利用重组工程技术构建多价DNA疫苗载体及大肠杆菌活菌疫苗载体的分析比较
|
摘要
疾病预防的有效途径之一是接种疫苗，继第一代减毒或无毒疫苗和第二代亚单位疫苗之后出现了第三代DNA疫苗。疫苗效果在很大程度上依赖于输送抗原的载体。为研究更有效的疫苗载体形式，本研究应用噬菌体Red重组酶介导的新型重组工程技术，构建了多价DNA疫苗载体前体及携带禽流感病毒抗原基因的多价DNA疫苗载体，为了更好的输送多价DNA疫苗，探索构建了具有哺乳动物上皮细胞黏附能力的无毒性重组大肠杆菌活菌疫苗菌株。
在本室前期工作基础上，本实验应用如下Red重组工程系统及技术：①大肠杆菌染色体上Red重组系统及kan-sacB选择反选择筛选方法②pKD46重组系统及kan-kil选择反选择筛选方法③pYM-Red重组系统及Gap-Repair缺口修复技术，进行染色体上基因的无痕敲入、替换及质粒构建。本实验解决基因融合打靶问题时，在重组技术上有创新应用，利用Red重组酶工作原理，将外源片段体外重叠PCR法制各改为片段体内重组融合，片段融合及重组均在体内完成，省略了体外PCR的步骤加快实验进程。
以pcDNA3.1(-)B为基础，以酶切连接方法构建了四价DNA疫苗载体pWYⅠ-Ⅱ-Ⅲ-Ⅳ，瞬时转染实验证明其具有蛋白表达能力，真核表达的多价DNA疫苗载体构建成功，可用于携带多价抗原基因，避免了多质粒操作带来的不便。然后利用重组技术试图将禽流感病毒基因组抗原基因HA头部、NA全长分别加载到其Ⅰ、Ⅱ位，构建二价DNA疫苗。
将南美锥虫(trypanosome cruzi)gp82基因的P4及P8 DNA区段融合构建成P4／8基因片段，将其敲入大肠杆菌染色体的外膜蛋白LamB基因内形成融合蛋白，并展示在细胞外膜上，构建出带有细胞黏附能力的Ecoli DY330 P4／8<>lamb菌株。通过体外黏附实验证明，以前构建的Ecoli DY330 P4<>lamb比新构建的Ecoli DY330 P4／8<>lamb菌株黏附哺乳动物上皮细胞的能力要强；同时体外细胞毒性实验证明DY330 P4<>lamb菌株安全性良好，可做为活菌疫苗载体的候选株进行深入研究。
研究表明，重组工程技术在构建多种形式疫苗载体中具有灵活性及有效性，构建的多价DNA疫苗载体具备通用性，可以加载多种抗原基因的组合，作为快速制备疫苗的模板，有效应对突发疫情；构建的黏附性活菌疫苗载体可以携带DNA疫苗，也可以直接在染色体上携带抗原基因，高效运送抗原基因或蛋白。在应用中不断尝试各种构建方案也使我们更深入理解了重组酶工作原理，希望通过技术的拓展加速疫苗的研制与开发进程，为疾病的防治作出贡献。
Vaccine inoculation is the most effective way for disease prophylaxis. DNA vaccine as the third generation of vaccine emerged from the first and the second generation of vaccine. The effct of vaccine fairly depends on vectors used to transport antigens.To research more effective vaccine vectors, we constructed precursor for multivalent DNA vaccine vector and multivalent DNA vaccine with AIV antigen genes using recombineering mediated by phageλRed recombinase. In order to transport multivalent DNA vaccine better, we explored to construct avirulence recombineering E.coli as live bacterial vaccine vectors, which can adhere to endothelial cell of mammalian.
On the base of our early work, we use Red recombineering technologies as follows:①Red recombineering system on E.coli chromosome and kan-sacB positive and negative screening.②pKD46 plasmid recombineering system and kan-kil positive and negative screening.③)pYM-Red plasmid recombineering system and Gap-Repair. Some technology improvements on solving gene fusion in vivo have been established based on the principle of Red recombinases. We made two DNA fragments fuse in vivo by recombineering insteading of over-lapping PCR in vitro, which speed up the process of our experiment.
