摘要
鸡球虫病(Coccidiosis)是由艾美耳属(Eimeria)的各种球虫寄生在鸡的肠道引起的寄生性原虫病,所有日龄和品种的鸡都易感,严重影响了养殖业的发展。卡介苗(BCG)集佐剂与抗原于一身,是表达外源基因的理想活菌疫苗载体。为探讨重组卡介苗(rBCG)对鸡球虫感染的免疫保护作用,本研究首先构建了E.tenella rhomboid基因穿梭表达载体pMV261-Rho和整合表达载体pMV361-Rho,并在BCG内进行了rhomboid基因的诱导表达和免疫原性分析;然后将两种rBCG疫苗通过不同途径免疫雏鸡,观察其对动物免疫保护效果;在此基础上构建EGFP标记的rBCG,对Rhomboid蛋白在鸡体内各组织器官中的分布及稳定性和消长规律进行了研究;此外还将rhomboid基因与鸡IL-2基因(ChIL-2)联合构建rBCG,观察抗球虫效果。结果表明在鸡肝、脾、肺、肾、盲肠各组织器官均检测到rhomboid基因表达,雏鸡免疫14天后基因表达量达到最高,随后开始下降,至28天后消失;动物免疫保护性实验表明rBCG疫苗对E.tenella卵囊的攻击具有一定的免疫保护作用,诱导产生细胞免疫和体液免疫反应,且以滴鼻方式免疫效果最佳,抗球虫指数为174.6;将rhomboid基因与ChIL-2基因联合在卡介苗中表达,结果显示IL-2具有明显增强Rhomboid蛋白的免疫保护作用,其中pMV261-Rho-IL-2滴鼻免疫组保护效果最为明显,抗球虫指数ACI达到180以上,已达到临床使用的要求。本研究为球虫病的预防奠定了基础。
Coccidiosis, caused by intestinal protozoan parasites of the genus Eimeria, is a ubiquitous disease in poultry. E.tenella is one of the most important and serious species causing coccidiosis in chickens. Currently the control strategies of coccidiosis rely on chemotherapy and live parasite vaccines. However, due to the continual emergence of drug resistant strains, higher costs for new medicine development and the risks of reversion to highly virulent strains, novel vaccines are urgently needed to prevent further economic loss from the E.tenella infection. Rhomboid protein is the signal-generating component of epidermal growth factor (EGF) receptor signaling during development. Apicomplexan Rhomboid protein, having a potential role in microneme protein cleavage during host cell invasion, is a candidate antigen for development of a coccidiosis vaccine. BCG, the attenuated strain of Mycobacterium bovis, has been considered a promising candidate for the delivery of foreign antigens to the immune system. Several features have encouraged its use as a live carrier for recombinant antigens, including its known adjuvant properties, its ability to elicit humoral or cellular immune responses toward heterologous antigens, its thermostability, its low frequency of side effects and it is inexpensive to produce. A wide range of recombinant BCG vaccine candidates containing foreign viral, bacterial, parasitic or immunomodulatory genetic materials have been developed and evaluated for stimulation of immune response to the foreign antigen, but there is no report has been published on rBCG used in avian species against coccidiosis.
Based on our aim to combine the two, two rBCG strains pMV261-Rho and pMV361-Rho expressing rhomboid gene of E.tenella were constructed in this study. Animal experiments via intranasal, oral and subcutaneous route in chickens were carried out to evaluate the immune efficacy of the vaccines. The protective effects of immunization were evaluated by mortality, cecal lesion scores, oocysts output and growth performance after a challenge with E.tenella oocysts. We also constructed M. bovis BCG recombinants expressing enhance green fluorescent protein (EGFP) as a fusion protein with Rhomboid antigen and evaluate the stability and the localization of the rBCG in tissues of chickens. For enhancement of the protective efficacy of E. tenella infection in chickens, recombinant BCG (rBCG) co-expressing rhomboid and ChIL-2 gene were constructed, and its efficacy against coccidiosis in chickens were evaluated. This report should provide a new method for the future development of vaccines against coccidiosis.
Construction of pMV261-Rho and pMV361-Rho expression vectors and expression in BCG
The rhomboid gene segment (GenBank accession No. DQ323509) of E.tenella F2 strains was generated by PCR with primers, and was subcloned into the PstI/ClaI sites of pMV261 and PvuII/ClaI sites of pMV361, respectively. The resulting plasmids pMV261-Rho and pMV361-Rho were electrotransfected into BCG and selected by kanamycin. The results showed that the two vectors pMV261-Rho and pMV361-Rho containing rhombid gene were successful constructed, and Western blotting indicated that the Rhomboid protein was successfully expressed in rBCG. This study has established foundation for further study of E.tenella rBCG.
Protective immunity of rBCG pMV261-Rho and pMV361-Rho against E.tenella challenge
Chickens were immunized with rBCG pMV261-Rho and pMV361-Rho via intranasal, oral and subcutaneous route, with BCG immunized as control. After 1 and 2 week intervals, each sample was boosted. Chickens were then challenged with E.tenella sporulated oocysts and the efficacy of immunization was evaluated on the basis of oocyst output, cecal lesion scores, ACI, response immunity of cellular and antibody. All the rBCG immunized chickens developed specific immune responses, and there was a significant increases of the percentages of CD4+ and CD8+ cells compared to the control(P<0.01). Vaccination via intranasal and oral produced better protective effect, The anticoccidiosis index(ACI) of intranasal immunized rBCG pMV261-Rho-IL-2 groups was 174.6. Challenge experiments demonstrated that the two rBCG strains could provide significant protection against E. tenella challenge.
