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1 生物工程学报 Chin J Biotech 2009, April 25; 25(4): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 动物及兽医生物技术 DNA 马鸣潇 1, 金宁一 2, 尹革芬 3, 鲁会军 2, 李昌 2, 金扩世 2, 曲祖乙 1, , , : 以 O 型 A 型口蹄疫病毒 (FMDV) 结构蛋白 VP1 全基因和 AsiaI 型 FMDV 两个基因拓扑型的结构蛋白 VP1 基因上的 5 个抗原表位基因作为主要免疫原基因, 以来源于非结构蛋白 3ABC 和结构蛋白 VP4 上的 3 个 Th2 细胞表位基因作为辅助基因, 构建了 O 型 A 型和 Asia1 型 FMDV 复合多表位基因工程疫苗表达盒 OAAT, 在此基础上, 以金黄色葡萄球菌肠毒素 A(SEA) 为基因佐剂, 通过分子设计构建了 SEA 与 OAAT 融合表达基因将构建好的表达盒 OAAT 与 SEA 融合表达基因克隆至真核表达载体 PVAX1 PCMV 启动子下游, 构建了口蹄疫三价基因佐剂 DNA 疫苗 pea 经 Western blotting 和 IFA 检测, 目的蛋白在 Hela 细胞中获得正确表达小鼠免疫实验表明, pa 和 pea 免疫组的血清抗体均能分别与 O 型 A 型和 AsiaI 抗原反应, 与对照组相比差异较显著, 且 pea 免疫组和灭活疫苗免疫组抗体水平均显著高于 pa 免疫组 ; 同时 pa 和 pea 免疫小鼠细胞因子 IL-2 IFN-γ IL-4 和 IL-10 较对照组显著提高, 且 pea 免疫组的 IL-2 IFN- γ 和 IL-4 水平明显高于 pa 免疫组用 O/NY00 和 Asia1/YNBS/58 株 FMDV 进行豚鼠攻毒免疫保护试验, 结果表明 O 型和 Asia1 型 FMDV 二联灭活疫苗免疫组均获得完全保护, pa 免疫组均获得 2/4 保护, pea 免疫组均获得 3/4 保护, 而 pvax1 和 PBS 免疫组完全没有保护实验结果表明, SEA 与 OAAT 融合表达蛋白具有较好免疫原性, 且 SEA 可以作为 DNA 疫苗的有效基因佐剂 : 口蹄疫病毒, 金黄色葡萄球菌肠毒素 A(SEA), DNA 疫苗 1 Molecular design and immunogenicity of a multiple-epitope foot-and-mouth disease virus antigen, adjuvants, and DNA vaccination Mingxiao Ma 1, Ningyi Jin 2, Gefen Yin 3, Huijun Lu 2, Chang Li 2, Kuoshi Jin 2, and Zuyi Qu 1 1 College of Animal Science and Veterinary Medicine, Liaoning Medical University, Jinzhou , China 2 Genetic Engineering Laboratory, Academy of Military Medical Sciences, Changchun , China 3 College of Animal Science and Veterinary Medicine, Yunnan Agricultural University, Kunming , China Received: November 18, 2008; Accepted: January 21, 2009 Supported by: National High Technology Research and Development Program of China (863 Program) (No. 2006AA10A204), Ph. D Programs Foundation of Liaoning Province (No ). Corresponding author: Ningyi Jin. Tel: ; ; Fax: ; ningyik@126.com 国家高技术研究发展计划 (863 计划 ) (No. 2006AA10A204), 辽宁省博士启动资金 (No ) 资助