Based on pcDNA3.1(-)B, we constructed quadrivalence DNA vaccine vector pWYⅠ-Ⅱ-Ⅲ-Ⅳusing restriction enzyme and T_4 ligase, then transient transfection proved that it can express proteins. It is convenient to load several antigen genes only in one plasmid. Then we try to construct bivalence DNA vaccine by loading HA and NA gene of AIV onⅠandⅡsite of pWYⅠ-Ⅱ-Ⅲ-Ⅳ.
First, P4 and P8 DNA fragments of trypanosome cruzi gp82 gene are fused into P4/8, then knock the fused fragment P4/8 into E.coli outer membrane protein LamB gene presenting on outer membrane of bacteria and obtain E.coli DY330 P4/8<>lamb. In vitro adhesion experiments proved that E.coli DY330 P4<>lamb constructed before is stronger and safer than DY330 P4/8<>lamb when they adhere to endothelial cell of mammalian. As a result, E.coli DY330 P4<>lamb can be the candidate as live bacterial vaccine vectors.
Our research indicates that recombineering technology has its flexibility and efficacy in the process of constructing vaccine vectors. Multivalent DNA vaccine vectors are provided by their versatility, which can load several antigen genes only in one plasmid and make fast reaction to outburst epidemic situation. Adhesive live bacterial vaccine vector can take along DNA vaccine or load antigen genes on their chromosomes, which can effectively transport antigen genes or protein. In the application of different protocols, we understand the principle of recombinase more deeply. We hope technology improvements can speed up research and development of vaccine and can contribute to prophylaxis of diseases.
在本室前期工作基础上，本实验应用如下Red重组工程系统及技术：①大肠杆菌染色体上Red重组系统及kan-sacB选择反选择筛选方法②pKD46重组系统及kan-kil选择反选择筛选方法③pYM-Red重组系统及Gap-Repair缺口修复技术，进行染色体上基因的无痕敲入、替换及质粒构建。本实验解决基因融合打靶问题时，在重组技术上有创新应用，利用Red重组酶工作原理，将外源片段体外重叠PCR法制各改为片段体内重组融合，片段融合及重组均在体内完成，省略了体外PCR的步骤加快实验进程。
以pcDNA3.1(-)B为基础，以酶切连接方法构建了四价DNA疫苗载体pWYⅠ-Ⅱ-Ⅲ-Ⅳ，瞬时转染实验证明其具有蛋白表达能力，真核表达的多价DNA疫苗载体构建成功，可用于携带多价抗原基因，避免了多质粒操作带来的不便。然后利用重组技术试图将禽流感病毒基因组抗原基因HA头部、NA全长分别加载到其Ⅰ、Ⅱ位，构建二价DNA疫苗。
将南美锥虫(trypanosome cruzi)gp82基因的P4及P8 DNA区段融合构建成P4／8基因片段，将其敲入大肠杆菌染色体的外膜蛋白LamB基因内形成融合蛋白，并展示在细胞外膜上，构建出带有细胞黏附能力的Ecoli DY330 P4／8<>lamb菌株。通过体外黏附实验证明，以前构建的Ecoli DY330 P4<>lamb比新构建的Ecoli DY330 P4／8<>lamb菌株黏附哺乳动物上皮细胞的能力要强；同时体外细胞毒性实验证明DY330 P4<>lamb菌株安全性良好，可做为活菌疫苗载体的候选株进行深入研究。
研究表明，重组工程技术在构建多种形式疫苗载体中具有灵活性及有效性，构建的多价DNA疫苗载体具备通用性，可以加载多种抗原基因的组合，作为快速制备疫苗的模板，有效应对突发疫情；构建的黏附性活菌疫苗载体可以携带DNA疫苗，也可以直接在染色体上携带抗原基因，高效运送抗原基因或蛋白。在应用中不断尝试各种构建方案也使我们更深入理解了重组酶工作原理，希望通过技术的拓展加速疫苗的研制与开发进程，为疾病的防治作出贡献。
Vaccine inoculation is the most effective way for disease prophylaxis. DNA vaccine as the third generation of vaccine emerged from the first and the second generation of vaccine. The effct of vaccine fairly depends on vectors used to transport antigens.To research more effective vaccine vectors, we constructed precursor for multivalent DNA vaccine vector and multivalent DNA vaccine with AIV antigen genes using recombineering mediated by phageλRed recombinase. In order to transport multivalent DNA vaccine better, we explored to construct avirulence recombineering E.coli as live bacterial vaccine vectors, which can adhere to endothelial cell of mammalian.
