摘要
目的(1)构建定位于溶酶体、泛素的PrP表达载体,并进行蛋白表达特点及定位的鉴定;(2)动物免疫后,初步研究此融合表达载体在小鼠体内激起的细胞免疫反应和体液免疫反应,得到一组最基础的免疫数据,为以后朊蛋白疫苗的研究提供基础数据。
方法(1)将泛素基因、溶酶体膜定位信号序列基因和PrP基因连接,克隆至pcDNA3.1载体中,构建表达载体pcDNA3.1-UPrP、pcDNA3.1-PrPL;瞬时转染真核表达细胞,经Western Blot和间接免疫荧光技术检测PrP表达特点;(2)小鼠分八组,其中四组小鼠按0、14、28天分三次分别用质粒pcDNA3.1、pcDNA3.1-HuPrP、pcDNA3.1-UbPrP、pcDNA3.1-PrPL免疫;另四组小鼠中,有一组按0、14、28天分三次免疫重组PrP,另三组分别用质粒pcDNA3.1-HuPrP、pcDNA3.1-UbPrP、pcDNA3.1-PrPLⅡ首次免疫,PrP蛋白加强两次。最后一次免疫后2周,取脾脏及血液进行ELISPOT、ELISA、WesternBlot检测。
结果(1)构建的各种PrP定位表达载体均可定位表达具有三种类型的糖基化分子的PrP,以双糖基化分子类型最多。带有泛素、溶酶体信号尾的质粒pcDNA3.1-UbPrP、pcDNA3.1-PrPLⅡ的PrP表达随着时间的延长蛋白表达量下降,提示泛素、溶酶体信号能加速表达的PrP在细胞内的降解。(2)动物免疫后,重组蛋白增强免疫的四组小鼠脾淋巴细胞在全长人PrP的刺激下,UbPrP+rPrP组有两只小鼠的脾淋巴细胞分泌特异性IFN-γ,分别为215、50SFCs/106/PBMCs;PrP+rPrP组中有一只小鼠为113.5 SFCs/106/PBMCs,PrPLⅡ+rPrP组和rPrP组的小鼠脾淋巴细胞不分泌特异性IFN-γ;在DNA核酸疫苗免疫的四组小鼠脾淋巴细胞中,仅UbPrP组的淋巴细胞两只小鼠淋巴细胞能产生明显的斑点,分别为56、50 SFCs/106/PBMC,其他组的脾淋巴细胞在重组蛋白的刺激下均为阴性。小鼠抗血清ELISA结果显示,重组蛋白组免疫小鼠的抗血清的滴度为1∶51200,经质粒初免、重组蛋白增强小鼠抗血清效价在大于1∶102400,明显高于单纯重组蛋白免疫组;UbPrP组及PrPLⅡ组质粒免疫小鼠抗血清滴度大于1∶4 00;未修饰PrP组及空载体pcDNA3.1组小鼠抗血清与免疫前血清的P/N值小于2.1。各组抗血清特异性检测的WB结果显示:各组免疫小鼠的抗血清均可特异性识别重组人PrP。
结论:成功构建了溶酶体、泛素定位表达的PRNP核酸疫苗载体pcDNA3.1-UbPrP、pcDNA3.1-PrPLⅡ;与泛素融合表达及定位于溶酶体的PrP能一定程度地打破免疫耐受,诱导特异性免疫反应。
Objective: To evaluate PrP expression characteristic and the basic data of PRNP nucleic acid vaccine with ubiquitin or the lysosome-targeting signal.
Method: The gene of ubiquitin and lysosome-targeting signal were ligated to PRNP and pcDNA3.1 vector, that is, pcDNA3.1-UbPrP and pcDNA3.1-PrPLⅡwere constructed. The expression characteristics of PrP with two signals were evaluated by western blot and the localization was observed by indirect immune fluorescence. The first four group mice were immuned by DNA vaccine. Each group was made of four mice, and each mouse received three DNA immunise at 14-day intervals. The other four group mice were immuned by DNA vaccine and boosted by recombinant prion protein. The immune responses of different groups were analyzed in 2 weeks after the last immunization. Specificity and sensitivity of antibodies were detected by both ELISA and Western blotting. Lymphocyte were collected and stimulated with recombinant human PrP protein from different groups, IFN-r was detected by ELISPOT.