2 : DNA 515 Abstract: We designed and constructed a fuse expression gene OAAT and staphylococcal enterotoxin A (SEA) on the basis of the OAAT designed and constructed which consists of the structural protein VP1 genes from serotypes A and O FMDV, 5 major VP1 immunodominant epitopes from two genotypes of Asia1 serotype, and 3 Th2 epitopes originating from the non-structural protein, 3ABC gene and structural protein VP4 gene. The recombinant plasmids pea was constructed using SEA as a genetic adjuvant. Expressions of target gene from the pea in Hela cell were verified by IFA and Western blotting. The experiment of BALB/c mice immunized with the DNA vaccines showed that pa and pea could induce simultaneously specific antibodies against serotypes A, Asia1, and O FMDV, and the highest antibody titres were found in the pea and inactivated vaccine groups compared to pa vaccinating mice. Compared with the control, the levels of IL-2, IFN-γ, IL-4, and IL-10 expression by splenic lymphocytes from mice immunized with pa and pea were significantly increased. In addition, we found that the levels of IL-2, IFN-γand IL-4 from the mice immunized with pea was higher than mice immunized with pa did. The results of viral challenge in guinea pigs showed the pa, pea and inactivated vaccine provided full protection in 2/4, 2/4, 3/4, 3/4 and 4/4, 4/4 guinea pigs from challenge with FMDV O/NY00 and Asia1/YNBS/58, respectively. The results demonstrated fuse protein OAAT and SEA may be potential immunoge against FMDV, furthermore, SEA may be an effective genetic adjuvant for DNA vaccine. Keywords: foot and mouth disease virus, staphylococcal enterotoxin A(SEA), DNA vaccine (Foot-and-mouth disease, FMD) (Foot-and-mouth disease virus, FMDV) FMDV 7,, FMDV 60 4 VP1 VP2 VP3 VP4 FMDV B, 1 VP1 141~160aa 200~213aa, 2, ; 3 VP1 40~60aa, 1 ; 2( VP2 70~78aa 131~134aa), 4( VP3 56~58aa) 5( 149aa) DiMarchi [1], FMDV VP1 140~ ~213,,, [2], T B A T (VP ) [3], FMDV T [4], T MHC,, FMDV T,,,,, DNA FMDV, O A FMDV VP1 Asia1 FMDV VP1 5, DNA B, FMDV T, B, 3ABC VP4 3 Th2,, liker, T AsiaI FMDV A O FMDV VP1 OAAT,, SEA, O A AsiaI DNA, FMDV,, FMDV, 1 材料与方法 1.1 主要试剂 ExTaq RNA T4 DNA TaKaRa, DNA, M-MLV Promega, FITC IgG SBA T(AAIEFFEGMVHFDIK) ( )

3 516 ISSN CN /Q Chin J Biotech April 25, 2009 Vol.25 No.4,,, 95% 1.2 质粒和毒株 pvax1, O A AsiaI DNA pa [5] (paxi-oaat), SEA pet-sea, FMDV O/NY00(GenBank Accession No. AY333431) Asial/YNBS/58(GenBank Accession No. AY390432), BHK21, TCID 50 /0.01mL TCID 50 /0.01 ml O Asia1 FMDV 1.3 检测试剂盒 Mouse Th1/Th2 ELISA ebioscience Co. Ltd, USA 1.4 实验动物 BALB/c (4~5 ) (400~500 g) 1.5 FMDV 多表位佐剂 DNA 疫苗分子设计及构建 pet-sea, SEA (sense: 5 - CG GGT ACC ATG AGC GAG AAA AGC GAA G -3 ; antisense: 5 -CG GGA TCC AGA CGG CGG TGCGCCACC ACT TGT ATA TAAA-3 ) PCR, SEA pmd18-t pmd18-t-sea, pmd18-t-sea Kpn I SEA, SEA paxi-oaat Kpn I, DNA paxi-sea-oaat( p-sea), BamH I Kpn I SEA-OAAT, (Homological modelling), PDB, OAAT-SEA 1.6 Western blotting 检测 pvax1 pea HeLa, 72 h, SDS-PAGE, FMDVO, IgG Western blotting 1.7 间接免疫荧光检测 (IFA) pvax1 pea, 60%~70% Hela, 72 h, PBS 10% BSA PBS, ( FMDV ), FITC, 37 o C 1 h ; PBS, 1.8 小鼠免疫及免疫指标检测 60 5, 12, : 1 pvax1, 2 PBS, 3 pa, 4 pea, 5 O AsiaI FMDV, 100 μl ( 1 μg/μl) PBS O AsiaI FMDV, 3, 2, 0 d 10 d 21 d 28 d 38 d 50, O AsiaI A FMDV VP1, ELISA O AsiaI A FMDV,,, 20 μg/ml T 10 ng/ml IL-2, 4 h, 80 μg/ml C 2 h, /ml, 10:1, 2 μg/ml CoA 20 ng/ml IL d, Mouse Th1/Th2 ELISA 1.9 豚鼠免疫攻毒试验 40 5, 8 (n=8) μl PBS pvax1 (200 μg/200 μl PBS), μg/μl pa pea 200 μl μl O Asia1 FMDV 200 μl, ,, 100 TCID 50 FMDV O/NY00 Asia1/YNBS/ 58 O Asia1 FMDV 2, 2, 4, 0.2 ml 250 ID 50 O/NY00 Asia1/YNBS/58 FMDV,