On the base of our early work, we use Red recombineering technologies as follows:①Red recombineering system on E.coli chromosome and kan-sacB positive and negative screening.②pKD46 plasmid recombineering system and kan-kil positive and negative screening.③)pYM-Red plasmid recombineering system and Gap-Repair. Some technology improvements on solving gene fusion in vivo have been established based on the principle of Red recombinases. We made two DNA fragments fuse in vivo by recombineering insteading of over-lapping PCR in vitro, which speed up the process of our experiment.
Based on pcDNA3.1(-)B, we constructed quadrivalence DNA vaccine vector pWYⅠ-Ⅱ-Ⅲ-Ⅳusing restriction enzyme and T_4 ligase, then transient transfection proved that it can express proteins. It is convenient to load several antigen genes only in one plasmid. Then we try to construct bivalence DNA vaccine by loading HA and NA gene of AIV onⅠandⅡsite of pWYⅠ-Ⅱ-Ⅲ-Ⅳ.
First, P4 and P8 DNA fragments of trypanosome cruzi gp82 gene are fused into P4/8, then knock the fused fragment P4/8 into E.coli outer membrane protein LamB gene presenting on outer membrane of bacteria and obtain E.coli DY330 P4/8<>lamb. In vitro adhesion experiments proved that E.coli DY330 P4<>lamb constructed before is stronger and safer than DY330 P4/8<>lamb when they adhere to endothelial cell of mammalian. As a result, E.coli DY330 P4<>lamb can be the candidate as live bacterial vaccine vectors.
Our research indicates that recombineering technology has its flexibility and efficacy in the process of constructing vaccine vectors. Multivalent DNA vaccine vectors are provided by their versatility, which can load several antigen genes only in one plasmid and make fast reaction to outburst epidemic situation. Adhesive live bacterial vaccine vector can take along DNA vaccine or load antigen genes on their chromosomes, which can effectively transport antigen genes or protein. In the application of different protocols, we understand the principle of recombinase more deeply. We hope technology improvements can speed up research and development of vaccine and can contribute to prophylaxis of diseases.