Result: The protein expressed by pcDNA3.1-UbPrP and pcDNA3.1-PrPLⅡwith ubiquitin and lysosome-targeting signal can be recognized by prion-specific antibody. The protein has three glycosylation molecules form as native PrP. PrP with ubiquitin was degraded gradually with time extension, whereas quantity of PrP with lysosome signal reduced in 48h after transfection. The protein with two location signals can direct fusion prion proteins into cytoplasm. The ELISPOT result show, there are 56、50 SFCs/106/PBMC in two mice of the UbPrP group respectively, 215、50SFCs/106/PBMCs in two mice of the UbPrP+rPrP group respectively, 113.5 SFCs/106/PBMCs in one mouse of the PrP+rPrP group. Lymphocyte from the other mice do not secreated PrP-specific IFN-γ. The ELISA results found that the titer of serum from mice immunized with a plasmid encoding both ubiquitin-tagged and lysosome-targeting signal PrP were above 1:400. But P/N levels of the serum titers from mice immunized with pcDNA3.1-PrP or pcDNA3.1 were below 2.1 The titer of serum from the protein boost group were much higher than the DNA immune group. The titers were above 1:51200 in the rPrP group, and the titers of mice with plasmid Primary Immunizied and protein boosted were above 1:102400. Antibodies induced after immunized with pcDNA3.1-UbPrP, pcDNA3.1-PrPLⅡ, pcDNA3.1-UbPrP+rPrP, pcDNA3.1-PrP+rPrP, pcDNA3.1-PrPLⅡ+rPrP could recognize the recombinant PrP proteinby Western bloting. The pcDNA3.1-UbPrP can breaked immune tolerance and enhanced both humoral and cellular responses against human prion protein.
Conclusion: The PRNP vaccine with ubiquitin or the lysosome-targeting signal were constructed and expressed in eukaryocyte successfully. The vaccine with ubiquitin can break immune tolerance against the prion protein in wild-type mice, resulting in the induction of PrP-specific antibody and T-cell responses.
引文
[1] Hill A F. Antoniou M. Collinge J. Protease-resistant prion protein produced in vitro lacks detectable infectivity. J Gen Virol, 1999; 80(pt 1): 11–14.
[2] Kocisko D A. Come J H. Priola S A. et al. Cell-free formation of protease-resistant prion protein. Nature, 1994, 370(6489): 471–474.
[3] Saborio G P. Permanne B. Soto C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature, 2001, 411(6839): 810–813.
[4] Castilla J. Saa′P. Hetz C. Soto C. In vitro generation of infectious scrapie prions. Cell, 2005, 121(2): 195–206.
[5] Prusiner S B. Prions. Proc Natl Acad Sci U S A, 1998, 95(23): 13363-13383.
[6] Bueler H. Aguzzi A. Sailer A. et al. Mice devoid of PrP are resistant to scrapie. Cell, 1993, 73(7): 1339–1347.
[7] astilla J. Saa′P. Soto C. Detection of prions in blood. Nat Med, 2005, 11: 982-985.
[8] Kretzschmar H A. Prusiner S B. Stowring L E. DeArmond S J. Scrapie prion proteins are synthesized in neurons. Am J Pathol, 1986, 122(1): 1-5.
[9] Moudjou M. Frobert Y. Grassi J. La Bonnardiere C. Cellular prion protein status in sheep: tissue-specific biochemical signatures. J Gen Virol, 2001, 82(pt 8): 2017-2024.
[10] Prusiner S B. Novel proteinaceous infectious particles cause scrapie. Science, 1982, 216(4542): 136–144.