609~622aa, VP1 141~160aa 200~213aa),,, OAAT-SEA 2.4 ELISA 抗体检测 ELISA 4~6, 10 d, pa pea O A Asia1 FMDV, 38 d, pvaxi PBS, (P<0.05), pea pa, (P<0.

图 1 FMDV 主要抗原表位在 OAAT-SEA 三维结构中的位置 Fig. 1 Spacial position of FMDV dominant epitopes in OAAT -SEA expression box. 2.

4 : DNA 517 1,, 2 结果 2.1 分子模拟 0AAT-SEA 1, AsiaI FMDV (94~113aa, 123~142aa 164~176aa, VP1 141~160aa 200~213aa)O FMDV (329~348aa, 388~401aa, VP1 141~160aa 200~213aa)A FMDV (550~569aa, 609~622aa, VP1 141~160aa 200~213aa),,, OAAT-SEA 2.4 ELISA 抗体检测 ELISA 4~6, 10 d, pa pea O A Asia1 FMDV, 38 d, pvaxi PBS, (P<0.05), pea pa, (P<0.05), (A FMDV ) (P>0.05) 图 2 pea 表达产物的 Western blotting 检测 Fig. 2 Western blotting of expressed product of pea. 1: pvax1; 2: pea; M: protein marker. 图 1 FMDV 主要抗原表位在 OAAT-SEA 三维结构中的位置 Fig. 1 Spacial position of FMDV dominant epitopes in OAAT -SEA expression box. 2.2 目的基因的 Western blotting 检测 3 d Hela, SDS-PAGE,, : pea 95 kd,, 目的基因的间接免疫荧光检测 (IFA) IFA 3, Hela, FMDV, pvaxi pea 图 3 核酸疫苗质粒转染 Hela 细胞的荧光图片 ( 伊文斯蓝染色 ) Fig. 3 Fluorescent photos of Hela cells transfected by plasmids DNA(stained by azovan blue). (A) Hela cell. (B) pvax1. (C) pea. 图 4 免疫小鼠抗体检测结果 (A 型 ) Fig. 4 Specific ELISA antibody to VP1 of type A FMDV after vaccination.

518 ISSN1000-3061 CN11-1998/Q Chin J Biotech April 25, 2009 Vol.25 No.4 2.5 细胞因子检测 7, IL-10, pa pea IL-2 IL-4 IFN-γ, (P<0.05), pea IFN- γ, (P<0.05), IL-2 IL-4 pa (P<0.05), pa, (P>0.

5 518 ISSN CN /Q Chin J Biotech April 25, 2009 Vol.25 No 细胞因子检测 7, IL-10, pa pea IL-2 IL-4 IFN-γ, (P<0.05), pea IFN- γ, (P<0.05), IL-2 IL-4 pa (P<0.05), pa, (P>0.05) 图 5 免疫小鼠 ELISA 抗体检测结果 (O 型 ) Fig. 5 Specific ELISA antibody to VP1 of type O FMDV after vaccination. 2.6 豚鼠免疫攻毒试验 O Asial FMDV, 1 2,, pea pa, O/NY00 Asia1/YNBS/58 FMDV, 1, O Asial FMDV, pa 2/4, pea 3/4, pvax1 PBS 表 1 O/NY00 株 FMDV 攻毒保护试验结果 Table 1 Protection of guinea pigs from O/NY00 strain FMDV challenge Guinea pig (No.) pvax1 PBS pa pea Inactive vaccine 1 None None Total Partial Total 2 None None Total Total Total 3 None None Partial Total Total 4 None None None Total Total Rate of protection (%) 0 (0/0) 0 (0/0) 50(2/4) 75(3/4) 100 (4/4) 图 6 免疫小鼠抗体检测结果 (Asia1 型 ) Fig. 6 Specific ELISA antibody to VP1 of type Asia1 FMDV after vaccination. 图 7 细胞因子 IFN-γ IL-2 IL-4 和 IL-10 检测结果 Fig. 7 ELISA results of the cytokines, IFN-γ, IL-2, IL-4 and IL-10. 表 2 Asia1/YNBS/58 株 FMDV 攻毒保护试验结果 Table 2 Protection of guinea pigs from Asia1/YNBS/58 strain FMDV challenge Guinea pig (No.) pvax1 PBS pa pea Inactive vaccine 1 None None Total Total Total 2 None None Total Partial Total 3 None None Partial Total Total 4 None None None Total Total Rate of protection (%) 0 (0/0) 0 (0/0) 50(2/4) 75 (3/4) 100 (4/4) 3 讨论 O A Asia1 FMDV, 3 FMDV,, FMD,, FMD [6,7] DNA, FMDV DNA [8 11],