引文
[1] Copeland NG, Jenkins NA, Court DL. Recombineering: a powerful new tool for mouse functional genomics[J]. Nat Rev Genet, 2001, 2(10): 769-79
[2] Zhang Y, Buchholz F, Muyrers JP, et al. A new logic for DNA engineering using recombination in Escherichia coli[J]. Nat Genet, 1998, 20(2):123-8
[3] Yu D, Ellis HM, Lee EC, et al. An efficient recombination system for chromosome engineering in Escherichia coli[J]. Proc.Natl.Acad.Sci USA, 2000, 97(11):5978-83
[4] Court DL, Sawitzke JA, Thomason LC. Genetic engineering using homologous recombination[J]. Annu Rev Genet, 2002, 36:361-88
[5] ZhouJG (周建光 ) , HongX (洪鑫) , HuangCF (黄翠芬) .Recombineering and its application[J]. Acta Genetica Sinica (遗传学报) , 2003, 30(10):983-988
[6] Little JW. An exonuclease induced by bacteriophage lambda. Ⅱ. Nature of the enzymatic reaction[J]. J Biol Chem, 1967, 242(4): 679-86
[7] Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J]. Proc Natl Acad Sci USA, 2000, 97(12):6640-5
[8] YuM(于梅)，ZhouJG(周建光)，ChenW(陈伟)，LiSH(李山虎)，HuangCF(黄翠芬)．The construction of a new mobile recombineering system of pYM-Red[J]．Prog Biochem BiopHys(生物化学与生物物理进展)，2005，32(4)：359-364
[9] ChenW(陈伟)，YuM(于梅)，LiSH(李山虎)，WangMG(王鸣刚)，ZhouJG(周建光)．Gene knockout and knockin in the Escherichia coli lac operon locui using pBR3 22-Red system[J]．Chinese Journal of Biotechnology(生物工程学报)，2005，21：192—197
[10] Liu P, Jenkins NA, Copeland NG. A highly efficient recombineering-based method for generating conditional knockout mutations[J]. Genome Res, 2003, 13(3): 476-484
[11] Muyrers JP, Zhang Y, Benes V. Point mutation of bacterial artificial chromosomes by ET recombina2tion[J]. EMBO Rep, 2000, 1:239-243
[12] Muyrers JP, Zhang Y, Testa G, et al. Rapid modification of bacterial artificial chromosomes by ET-recombination[J]. Nucleic Acids Res, 1999, 27(6): 1555-1557
[13] Wolff JA, Malone RW, Williams P, et al. Direct gene transfer into mouse muscle in vivo[J]. Science, 1990, 247:1465-1469
[14] Williams RS, Johnston SA, Riedy M, et al. Introduction of foreign genes into tissues of living mice by DNA-coated microprojectile[J]. Proc Natl Acad Sci USA, 1991, 882726-2732
[15] Tang DC, DeVit M, Johnston SA, et al. Genetic immunization is a simple method for eliciting an immune response[J]. Nature, 1992, 356:152-157
[16] ShenJJ(沈际佳)，JiangZJ(蒋作君)，YuXB(余新炳)．Nucleic acid vaccine and its application in parasitosis[J]．Chinese Journal of Disease Control&Prevention(疾病控制杂志)，2000，4(1)：84-87
[17] Donnelly JJ, Friedman A, Martinez D, et al. Preclinical efficacy of a prototype DNA vaccine: enhanced protection against antigenic drift in influenza virus[J]. Nat Med, 1995, 1(6): 583-587
[18] Xiang ZQ, Spitalnik SL, Cheng J, et al. Immune responses to nucleic acid vaccines to rabies virus[J]. Virology, 1995, 209(2): 569-579
[19] WolffJA, Danko I. Direct gene transfer into muscle[J]. Vaccine, 1994, 12(16): 1499-1502
[20] Kuhober A, Pudollek HP, Reifenberg K, et al. DNA immunization induces antibody and cytotoxic T cell responses to hepatitis B core antigen in H-2b mice[J]. J Immunol, 1996, 156(10): 3687-3695
[21] Sato Y, Roman M, Tighe H, et al. Immunostimulatory DNA sequences necessary for effective intradermal gene immunization[J]. Science, 1996, 273(5273): 352-354
[22] Yamamoto S, Yamarnoto T, Kataoka T, et al. Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN [correction of INF] and augment IFN-mediated [correction of INF] natural killer activity[J]. J Immunol, 1992, 148(12): 4072-4076
[23] Krieg AM, Yi AK, Matson S, et al. CpG motifs in bacterial DNA trigger direct B-cell activation[J]. Nature, 1995, 374(6522): 546-569
[24] Ulmer JB, Donnelly JJ, Parker SE, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein[J]. Science, 1993,259:1745-1749