[11] Prusiner S B. Groth D. Serban A. Koehler R. Foster D. Torchia M. Burton D. Yang S L. DeArmond S J. Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies. Proc Natl Acad Sci U S A, 1993, 90(22): 10608-10612.
[12] Sailer A. Bueler H. Fischer M. Aguzzi A. Weissmann C. No propagation of prions in mice devoid of PrP. Cell, 1994, 77(7): 967-968.
[13] Bruce M E. Will R G. Ironside J W. McConnell I. et al. Transmissions to mice indicate that‘new variant’CJD is caused by the BSE agent. Nature, 1997, 389(6650):498–501.
[14] Ricketts M N. Public health and the BSE epidemic.Curr Top Microbiol Immunol, 2004, 284:99–119.
[15] Llewelyn C A. Hewitt P E. Knight R S. Amar K. Cousens S. Mackenzie J. Will R G. Possible transmission of variant Creutzfeldt–Jakob disease by blood transfusion. Lancet., 2004, 363(9407): 417–424.
[16] Citron M. Strategies for disease modification in Alzheimer’s disease. Nat Rev Neurosci, 2004, 5(9): 677–685.
[17] Monsonego A. Weiner H L. Immunotherapeutic approaches to Alzheimer’s disease. Science, 2003, 302(5646):834–838.
[18] Caughey B. Lansbury P T. Protofibrils pores fibrils and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Annu Rev Neurosci, 2003, 26: 267–298.
[19] Prusiner S B. Early evidence that a protease-resistant protein is an active component of the infectious prion. Cell, 2004. 116 (2 Suppl):S109, 1 p following S113.
[20] Flechsig E. Weissmann C. The role of PrP in health and disease. Curr Mol Med. 2004, 4(4): 337–353.
[21] Ford M J. Burton L J. Morris R J. Hall S M. Selective expression of prion protein in peripheral tissues of the adult mouse.. Neuroscience , 2002, 113(1): 177–192.
[22] Magri G. Clerici M. Dall’Ara P. et al. Decrease in pathology and progression of scrapie after immunisation with synthetic prion protein peptides in hamsters. Vaccine, 2005. 23(22): 2862–2868.
[23] Schwarz A. Kratke O. Burwinkel M. Riemer C. Schμltz J. Henklein P. Bamme T. Baier M. Immunisation with a synthetic prion protein-derived peptide prolongs survival times of mice orally exposed to the scrapie agent. Neurosci Lett, 2003, 350(3): 187–189.
[24] Koller M F. Grau T. Christen P. Induction of antibodies against murine full-length prion protein in wild-type mice. J Neuroimmunol, 2002, 132(1-2): 113–116.
[25] Sigurdsson E M. Brown D R. Daniels M. Kascsak R J. Kascsak R. Carp R. Meeker H C. Frangione B. Wisniewski T. Immunization delays the onset of prion disease in mice. Am J Pathol, 2002, 161(1): 13–17.
[26] Gilch S. Wopfner F. Renner-Muller I. Kremmer E. Bauer C. et al. Polyclonal anti-PrP auto-antibodies induced with dimeric PrP interfere efficiently with PrPSc propagation in prion-infected cells. J Biol Chem, 2003, 278(20): 18524–18531.
[27] Polymenidou M. Heppner F L. Pellicioli E C. Urich E. Miele G. Braun N. Wopfner F. Schatzl H M. Becher B. Aguzzi A. Humoral immune response to native eukaryotic prion protein correlates with anti-prion protection. Proc Natl Acad Sci U S A. 2004, 101 Suppl 2 : 14670–14676.
[28] Nikles D. Bach P. Boller K. Merten C A. Montrasio F. Heppner F L. Aguzzi A. Cichutek K. Kalinke U. Buchholz C J. Circumventing tolerance to the prion protein (PrP):vaccination with PrP-displaying retrovirus particles induces humoral immune responses against the native form of cellular PrP. J Virol. 2005, 79(7): 4033–4042.
