摘要
流行性感冒是由流感病毒引起的急性呼吸道传染病,对人群健康,社会稳定均产生重大影响。降低流感流行的最有效手段仍是接种流感疫苗,然而,由于流感病毒血清亚型众多,变异性强,因此设计开发安全的且可提供长久保护的,对多亚型流感共保护的新型流感疫苗势在必行。
根据当前流感的流行现状和高致病性禽流感病毒跨种间传播的现状,本研究在本实验室已构建好的表位盒基础上,加以改进并合成了两个表位盒(H5亚型Th／B,CTL表位盒)。另外,采用基因串联的方式,构建了两种核酸疫苗质粒(pVAX1-NP-ECH5、PVAX1-H3-ETH5-H1HA1)。为了验证其能否用于进一步的体内试验,本研究以脂质体法转染BHK细胞,分别以RT-PCR和IFA对外源基因转录和蛋白表达及表达产物生物学活性进行了鉴定。结果显示RT-PCR均检测到外源基因的mRNA转录情况；IFA检测到了特异性荧光,说明表达产物具有相应的生物学活性。这说明各抗原组分可以在真核细胞中有效表达,为进一步动物实验免疫研究奠定了基础。
本研究以小鼠模型作为哺乳动物模型,对之前构建的多表位核酸疫苗进行了免疫研究,对疫苗的免疫原性进行了评估。血清ELISA抗体检测结果显示：添加表位疫苗组(ThB、CTL表位)激发了与抗原单表达组相当的抗体水平,且并在H5亚型流感病毒特异性ELISA抗体水平显示了优势。细胞因子IL-4、IFN-γ检测结果显示：表位组分的加入显著上调了小鼠Th2细胞免疫功能,且CTL表位与NP串联免疫,可有效刺激了T淋巴细胞IFN-γ分泌。淋巴细胞转化试验结果表明：添加表位的DNA疫苗可刺激小鼠产生较强的特异性和非特异性增殖能力,免疫组小鼠的脾脏T淋巴细胞无论是特异性刺激指数还是非特异性刺激指数均明显高于对照组,且优于未添加表位DNA疫苗组。
以上实验结果显示,本研究构建的以H3HA和H1HA1融合表达基因为骨架嵌合加入多表位基因的核酸疫苗,小鼠免疫实验显示,其不仅产生针对H3、H1 HA产生了特异性免疫应答(抗体和特异性细胞免疫),还增加了其抗H5亚型流感感染的可能性,为最终获得通用型流感病毒多表位基因工程疫苗进行了有意义的尝试。
Influenza is caused by the influenza virus, acute respiratory infections, on human health, have a major impact on social stability. Reduce influenza pandemic remains the most effective means of influenza vaccination, however, due to a number of influenza virus subtypes, variability, and therefore design and development of safe and provide long-term protection against multiple subtypes of influenza of a new influenza vaccine to protect imperative.
Based on the current status and the prevalence of influenza highly pathogenic avian influenza virus transmission across species status, this study has been constructed in the laboratory on the basis of a good table-bit box, to improve and synthesized two epitope Box (H5 Asia Model Th/B, CTL epitopes box). In addition, the manner by gene tandem construct two plasmid DNA vaccine (pVAX1-NP-ECH5, pVAX1-H3-ETH5-H1HA1). To test its ability to be used for further in vivo tests, this study liposome transfected BHK cells, RT-PCR and IFA exogenous gene transcription and protein expression and biological activity of expression products were identified respectively. The results showed that both RT-PCR detected mRNA transcription of exogenous genes situation; IFA to detect the specific fluorescence, indicating expression products with the corresponding biological activity. This indicates that the antigen components can be efficiently expressed in eukaryotic cells, in order to further animal experiments laid the foundation for Immunization Research.
In this study, mice as a mammalian model, prior to construction of multi-epitope DNA vaccine immunization study, the immunogenicity of the vaccine was evaluated. ELISA serum antibody test results showed that:Add-epitope vaccine group (ThB, CTL epitopes) to stimulate expression of group with a single antigen-antibody level considerably, and in the H5 subtype of influenza A virus-specific ELISA antibody levels showed an advantage. Cytokines IL-4, IFN-γtest results showed:Table-bit components by adding significantly up-regulated Th2 cellular immune function in mice, and the CTL epitope immunization with the NP series, which can effectively stimulate T-lymphocyte IFN-γsecretion. Lymphocyte transformation test results showed that:Add the epitope DNA vaccine can stimulate the mice produced strong specific and non-specific proliferation and immune groups of mice spleen T lymphocytes, whether specific or non-specific stimulation index stimulation index significantly higher than, and superior to add-epitope DNA vaccine group.