[25] Robinson H L, et al. Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA[J]. Vaccine, 1993, 11:957-960
[26] Fynan EF, Webster RG, Fuller DH, et al. DNA vaccines: Protective immunizations by parenteral, mucosal, and gene-gun inoculations[J]. Proc Natl Acad Sci USA, 1993, 90:11478-11482
[27] Pertmer TM, Eisenbraun MD, MacCabe D,et al. Gene gun-based nucleic acid immunization: elicitation of humoral and cytotoxic T lympHocyte responses following epidermal delivery of nanogram quantities of DNA[J]. Vaccine, 1995, 13:1427-1430
[28] Sha l, Vincent MJ, Compans RW. Enhancement of mucosal immune responses to the influenza virus HA protein by alternative approaches to DNA immunization[J]. Immunobiology, 1999, 200(1):21-30
[29] Kodihalli S, Goto H, Kobasa DL. DNA vaccine encoding hemagglutinin provides protective immunity against H5N1 influenza virus infection in mice[J]. J Virol, 1999, 73(3):2094-2098
[30] PengJM (彭金美) , TongGZ (童光志) , WangYF (王云峰) , et al. Multi-epitope DNA Vaccines Against Avian Influenza in Chickens[J]. Chinese Journal of Biotechnology (生物工程学报), 2003, 19 (5) :623-627
[31] 余为一，仲大莲，李槿年．一种含多种病毒抗原表位的禽多价核酸疫苗制各方法：中国，CN1640494[P]．2005—07—20
[32] Simone Spreng, Guido Dietrich, Stefan Niewiesk, et al .Novel bacterial systems for the delivery of recombinant protein or DNA[J].FEMS Immunology and Medical Microbiology.2000, 27:299
[33] Gabriele Neumann, Ken Fujii, Yoichiro Kino, et al. An improved reverse genetics system for influenza A virus generation and its implications for vaccine production[J].PNAS. 2005, 102(46):16825-16829
[34] Grillot-Courvalin C, Goussard S, Huetz F, et al. Fracncois.Functional gene transfer from intracellular bacteria to mammalian cells[J]. Natuer Biotechnology, 1998, 16:862-866
[35] RJ Critchley ,S Jezzaed,KJ Radford, et al. Potential therapeutic applications of recombinant, invasive E. coli[J].Gene Therapy, 2004, 11, 1224
[36] I Castagliuolo, E Beggiao, P Brun, et al. Engineered E. coli delivers therapeutic genes to the colonic mucosa[J]. Gene Therapy, 2005, 1-9
[37] CA'TIA M. PEREIRA, SI'LVIO FAVORETO, JR. et al. Adhesion of Escherichia coli to HeLa Cells Mediated by Trypanosoma cruzi Surface Glycoprotein-Derived Peptides Inserted in the Outer Membrane Protein LamB[J]. Infection and Immunity, 1999, 67(9):4908-4911
[38] Silvio FJ, Miriam LD, Nobuko Y. Trypanosoma cruzi 175-KDa pHospHorylation is associated with host cell invasion[J]. Experimental Parasitology, 1998, 89:188
[39] Hildegard E, Duc BM, Carola S et al. Bacterial PHage Receptors, Versatile Tools for Display of Polypeptides on the Cell Surface[J]. Journal of Bacteriology. 2001, 183:6924
[40] Patricio M. Manque, Daniel Eichinger, Maria A. Juliano, et al. Characterization of the Cell Adhesion Site of Trypanosoma cruzi Metacyclic Stage Surface Glycoprotein gp82[J]. Infection and Immunity, 2000, 68(2):478-484
[41] Subbarao K,Klimov A,Katz J, et al.Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness[J]. Science, 1998,279:393-396.
[42] J. Liu. H. Xiao, F. Lei, et al.Highly Pathogenic H5N1 InfluenzaVirus Infection in Migratory Birds[J]. Science, 2005，309：1206
[43] 萨姆布鲁克，弗里奇，曼尼阿蒂斯．分子克隆实验指南，第二版[M]．北京：科学出版社，1998,852—897．
[44] YanRQ(闰若潜)，DuXQ(杜向党)．Research Progress in Genomic Structures and Proteins of Influenza virus[J]. Progress in Veterinary Medicine, 2004, 25 (1): 32-35
[45] Matthew PHilpott, Bernard C. Easterday, Virginia S. Hinshaw. Neutralizing Epitopes of the H5 Hemagglutinin from a Virulent Avian Influenza Virus and Their Relationship to Pathogenicity[J]. Virology, Aug. 1989, 3453-3458
[46] Tonegawa K, Nobusawa E, N akajima 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.