[29] Goni F. Knudsen E. Schreiber F. Scholtzova H. Pankiewicz J. Carp R. Meeker H C. Rubenstein R. Brown D R. Sy M S. Chabalgoity J A. Sigurdsson E M. Wisniewski T. Mucosal vaccination delays or prevents prion infection via an oral route. Neuroscience, 2005, 133(2): 413–421.
[30] Sethi S. Lipford G. Wagner H. Kretzschmar H. Postexposure prophylaxis against prion disease with a stimμlator of innate immunity. Lancet, 2002, 360(9328): 229–230.
[31] Tal Y. Souan L. Cohen I R. Meiner Z. Taraboulos A. Mor F. Complete Freund’s adjuvantimmunization prolongs survival in experimental prion disease in mice. J Neurosci Res, 2003, 71(2): 286–290.
[32] Fernandez-Borges N. Brun A. Whitton J L. Parra B. Diaz-San Segundo F. Salguero F J. Torres J M. Rodriguez F. DNA vaccination can break immunological tolerance to PrP in wild-type mice and attenuates prion disease after intracerebral challenge. J Virol, 2006, 80(20): 9970–9976.
[33] Ishibashi D. Yamanaka H. Yamaguchi N. Yoshikawa D. Nakamura R. Okimura N. Yamaguchi Y. Shigematsu K. Katamine S. Sakaguchi S. Immunization with recombinant bovine but not mouse prion protein delays the onset of disease in mice inoculated with a mouse-adapted prion. Vaccine , 2007, 25(6): 985–992.
[34] Cindy Nitschke. Eckhard Flechsig. Jens van den Brandt. Nele Lindner. Thorsten Lu¨hrs.ΜLf Dittmer. Michael A. Klein a. Immunisation strategies against prion diseases: prime-boost immunisation with a PrP DNA vaccine containing foreign helper T-cell epitopes does not prevent mouse scrapie. Veterinary Microbiology, 2007, 123 (4):367–376.
[35] Gabizon R. McKinley M P. Groth D. Prusiner S B. Immunoaffinity purification and neutralization of scrapie prion infectivity. Proc Natl Acad Sci U S A, 1988:85(18): 6617–6621.
[36] Donofrio G. Heppner F L. Polymenidou M. Musahl C. Aguzzi A. Paracrine inhibition of prion propagation by anti-PrP single-chain Fv miniantibodies. J Virol, 2005, 79(13): 8330–8338.
[37] Enari M. Flechsig E. Weissmann C. Scrapie prion protein accumμlation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody. Proc Natl Acad Sci USA, 2001, 98(16): 9295–9299.
[38] Peretz D. Williamson R A. Kaneko K. Vergara J. Leclerc E. Schmitt-ΜLms G. Mehlhorn I R. Legname G. Wormald M R. Rudd P M. Dwek R A. Burton D R. Prusiner S B. Antibodies inhibit prion propagation and clear cell cultures of prion infectivity. Nature, 2001, 412(6848): 739–743.
[39] Heppner F L. Musahl C. Arrighi I. Klein M A. Ru¨licke T. Oesch B. Zinkernagel R M. Kalinke U. Aguzzi A. Prevention of scrapie pathogenesis by transgenic expression of anti-prion protein antibodies. Science , 2001, 294(5540): 178–182.
[40] Schwarz A. Kratke O. Burwinkel M. Riemer C. Schultz J. Henklein P. Bamme T. Baier M. Immunisation with a synthetic prion protein-derived peptide prolongs survival times of mice orally exposed to the scrapie agent. Neurosci Lett, 2003, 350(3): 187–189.
[41] White A R. Enever P. Tayebi M. Mushens R. et al. Monoclonal antibodies inhibit prion replication and delay the development of prion disease. Nature, 2003, 422(6927):80-83.
[42] Jian Zhang. Ubiquitin ligases in T cell activation and autoimmunity [J]. Clin Immunology, 2004, 111(3):234 - 240.