These results show that this study was constructed in order to H3HA and H1HA1 fusion gene expression for the skeleton, adding multi-epitope chimeric gene DNA vaccine, immunization experiments in mice showed that they not only produce for the H3, H1 HA produced a specific immune response (antibodies and specific cell-mediated immunity) and also increased its anti-H5 subtype of influenza infection in the possibility of ultimately universal influenza virus multi-epitope genetically engineered vaccines meaningful attempt.
引文
[1]TAUBENBERGER JK. The virulence of the 1918 pandemic influenza virus:unraveling the enigma[J]. Arch Virol Suppl,2005 (19):101-115.
[2]VIBOUD C, GRAIS RF, LAFONT BA, MILLER MA, SIMONSEN L. Multinational impact of the 1968 Hong Kong influenza pandemic:evidence for a smoldering pandemic[J]. J Infect Dis,2005,192 (2):233-248.
[3]LAVER G, GARMAN E. Virology. The origin and control of pandemic influenza[J]. Science, 2001,293 (5536):1776-1777.
[4]OWENS SR. Being prepared. Preparations for a pandemic of influenza[J]. EMBO Rep,2001, 2 (12):1061-1063.
[5]SIMS LD, DOMENECH J, C B, AL E. Originan devolution of highly pathogenic H5N1 avian in fluenzain Asia. [J]. Vet Rec,2005,15 (6):159-164.
[6]ZELL R, MOTZKE S, KRUMBHOLZ A, et al. Novel rea.ssortant of swine influenza H1N2 virus in Germany [J]. J Gen Virol,2008,89 (Pt 1):271-276.
[7]WAN XF, OZDEN M, LIN G. Ubiquitous reassortments in influenza A viruses [J]. J Bioinform Comput Biol,2008,6 (5):981-999.
[8]KODIHALLI S, GOTO H, KOBASA DL, et al. DNA vaccine encoding hemagglutinin provides protective immunity against H5N1 influenza virus infection in mice[J]. J Virol,1999,73 (3): 2094-2098.
[9]WILEY DC, SKEHEL JJ. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus[J]. Annu Rev Biochem,1987,56:365-394.
[10]TONEGAWA K, NOBUSAWA E, NAKAJIMA K, et al. Analysis of epitope recognition of antibodies induced by DNA immunization against hemagglutinin protein of influenza A virus[J]. Vaccine, 2003,21 (23):3118-3125.
[11]STEINER DF, SMEEKENS SP, OHAGI S, CHAN SJ. The new enzymology of precursor processing endoproteases[J]. J Biol Chem,1992,267 (33):23435-23438.
[12]SUZUKI T, TAKAHASHI T, SAITO T, et al. Evolutional analysis of human influenza A virus N2 neuraminidase genes based on the transition of the low-pH stability of sialidase activity[J]. FEBS Lett,2004,557 (1-3):228-232.
[13]SAKAGUCHI T, TU Q, PINTO LH, LAMB RA. The active oligomeric state of the minimalistic influenza virus M2 ion channel is a tetramer[J]. Proc Natl Acad Sci U S A,1997,94 (10): 5000-5005.
[14]BELZ GT, XIE W, ALTMAN JD, DOHERTY PC. A previously unrecognized H-2D(b)-restricted peptide prominent in the primary influenza A virus-specific CD8(+) T-cell response is much less apparent following secondary challenge[J]. J Virol,2000,74 (8):3486-3493.
[15]HALE BG, BARCLAY WS, RANDALL RE, RUSSELL RJ. Structure of an avian influenza A virus NS1 protein effector domain [J]. Virology,2008,378 (1):1-5.
[16]TREANOR J. Influenza vaccine--outmaneuvering antigenic shift and drift[J]. N Engl J Med,2004,350 (3):218-220.
[17]杨春,余佳,徐凤琴,等.甲型流感病毒抗原变异的机理[J].国外医学病毒学分册,1999,6(3):88-90.
[18]SCHOLTISSEK C, STECH J, KRAUSS S, WEBSTER RG. Cooperation between the hemagglutinin of avian viruses and the matrix protein of human influenza A viruses [J]. J Virol,2002,76 (4): 1781-1786.