[47] Zebedee S L, Lamb R A. Growth restriction of influenza A virus by M2 protein antibody is genetically linked to the Ml protein[J]. Proc Natl Acad Sci USA, 1989, 86(3): 1061-1065
[48] Nathalie M, Sylvie A, Camille L. Nasal vaccination using live bacterial vectors[J]. Advanced Drug Delivery .2001, 51:55
[49] Mari OH, Masafumi Y, Yoshikazu Y et al. Non-toxic Stx derivatives from E.coli possess adjuvant activity for mucosal immunity[J]. Vaccine 2004, 22:3751
[50] Bermudes D, Zheng L M, King I C. Live bacteria as anticancer agents and tumor selective protein delivery vectors[J]. Curr Opin Drug Discov Devel, 2002, 5(2): 194
[51] Drabner B, Guzman CA. Elicitation of predictable immune responses by using live bacterial vectors[J]. Biomolecular Engineering, 2001, 17:75
[52] Christine Hale, Ian RH, Tracy Hussell, et al. Mucosal immunization of murine neonates using whole cell and acellular Pertussis vaccines[J]. Vaccine, 2004, 22:3595
[2] Zhang Y, Buchholz F, Muyrers JP, et al. A new logic for DNA engineering using recombination in Escherichia coli[J]. Nat Genet, 1998, 20(2):123-8
[3] Yu D, Ellis HM, Lee EC, et al. An efficient recombination system for chromosome engineering in Escherichia coli[J]. Proc.Natl.Acad.Sci USA, 2000, 97(11):5978-83
[4] Court DL, Sawitzke JA, Thomason LC. Genetic engineering using homologous recombination[J]. Annu Rev Genet, 2002, 36:361-88
[5] ZhouJG (周建光 ) , HongX (洪鑫) , HuangCF (黄翠芬) .Recombineering and its application[J]. Acta Genetica Sinica (遗传学报) , 2003, 30(10):983-988
[6] Little JW. An exonuclease induced by bacteriophage lambda. Ⅱ. Nature of the enzymatic reaction[J]. J Biol Chem, 1967, 242(4): 679-86
[7] Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J]. Proc Natl Acad Sci USA, 2000, 97(12):6640-5
[8] YuM(于梅)，ZhouJG(周建光)，ChenW(陈伟)，LiSH(李山虎)，HuangCF(黄翠芬)．The construction of a new mobile recombineering system of pYM-Red[J]．Prog Biochem BiopHys(生物化学与生物物理进展)，2005，32(4)：359-364
[9] ChenW(陈伟)，YuM(于梅)，LiSH(李山虎)，WangMG(王鸣刚)，ZhouJG(周建光)．Gene knockout and knockin in the Escherichia coli lac operon locui using pBR3 22-Red system[J]．Chinese Journal of Biotechnology(生物工程学报)，2005，21：192—197
[10] Liu P, Jenkins NA, Copeland NG. A highly efficient recombineering-based method for generating conditional knockout mutations[J]. Genome Res, 2003, 13(3): 476-484
[11] Muyrers JP, Zhang Y, Benes V. Point mutation of bacterial artificial chromosomes by ET recombina2tion[J]. EMBO Rep, 2000, 1:239-243
[12] Muyrers JP, Zhang Y, Testa G, et al. Rapid modification of bacterial artificial chromosomes by ET-recombination[J]. Nucleic Acids Res, 1999, 27(6): 1555-1557
[13] Wolff JA, Malone RW, Williams P, et al. Direct gene transfer into mouse muscle in vivo[J]. Science, 1990, 247:1465-1469
[14] Williams RS, Johnston SA, Riedy M, et al. Introduction of foreign genes into tissues of living mice by DNA-coated microprojectile[J]. Proc Natl Acad Sci USA, 1991, 882726-2732
[15] Tang DC, DeVit M, Johnston SA, et al. Genetic immunization is a simple method for eliciting an immune response[J]. Nature, 1992, 356:152-157