[43] Rodriguez F. Zhang J. Whitton J L. DNA immunization: ubiquitination of a viral proteinenhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody induction. J Virol, 1997, 71(11):8497-8503.
[44] Delogu G. Howard A. Collins F M. Morris S L. DNA vaccination against tubercμlosis: expression of a ubiquitin-conjugated tuberculosis protein enhances antimycobacterial immunity. Infect Immun, 2000, 68(6):3097-3102.
[45] Zhang M. Obata C. Hisaeda H. Ishii K. Murata S. Chiba T. et al. A novel DNA vaccine based on ubiquitinproteasome pathway targeting 'self'-antigens expressed in melanoma/melanocyte. Gene Ther, 2005, 12(13):1049-1057.
[46] Wu Y. Kipps T J. Deoxyribonucleic acid vaccines encoding antigens with rapid proteasome-dependent degradation are highly efficient inducers of cytolytic T lymphocytes. J Immunol, 1997, 159(12):6037-6043.
[47] Leachman S A. Shylankevich M. Slade M D. Levine D. Sundaram R K. Xiao W. et al. Ubiquitin-fused and/or mμltiple early genes from cottontail rabbit papillomavirus as DNA vaccines. J Virol, 2002, 76(15):7616-7624.
[48] Fu T M. Guan L. Friedman A. ULmer J B. Liu M A. Donnelly J J. Induction of MHC class I-restricted CTL response by DNA immunization with ubiquitin-influenza virus nucleoprotein fusion antigens. Vaccine, 1998, 16(18):1711-1717.
[49] Loison F. Nizard F. Sourissean T. et al. A ubiquitin - based assay for the cytosolic uptake of protein transduction domains[J]. Molecμlar Therapy, 2005, 11[2] : 205 - 214.
[50] Leachman S A. Shylankerich M. Slade M D. et al. Ubiquitin-fused and /or multiple early genes from cottontail rabbit papillomavirus as DNA Vaccines [J]. Journal of Virology, 2002, 76 [ 15 ] :7616 - 7624.
[51]王庆敏,胡振林,孙树汉.结核杆菌保护性抗原—泛素系统的建立[J].第二军医大学学报,2003,24[1] : 61 - 63.
[52] Bauer R. Scheiblhofer S. Kern K. et al. Generation of hypoallergenic DNA vaccines by forced ubiquitination: Preventive and therapeutic effects in a mouse model of allergy . J Allergy Clin Immunol, 2006, 118[1] : 269 - 276.
[53] Adorini L. Moreno J. Momburg F. Hammerling G J. Guery J C. Valli A. Fuchs S. Exogenous peptides compete for the presentation of endogenous antigens to major histocompatibility complex class II-restricted T cells. J Exp Med, 1991, 174(4):945–948.
[54] Bonifaz L C. Arzate S. Moreno J. Endogenous and exogenous forms of the same antigen are processed from different pools to bind MHC class II molecμles in endocytic compartments. Eur J Immunol, 1999, 29(1):119–131.
[55] Calin-Laurens V. Forquet F. Lombard-Platet S.et al. High efficiency of endogenous antigen presentation by MHC class II molecules. Int Immunol, 1992, 4(10):1113–1121.
[56] Moreno J. Vignali D A. Nadimi F. Fuchs S. Adorini L. Hammerling G J. Processing of an endogenous protein can generate MHC class II-restricted T cell determinants distinct fromthose derived from exogenous antigen. J Immunol, 1991, 147(10):3306–3313.
[57] Parra-Lopez C A. Lindner R. Vidavsky.I Gross M. Unanue E R. Presentation on class II MHC molecules of endogenous lysozyme targeted to the endocytic pathway. J Immunol, 1997, 158(6):2670–2679.
[58] Scheffner M. Werness B A. Huibregtse J M. Levine A J. Howley P M. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell, 1990, 63(6):1129–1136.