[19]RUBEN FL. Prevention and control of influenza. Role of vaccine [J]. Am J Med,1987, 82 (6A):31-34.
[20]CONNE P, GAUTHEY L, VERNET P, et al. Immunogenicity of trivalent subunit versus virosome-formulated influenza vaccines in geriatric patients[J]. Vaccine,1997,15 (15): 1675-1679.
[21]WAREING MD, TANNOCK GA. Live attenuated vaccines against influenza; an historical review[J].Vaccine,2001,19 (25-26):-3320-3330.
[22]POON LL, PRITLOVE DC, SHARPS J, BROWNLEE GG. The RNA polymerase of influenza virus, bound to the 5'end of virion RNA, acts in cis to polyadenylate mRNA[J]. J Virol,1998,72 (10): 8214-8219.
[23]PARKIN NT, CHIU P, COELINGH K. Genetically engineered live attenuated influenza A virus vaccine candidates [J]. J Virol,1997,71 (4):2772-2778.
[24]GLEZEN WP, PIEDRA PA, LONGINI IM, HALLORAN ME. Safety of cold-adapted live influenza vaccine [J]. Pediatr Infect Dis J,2004,23 (6):593-594; author reply 594.
[25]BEYER WE, PALACHE AM, DE JONG JC, OSTERHAUS AD. Cold-adapted live influenza vaccine versus inactivated vaccine:systemic vaccine reactions, local and systemic antibody response, and vaccine efficacy. A meta-analysis[J]. Vaccine,2002,20 (9-10):1340-1353.
[26]KEMBLE G, GREENBERG H. Novel generations of influenza vaccines [J]. Vaccine,2003,21 (16):1789-1795.
[27]戚凤春,盛军.流感疫苗的研究进展[J].中国生物制品学杂志,2004,17(3)：190-192.
[28]WOLFF JA, MALONE RW, WILLIAMS P, et al. Direct gene transfer into mouse muscle in vivo[J]. Science,1990,247 (4949 Pt 1):1465-1468.
[29]ROBINSON HL, HUNT LA, WEBSTER RG. Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA[J]. Vaccine,1993,11 (9): 957-960.
[30]DECK RR, DEWITT CM, DONNELLY JJ, LIU MA, ULMER JB. Characterization of humoral immune responses induced by an influenza hemagglutinin DNA vaccine[J]. Vaccine,1997,15 (1):71-78.
[31]KADOWAKI S, CHEN Z, ASANUMA H, et al. Protection against influenza virus infection in mice immunized by administration of hemagglutinin-expressing DNAs with electroporation[J]. Vaccine,2000,18 (25):2779-2788.
[32]殷震,刘景华主编.动物病毒学[M](第二版).科技出版社1937年.
[33]BARDIYA N, BAE JH. Influenza vaccines:recent advances in production technologies[J]. Appl Microbiol Biotechnol,2005,67 (3):299-305.
[34]GSCHOESSER C, ALMANZAR G, HAINZ. U, et al. CD4+ and CD8+ mediated cellular immune response to recombinant influenza nucleoprotein[J]. Vaccine,2002,20 (31-32):3731-3738.
[35]KODIHALLI S, KOBASA DL, WEBSTER RG. Strategies for inducing protection against avian influenza A virus subtypes with DNA vaccines [J]. Vaccine,2000,18 (23):2592-2599.
[36]ULMER JB, DECK RR, DEWITT CM, et al. Induction of immunity by DNA vaccination: application to influenza and tuberculosis[J]. Behring Inst Mitt,1997 (98):79-86.
[37]ROBINSON HL, BOYLE CA, FELTQUATE DM, et al. DNA immunization for influenza virus: studies using hemagglutinin-and nucleoprotein-expressing DNAs[J]. J Infect Dis,1997,176 Suppl 1:S50-55.
[38]CHEN Z, MATSUO K, ASANUMA H, et al. Enhanced protection against a lethal influenza virus challenge by immunization with both hemagglutinin-and neuraminidase-expressing DNAs[J]. Vaccine,1999,17 (7-8):653-659.
[39]LJUNGBERG K, KOLMSKOG C, WAHREN B, et al. DNA vaccination of ferrets with chimeric influenza A virus hemagglutinin (H3) genes[J]. Vaccine,2002,20 (16):2045-2052.