[16] ShenJJ(沈际佳)，JiangZJ(蒋作君)，YuXB(余新炳)．Nucleic acid vaccine and its application in parasitosis[J]．Chinese Journal of Disease Control&Prevention(疾病控制杂志)，2000，4(1)：84-87
[17] Donnelly JJ, Friedman A, Martinez D, et al. Preclinical efficacy of a prototype DNA vaccine: enhanced protection against antigenic drift in influenza virus[J]. Nat Med, 1995, 1(6): 583-587
[18] Xiang ZQ, Spitalnik SL, Cheng J, et al. Immune responses to nucleic acid vaccines to rabies virus[J]. Virology, 1995, 209(2): 569-579
[19] WolffJA, Danko I. Direct gene transfer into muscle[J]. Vaccine, 1994, 12(16): 1499-1502
[20] Kuhober A, Pudollek HP, Reifenberg K, et al. DNA immunization induces antibody and cytotoxic T cell responses to hepatitis B core antigen in H-2b mice[J]. J Immunol, 1996, 156(10): 3687-3695
[21] Sato Y, Roman M, Tighe H, et al. Immunostimulatory DNA sequences necessary for effective intradermal gene immunization[J]. Science, 1996, 273(5273): 352-354
[22] Yamamoto S, Yamarnoto T, Kataoka T, et al. Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN [correction of INF] and augment IFN-mediated [correction of INF] natural killer activity[J]. J Immunol, 1992, 148(12): 4072-4076
[23] Krieg AM, Yi AK, Matson S, et al. CpG motifs in bacterial DNA trigger direct B-cell activation[J]. Nature, 1995, 374(6522): 546-569
[24] Ulmer JB, Donnelly JJ, Parker SE, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein[J]. Science, 1993,259:1745-1749
[25] Robinson H L, et al. Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA[J]. Vaccine, 1993, 11:957-960
[26] Fynan EF, Webster RG, Fuller DH, et al. DNA vaccines: Protective immunizations by parenteral, mucosal, and gene-gun inoculations[J]. Proc Natl Acad Sci USA, 1993, 90:11478-11482
[27] Pertmer TM, Eisenbraun MD, MacCabe D,et al. Gene gun-based nucleic acid immunization: elicitation of humoral and cytotoxic T lympHocyte responses following epidermal delivery of nanogram quantities of DNA[J]. Vaccine, 1995, 13:1427-1430
[28] Sha l, Vincent MJ, Compans RW. Enhancement of mucosal immune responses to the influenza virus HA protein by alternative approaches to DNA immunization[J]. Immunobiology, 1999, 200(1):21-30
[29] Kodihalli S, Goto H, Kobasa DL. DNA vaccine encoding hemagglutinin provides protective immunity against H5N1 influenza virus infection in mice[J]. J Virol, 1999, 73(3):2094-2098
[30] PengJM (彭金美) , TongGZ (童光志) , WangYF (王云峰) , et al. Multi-epitope DNA Vaccines Against Avian Influenza in Chickens[J]. Chinese Journal of Biotechnology (生物工程学报), 2003, 19 (5) :623-627
[31] 余为一，仲大莲，李槿年．一种含多种病毒抗原表位的禽多价核酸疫苗制各方法：中国，CN1640494[P]．2005—07—20
[32] Simone Spreng, Guido Dietrich, Stefan Niewiesk, et al .Novel bacterial systems for the delivery of recombinant protein or DNA[J].FEMS Immunology and Medical Microbiology.2000, 27:299
[33] Gabriele Neumann, Ken Fujii, Yoichiro Kino, et al. An improved reverse genetics system for influenza A virus generation and its implications for vaccine production[J].PNAS. 2005, 102(46):16825-16829