6 : DNA 519,, FMDV,, ( CTL), O FMDV A FMDV VP1 AsiaI FMDV 2 VP1, FMDV Th2, FMDV OAAT, OAAT, OAAT A(Staphylococcus enterotoxin A, SEA), SEA-OAAT SEA, (Major histocompatibility complex, MHC)Ⅱ, Vβ T, CTLs,, SEA pea, SEA DNA pea (SEA-OAAT fusion) pa (OAAT alone) IL-2 IFN-γ IL-4 IL-10, IL-10,, IFN-γ pea pa Th1 Th2, IL-4 IgG1, Th1, IFN-γ [12,13], 2 DNA pa pea, O Asia1 FMDV, SEA DNA, pea SEA, FMDV REFERENCES [1] Marchi R, Brooke G, Gale C, et al. Protection of cattle against foot-and-mouth disease by a synthetic peptide. Science, 1986, 232(4750): [2] Taboga O, Tami C, Carrillo E. A large-scale evaluation of peptide vaccines against foot-and-mouth disease: Lack of solid protection in cattle and isolation of escape mutants. J Virol, 1997, 71(4): [3] Collen T, DiMarchi R, Doel TR. A T cell epitope in VP1 of foot-and-mouth disease virus is immunodominant for vaccinated cattle. J Immunol, 1991, 146(2): [4] Toja M, Escarmís C, Domingo E. Genomic nucleotide sequence of a foot-and-mouth disease virus clone and its persistent derivatives. Implications for the evolution of viral quasispecies during a persistent infection. Virus Res, 1999, 64(2): [5] Ma MX, Jin NY, Liu HJ, et al. Design and construction of multiple-epitope DNA vaccine against foot-and-mouth disease virus. Chin J Biologicals, 2007, 20(3): ,,,. O A Asia1 DNA., 2007, 20(3): [6] Beck E, Strohmaier K. Subtyping of European foot-and-mouth disease virus strains by nucleotide sequence determination. J Virol, 1987, 61(5), [7] King AMQ, Underwood BO, McCahon D, et al. Biochemical identification of viruses causing the 1981 outbreaks of foot-and-mouth disease in the UK. Nature, 1981, 293, [8] Wu L, Jiang L, Zhou Z, et al. Expression of foot-and-mouth disease virus epitopes in tobacco by a tobacco mosaic virus-based vector. Vaccine, 2003, 21(27-30): [9] Zhang HY, Sun SH, Guo YJ, et al. Immune response in mice inoculated with plasmid DNAs containing multiple-epitopes of foot-and-mouth disease virus. Vaccine, 2003, 21(32): [10] Cedillo-Barron L, Foster-Cuevas M, Cook A, et al. Immunogenicity of plasmids encoding T and B cell epitopes of foot-and-mouth disease virus (FMDV) in swine.vaccine, 2003, 21(27-30): [11] Wang QM, Sun SH, Hu ZL, et al. Improved immunogenicity of a tuberculosis DNA vaccine encoding ESAT6 by DNA priming and protein boosting. Vaccine, 2004, 22(27-28): [12] Blotta M, Dekruyff R, Umetsu D. Corticosteroids inhibit IL-12 production in human monocytes and enhance their capacity to induce IL-4 synthesis in CD4 + lymphocytes. J Immunol, 1997, 158(12): [13] Franchimon D, Galon J, Gadina M, et al. Inhibition of Th1 immune response by glucocorticoids: dexamethasone selectively inhibits IL-12-induced Stat4 phosphorylation in T lymphocytes. J Immunol, 2000, 164:

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