[40]KISTNER O, BARRETT N, MUNDT W, et al. [Development of a novel influenza vaccine derived from a continuous cell line][J]. ALTEX,2001,18 (1):50-54.
[41]YOUIL R, SU Q, TONER TJ, et al. Comparative study of influenza virus replication in Vero and MDCK cell lines[J]. J Virol Methods,2004,120 (1):23-31.
[42]AULT A. Vaccines. Shifting tactics in the battle against influenza[J]. Science,2004, 303 (5662):1280.
[43]YOUIL R, KISELEVA I, KWAN WS, et al. Phenotypic and genetic analyses of the heterogeneous population present in the cold-adapted master donor strain: A/Leningrad/134/17/57 (H2N2)[J]. Virus Res,2004,102 (2):165-176.
[44]JEON SH, BEN-YEDIDIA T, ARNON R. Intranasal immunization with synthetic recombinant vaccine containing multiple epitopes of influenza virus[J]. Vaccine,2002,20 (21-22): 2772-2780.
[45]陈义锋,金宁一,贾雷立,等.H3,H5,H7,H9亚型流感病毒通用多表位重组鸡痘病毒构建[J].免疫学杂志,2007,5(23)：524-527.
[46]LEVI R, ARNON R. Synthetic recombinant influenza vaccine induces efficient long-term immunity and cross-strain protection [J]. Vaccine,1996,14 (1):85-92.
[47]THOMSON SA, SHERRITT MA, MEDVECZKY J, et al. Delivery of multiple CD8 cytotoxic T cell, epitopes by DNA vaccination[J]. J Immunol,1998,160 (4):1717-1723.
[48]BRUMEANU TD, CASARES S, BOT A, BOT S, BONA CA. Immunogenicity of a contiguous T-B synthetic epitope of the A/PR/8/34 influenza virus[J]. J Virol,1997,71 (7):5473-5480.
[49]CROWE SR, MILLER SC, WOODLAND DL. Identification of protective and non-protective T cell epitopes in influenza[J]. Vaccine,2006,24 (4):452-456.
[50]OPERSCHALL E, PAVLOVIC J, NAWRATH M, MOLLING K. Mechanism, of protection against influenza A virus by DNA vaccine encoding the hemagglutinin gene[J]. Intervirology,2000,43 (4-6):322-330.
[51]KRETZSCHMAR E, BUONOCORE L, SCHNELL MJ, ROSE JK. High-efficiency incorporation of functional influenza virus glycoproteins into recombinant vesicular stomatitis viruses[J]. J Virol,1997,71 (8):5982-5989.
[52]TANG M, HARP JA, WESLEY RD. Recombinant adenovirus encoding the HA gene from swine H3N2 influenza virus partially protects mice fium challenge with heterologous virus. A/HK/1/68 (H3N2)[J]. Arch Virol,2002,147 (11):2125-2141.
[53]南文龙,金宁一,鲁会军,等.H5N1亚型禽流感病毒血凝素Th和B细胞表位预测及抗原性分析[J].中国免疫学杂志,2009,25(7)：630-634.
[54]FOUCHIER RA, SCHNEEBERGER PM, FW R, AL. E. Avian influenza A virus(H7N7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome [J]. Proc Natl Acad Sci USA,2004,101 (5):1356-1361.
[55]FRANCIS T, SALK JE, QUILLIGAN JJ. Experience with Vaccination Against Influenza in the Spring of 1947:A Preliminary Report[J]. Am J Public Health Nations Health,1947,37 (8): 1013-1016.
[56]HEINY AT, MIOTTO 0, SRINIVASAN KN, et al. Evolutionarily conserved protein sequences of influenza a viruses, avian and human, as vaccine targets [J]. PLoS One,2007,2 (11):e1190.
[57]OZAWA M, FUJII K, MURAMOTO Y, et al. Contributions of two nuclear localization signals of influenza A virus nucleoprotein to viral replication[J]. J Virol,2007,81 (1):30-41.
[58]VAN DEN BERG T, LAMBRECHT B, MARCHE S, et al. Influenza vaccines and vaccination strategies in birds[J]. Comp Immunol Microbiol Infect Dis,2008,31 (2-3):121-165.
[59]KREIJTZ JH, BODEWES R, VAN AMERONGEN G,et al. Primary influenza A virus infection induces cross-protective immunity against a lethal infection with a heterosubtypic virus strain in mice[J]. Vaccine,2007,25 (4):612-620.