[34] Grillot-Courvalin C, Goussard S, Huetz F, et al. Fracncois.Functional gene transfer from intracellular bacteria to mammalian cells[J]. Natuer Biotechnology, 1998, 16:862-866
[35] RJ Critchley ,S Jezzaed,KJ Radford, et al. Potential therapeutic applications of recombinant, invasive E. coli[J].Gene Therapy, 2004, 11, 1224
[36] I Castagliuolo, E Beggiao, P Brun, et al. Engineered E. coli delivers therapeutic genes to the colonic mucosa[J]. Gene Therapy, 2005, 1-9
[37] CA'TIA M. PEREIRA, SI'LVIO FAVORETO, JR. et al. Adhesion of Escherichia coli to HeLa Cells Mediated by Trypanosoma cruzi Surface Glycoprotein-Derived Peptides Inserted in the Outer Membrane Protein LamB[J]. Infection and Immunity, 1999, 67(9):4908-4911
[38] Silvio FJ, Miriam LD, Nobuko Y. Trypanosoma cruzi 175-KDa pHospHorylation is associated with host cell invasion[J]. Experimental Parasitology, 1998, 89:188
[39] Hildegard E, Duc BM, Carola S et al. Bacterial PHage Receptors, Versatile Tools for Display of Polypeptides on the Cell Surface[J]. Journal of Bacteriology. 2001, 183:6924
[40] Patricio M. Manque, Daniel Eichinger, Maria A. Juliano, et al. Characterization of the Cell Adhesion Site of Trypanosoma cruzi Metacyclic Stage Surface Glycoprotein gp82[J]. Infection and Immunity, 2000, 68(2):478-484
[41] Subbarao K,Klimov A,Katz J, et al.Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness[J]. Science, 1998,279:393-396.
[42] J. Liu. H. Xiao, F. Lei, et al.Highly Pathogenic H5N1 InfluenzaVirus Infection in Migratory Birds[J]. Science, 2005，309：1206
[43] 萨姆布鲁克，弗里奇，曼尼阿蒂斯．分子克隆实验指南，第二版[M]．北京：科学出版社，1998,852—897．
[44] YanRQ(闰若潜)，DuXQ(杜向党)．Research Progress in Genomic Structures and Proteins of Influenza virus[J]. Progress in Veterinary Medicine, 2004, 25 (1): 32-35
[45] Matthew PHilpott, Bernard C. Easterday, Virginia S. Hinshaw. Neutralizing Epitopes of the H5 Hemagglutinin from a Virulent Avian Influenza Virus and Their Relationship to Pathogenicity[J]. Virology, Aug. 1989, 3453-3458
[46] Tonegawa K, Nobusawa E, N akajima 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.
[47] Zebedee S L, Lamb R A. Growth restriction of influenza A virus by M2 protein antibody is genetically linked to the Ml protein[J]. Proc Natl Acad Sci USA, 1989, 86(3): 1061-1065
[48] Nathalie M, Sylvie A, Camille L. Nasal vaccination using live bacterial vectors[J]. Advanced Drug Delivery .2001, 51:55
[49] Mari OH, Masafumi Y, Yoshikazu Y et al. Non-toxic Stx derivatives from E.coli possess adjuvant activity for mucosal immunity[J]. Vaccine 2004, 22:3751
[50] Bermudes D, Zheng L M, King I C. Live bacteria as anticancer agents and tumor selective protein delivery vectors[J]. Curr Opin Drug Discov Devel, 2002, 5(2): 194
[51] Drabner B, Guzman CA. Elicitation of predictable immune responses by using live bacterial vectors[J]. Biomolecular Engineering, 2001, 17:75
[52] Christine Hale, Ian RH, Tracy Hussell, et al. Mucosal immunization of murine neonates using whole cell and acellular Pertussis vaccines[J]. Vaccine, 2004, 22:3595