[59] Vega M A. Segui-Real B. Garcia J A. Cales C. Rodriguez F. anderkerckhove J. Sandoval I V. Cloning sequencing and expression of a cDNA encoding rat LIMP II a novel 74-kDa lysosomal membrane protein related to the surface adhesion protein CD36. J Biol Chem, 1991, 266(25):16818–16824.
[60] Ogata S. Fukuda M. Lysosomal targeting of Limp II membrane glycoprotein requires a novel Leu-Ile motif at a particular position in its cytoplasmic tail. J Biol Chem, 1994, 269(7):5210–5217.
[61] Sandoval I V. Arredondo J J. Alcalde J. Gonzalez N A. Vandekerckhove J. Jimenez M A. Rico M. The residues Leu(Ile)475-Ile(Leu. Val. Ala)476 contained in the extended carboxyl cytoplasmic tail are critical for targeting of the resident lysosomal membrane protein LIMP II to lysosomes. J Biol Chem, 1994, 269(9):6622–6631.
[62] Vega M A F. Rodriguez B. Segui C. Cales J. Alcalde. and I V Sandoval. Targeting of lysosomal integral membrane protein LIMP II. The tyrosine- lacking carboxyl cytoplasmic tail of LIMP II is sufficient for direct targeting to lysosomes. J Biol Chem, 1991, 266(25):16269-16272.
[63] Rodriguez F. Harkins S. Redwine J M. de Pereda J M. Whitton J L. CD4(+) T cells induced by a DNA vaccine: immunological consequences of epitope-specific lysosomal targeting. J. Virol, 2001, 75(21):10421–10430.
[64] Müller-Schiffmann A. Korth C.Vaccine approaches to prevent and treat prion infection : progress and challenges.BioDrugs, 2008; 22(1): 45-52.
[65] Pankiewicz J. Prelli F. Sy M S. Kascsak R J. Kascsak R B. Spinner D S. Carp R I. Meeker H C. Sadowski M. Wisniewski T. Clearance and prevention of prion infection in cell cμlture by anti-PrP antibodies. Eur J Neurosci, 2006, 23(10):2635-2647.
[66] Nitschke C. Flechsig E. van den Brandt J. Lindner N. Lührs T. Dittmer U. Klein M A. Immunisation strategies against prion diseases: prime-boost immunisation with a PrP DNA vaccine containing foreign helper T-cell epitopes does not prevent mouse scrapie. Vet Microbiol, 2007,123(4): 367-376.
[67] Wolff J A, Malone R W, Williams P. et al. Direct gene transfer into mouse muscle in vivo [J]. Science, 1990,247(4949 pt 1):1465-1468.
[68] Bennett E J, Bence N F, Jayakumar R. Kopito R R. Global impairment of the ubiquitin proteasome system by nuclear or cytoplasmic protein aggregates precedesinclusion body formation [J]. Mol Cell, 2005, 17(3):351-365.
[69] Reinstein E. Immunologic aspects of protein degradation by the ubiquitin-proteasome system [J]. Isr Med Assoc J, 2004,6(7):420-424.
[70] Ciechanover A. Proteolysis: from the lysosome to ubiquitin and the proteasome [J]. Nat Rev Mol Cell Biol, 2005, 6(1):79-87.
[71] Esser C, Alberti S, Hohfeld J. Cooperation of molecular chaperones with the ubiquitin/proteasome system [J]. Biochim Biophys Acta, 2004, 1695(1-3):171-188.
[72] Rodriguez F, Whitton J L. Enhancing DNA immunization[J]. Virology, 2000, 268(2):233-238.
[73] Fynan E F, Webster R G, Fuller D H. et al. DNA vaccines: protective immunizations by parenteral, mucosal, and gene gun inoculations [J]. Proc Natl Acad Sci USA, 1993, 90(24):11478-11482.
[74] Davis H L, Whalen R G, Demeneix B A. Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression [J]. Hum Gene Ther, 1993, 4(2):151-159.