[60]CHEN C, OKAYAMA H. High-efficiency transformation of mammalian cells by plasmid DNA[J]. Mol Cell Biol,1987,7 (8):2745-2752.
[61]BETHESDA MD. Workshop on the control and standardization of nucleic acid vaccines[J]. Vaccine,1997,15:931-933.
[62]DE FELIPE P. Skipping the co-expression problem:the new 2A "CHYSEL" technology[J]. Genet Vaccines Ther,2004,2 (1):13.
[63]DE FELIPE P, HUGHES LE, RYAN MD, BROWN JD. Co-translational, intraribosomal cleavage. of polypeptides by the foot-and-mouth disease virus 2A peptide[J]. J Biol Chem,2003,278 (13): 11441-11448.
[64]DE FELIPE P, MARTIN V, CORTES ML, RYAN M, IZQUIERDO M. Use of the 2A sequence from foot-and-mouth disease virus in the generation of retroviral vectors for gene therapy [J]. Gene Ther,1999,6 (2):198-208.
[65]黄培堂,等译.分子克隆实验指南.科学出版社：北京2002：1271.
[66]段艳,等.阳离子脂质体转染He1a细胞的实验[J].中国组织工程研究与临床康复,2009,13(11)：2119-2122.
[67]AGARWAL SK, MARSHALL GD, JR. Dexamethasone promotes type 2 cytokine. production. primarily through inhibition of type 1 cytokines[J]. J Interferon Cytokine Res,2001,21 (3): 147-155.
[68]SELBY M, GOLDBECK C, PERTILE T, WALSH R, ULMER J. Enhancement of DNA vaccine potency by electroporation in vivo[J]. J Biotechnol,2000,83 (1-2):147-152.
[69]WIDERA G, AUSTIN M, RABUSSAY D, et al. Increased DNA vaccine delivery and immunogenicity by electroporation in vivo[J]. J Immunol,2000,164 (9):4635-4640.
[70]张强哲.H5亚型禽流感病毒HA DNA疫苗的研究. 西北农林科技大学硕士研究生毕业论文：陕西省2003.
[71]YANKAUCKAS MA, MORROW JE, PARKER SE, et al. Long-term anti-nucleoprotein cellular and humoral immunity is induced by intramuscular injection of plasmid DNA containing NP gene[J]. DNA Cell Biol,1993,12 (9):771-776.
[72]SHA Z, VINCENT MJ, COMPANS RW. Enhancement of mucosal (?)m(?)une (?)espo(?)ses to the influenza virus HA protein by alternative approaches to DNA immunization[J]. Immunobiology,1999,200 (1):21-30.
[73]COX RJ, MYKKELTVEDT E, ROBERTSON J, HAAHEIM LR. Non-lethal viral challenge of influenza haemagglutinin and nucleoprotein DNA vaccinated mice results in reduced viral replication[J]. Scand J Immunol,2002,55 (1):14-23.
[74]WEBSTER RG, KAWAOKA Y, TAYLOR J, WEINBERG R, PAOLETTI E. Efficacy of nucleoprotein and haemagglutinin antigens expressed in fowlpox virus as vaccine for influenza in chickens [J]. Vaccine,1991,9 (5):303-308.
[75]BOT A, BOT S, GARCIA-SASTRE A, BONA C. DNA immunization of newborn mice with a plasmid-expressing nucleoprotein of influenza virus[J]. Viral Immunol, 1996,9 (4):207-210.
[76]ALTSTEIN AD, GITELMAN AK, SMIRNOV YA, et al. Immunization with influenza A NP-expressing vaccinia virus recombinant protects mice against experimental infection with human and avian influenza viruses[J]. Arch Virol,2006,151 (5):921-931.
[77]HUA L, DING J, CHEN YH, AL. E. Recombinant protein comprising muli-neutralizing epitopes induced high titer of antibodies against Influecza A Virus[J]. lmmunobiol,2003,207: 305-313.
[78]彭金美,童光志,王云峰,等.抗禽流感多表位疫苗的构建及其免疫效力的研究[J].生物工程学报,2003,19(5)：623-627.
[79]贾雷立,金宁一,金扩世.猪流感复合多表位核酸疫苗的构建及其表达研究[J].中国生物制品学志,2006,4(19)：333-335.
[80]南文龙.流感功能表位筛选与复合多表位核酸疫苗设计及免疫研究[D].吉林大学博士研究生毕业论文：吉林省2009.