pore complex structure, assembly, and function. Dev. Cell,2003,(4):775-789.
[7] Ryan K J, Wente S R.The nuclear pore complex:a protein machineBridging the nucleus and cytoplasm[J].Curr Opin Cell Biol,2000,12(3):361-371
[8] Vasu S K, Forbes D J. Nuclear pores and nuclear assembly[J].CurrOpin Cell Biol,2001,13(3):363-375
[9] Rout M P, AitchisonJ D,Suprapto A,et al.The yeast nuclear pore Complex: composition, architecture, and transport mechanism[J].J Cell Biol,2000,148 (4):635-651
[10] Ribbeck K, Gorlich D. Kinetic analysis of translocation through Nuclear pore complexes[J].EMBO J,2001,20(6):1320-1330
[11] Kohler M, Fiebeler A, Hartwig M, et al. Differential expression of Classical nuclear transport factors during cellular proliferation and differentiation [J].Cell Physiol Biochem,2002,12(5-6):335-344
[12] Gasiorowski J Z, Dean D A. Mechanisms of nuclear transport and interventions[J].Adv Drug Deliv Rev, 2003, 55(6): 703-716
[13] Shah S, Forbes DJ: Separate nuclear import pathways converge on the nucleoporin Nup153 and can be dissected with dominant-negative inhibitors Curr Biol 1998, 8:1376-1386
[14] Rihs H P, Jans A, Fan H, et al. Therate of nuclear cytoplasmic protein transportis determined by the case in kinaseⅡsite flank in the nuclear localization sequence of the SV40 T-antigen.EMBOJ,1991,10(3):633~639
[15] Jans D A, Ackermann M J, Bischoff J R, et al. p34cdc2-mediate phosphorylation at T1 24 inhibits nuclear import of SV-40T antigen proteins. J Cell Biol, 1991,115(5):1203~1207
[2] Koralnik IJ. Overview of the cellular immunity against JC virus in progressive multifocal leukoencephalopathy. J Neurovirol 2002; 8 Suppl 2: 59-65.
[3] Padgett BL, Walker DL, ZuRhein GM, Eckroade RJ. Cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy. Lancet1971; 1: 1257-60.
[4] Gordon J, Khalili K. The human polyomavirus, JCV, and neurological diseases (review). Int J Mol Med 1998; 1:647-55.
[5] Khalili K, Gordon J, White MK. The polyomavirus, JCV, and its involvement in human disease. In Ahsan Ned. Polyomaviruses and human diseases . Landes Bioscience,2005: 21.1-21.14.
[6] Safak M, Khalili K. An overview: human polyomavirus JC virus and its associated disorders. J Neurovirol 2003; 9 Suppl 1: 3-9.
[7] Richardson EP. Progressive multifocal leukoencephalopathy. N Engl J Med 1961; 265: 815-23.
[8] White MK, Khalili K. Polyomaviruses and human cancer: molecular mechanisms underlying patterns of tumorigenesis. Virology 2004; 324: 1-16.
[9] White MK, Khalili K. Signaling pathways and polyomavirus oncoproteins: importance in malignant transformation.Gene Ther Mol Biol 2004; 8: 19-30.
[10] Khalili K,White MK, Sawa H, Nagashima K, Safak M. The agnoprotein of polyomaviruses: a multifunctional auxiliary protein. J Cell Physiol 2005; 204: 1-7.
[11] Ishii, N., N. Minami, E. Y. Chen, A. L. Medina, M. M. Chico, and H.Kasamatsu.. Analysis of a nuclear localization signal of simian virus 40 major capsid protein Vp1. J. Virol. 1996;70:1317–1322.
[12] Shishido-Hara, Y., Y. Hara, T. Larson, K. Yasui, K. Nagashima, and G. L.Stoner. Analysis of capsid formation of human polyomavirus JC (Tokyo-1 strain) by a eukaryotic expression system: splicing of late RNAs, translation and nuclear transport of major capsid protein VP1, and capsid assembly. J. Virol. 2000;74:1840–1853.
[13]查锡良。医学分子生物学(供研究生用)。北京:人民卫生出版社,2003.274
[1] Dingwall C,Sharnick S V, Laskey RA. A poly peptide domain that Specifies migration of nucleoplasmin into the nucleus[J].Cell,1982,30(2):449-458
[2] Jain A K, Bloom D A,Jaiswal A K.Nuclear import and export signals in control of Nrf2[J].J Biol Chem,2005,280(32):29158-29168
[3] Zhang X,Wang K S,Wang Z Q, et al.Nuclear localization signal of ING4 plays a key role in its binding to p53[J].Biochem BiophysRes Commun,2005,331(4): 1032-1038
[4] Cronshaw JM,Krutchinsky AN,Zhang W,et al.Proteomic analysis of the mammalian nuclear pore complex[J].J Cell Biol,2002,158 (5):915-927
[5] Rout M P,Aitchison J D,Suprapto A,et al.The yeast nuclear pore complex: composition,architecture,and transport mechanism[J].J Cell Biol,2000,148(4): 635-651
[6] Suntharalingam, M. & Wente, S. R. Peering through the pore: nuclear pore complex structure, assembly, and function. Dev. Cell,2003,(4):775-789.
[7] Ryan K J, Wente S R.The nuclear pore complex:a protein machineBridging the nucleus and cytoplasm[J].Curr Opin Cell Biol,2000,12(3):361-371
[8] Vasu S K, Forbes D J. Nuclear pores and nuclear assembly[J].CurrOpin Cell Biol,2001,13(3):363-375
[9] Rout M P, AitchisonJ D,Suprapto A,et al.The yeast nuclear pore Complex: composition, architecture, and transport mechanism[J].J Cell Biol,2000,148 (4):635-651
[10] Ribbeck K, Gorlich D. Kinetic analysis of translocation through Nuclear pore complexes[J].EMBO J,2001,20(6):1320-1330
[11] Kohler M, Fiebeler A, Hartwig M, et al. Differential expression of Classical nuclear transport factors during cellular proliferation and differentiation [J].Cell Physiol Biochem,2002,12(5-6):335-344
[12] Gasiorowski J Z, Dean D A. Mechanisms of nuclear transport and interventions[J].Adv Drug Deliv Rev, 2003, 55(6): 703-716
[13] Shah S, Forbes DJ: Separate nuclear import pathways converge on the nucleoporin Nup153 and can be dissected with dominant-negative inhibitors Curr Biol 1998, 8:1376-1386
[14] Rihs H P, Jans A, Fan H, et al. Therate of nuclear cytoplasmic protein transportis determined by the case in kinaseⅡsite flank in the nuclear localization sequence of the SV40 T-antigen.EMBOJ,1991,10(3):633~639
[15] Jans D A, Ackermann M J, Bischoff J R, et al. p34cdc2-mediate phosphorylation at T1 24 inhibits nuclear import of SV-40T antigen proteins. J Cell Biol, 1991,115(5):1203~1207