Construction of EGFP-tagged rBCG of E.tenella and distribution in chickens
The fragment of EGFP and rhomboid were subcloned into the pMV361 integrative expression vector, with correct structures was then transformed into BCG by electroporation. Chickens were immunized with this rBCG pMV361-Rho-EGFP, and one week after the immunization, various tissues containing liver, spleen, lung, kindey and caecum from rBCG pMV361-Rho-EGFP immunized chickens were removed and processed to frozen section. Expression of Rhomboid protein in liver, spleen, lung, kindey and caecum was observed by laser confocal microscopy. At the 7th, 14th, 21st, 28th day after the immunization, RNA from liver, spleen, lung, kindey, caecum tissues was extracted. The results showed that EGFP fluorescence expression of various tissues containing liver, spleen, lung, kindey and caecum from rBCG pMV361-Rho-EGFP immunized chickens was observed by laser confocal microscopy one week after the immunization. Real-time quantitative RT-PCR revealed that a rise of rhomboid expression level at 7th day and a peak at 14th day and a disappearance at the 28th day after immunization.
Construction of pMV261-Rho-IL-2 and pMV361-Rho-IL-2 and expression in BCG
ChIL-2 is an important cytokine with the ability of enhancement of the immune responses. ChIL-2 gene segment was ligated together with rhomboid fragment and subcloned into the pMV261 and pMV361 expression vector, separately. The recombinant plasmids pMV261-Rho-IL-2 and pMV361-Rho-IL-2 was electrotransfected into BCG and selected by kanamycin. After induction period at 45℃, the recombinant proteins were separated by SDS-PAGE and Western boltting was done for immunoblot analysis. A band with the molecular masses of about 40 kDa was detected from rBCG pMV261-Rho-IL-2 and pMV361-Rho-IL-2, suggesting that the fusion protein Rho-IL-2 had expressed successfully in rBCG
Protective immunity of rBCG pMV261-Rho-IL-2 and pMV361-Rho-IL-2 against Eimeria tenella challenge
Chickens were immunized with rBCG pMV261-Rho-IL-2 and pMV361-Rho-IL-2 via intranasal, oral and subcutaneous route, and boosted after 1 and 2 week intervals. Chickens were then challenged with E.tenella sporulated oocysts and the efficacy of immunization was evaluated on the basis of oocyst output, cecal lesion scores, ACI, cellular immunity response and antibody immunity. All the rBCG immunized chickens developed specific immune responses, and there was a significant increases of the percentages of CD4+and CD8+cells compared to the control(P<0.01). Challenge experiments demonstrated that the two rBCG strains could provide significant protection against E. tenella challenge. The level of CD4+,CD8+ lymphocyte and the ratio of CD4+/CD8+ of immunized chicken were increased significantly compared with controls(P<0.01). Chickens immunized rBCG revealed a significant decrease in oocyst output, especially in the group of intranasal immunized rBCG pMV261-Rho-IL-2. The ACI of rBCG pMV261-Rho-IL-2 and pMV361-Rho-IL-2 immunized groups were above 160. The highest ACI is 184.0 in the group of intranasal immunized rBCG pMV261-Rho-IL-2. The protective effect in pMV261-Rho-IL-2 and pMV361-Rho-IL-2 immunized groups are better than the groups received rBCG pMV261-Rho and pMV361-Rho.
引文
[1] Delogu G, Brennan MJ. Comparative antigens of Mycobacterium tuberculosis immune response to PE and PE PGRS[J]. Infect Immun, 2001, 69(9): 5606-5611.
[2] Miyaji EN, Mazzantini RP, Dias WO, et al. Induction of neutralizing antibodies against diphtheria toxin by priming with recombinant Mycobacterium bovis BCG expressing CRM (197), a mutant diphtheria toxin[J]. Infect Immun, 2001, 69: 869-874.
[3] Orem IM. Progress in the development of new vaccines against tuberculosis[J]. Int J Tuberc Lung Dis, 1997, 1(2): 95-100.
[4] Collins DM. New tuberculosis vaccines based on attenuated strains of the Mycobacterium tuberculosis complex[J]. Immunol Cell Biol, 2000, 78(10): 342-348.
[5] Hess J, Kaufmann SH. Live antigen carriers as tools for improved anti-tuberculosis vaccines[J]. Biolchem Mol Biol Int, 1999, 43 (6): 1117-1124.
[6]吴守芝,宋俊峰.结核分枝杆菌致病机制与免疫[J].中华卡介苗和呼吸杂志,2003,26(2):101-103.
[7] Janeway CA Jr. The immune system evolved to discriminate infectious nonself from noninfectious self[J]. Immunol Today, 1992, 13(1):11-16.
[8]王世若,王兴龙,韩文瑜.现代动物免疫学[M].长春:吉林科学技术出版社,2001, 142-154.
[9]刘丹.热休克蛋白在天然免疫和适应性免疫中的作用国外医学免疫学分册,2004, 27(3):121-124.
[10]焦新安,Lo-Mane R, Rueimonprez P, et al.重组卡介苗感染树突细胞的研究[J].扬州大学学报(农业与生命科学版),2002(23):1-5.
[11] Schlesinger LS, Bellinger-Kawahara CG, Payne NR, et al. Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3[J]. J Immunol, 1990, 144(7): 2771-2280.
[12] Schlesinger LS. Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is mediated by mannose receptors in addition to complement receptors[J]. J Immunol, 1993, 150(7): 2920-2930.
[13] Collins DM. Kawakami RP, Wards BJ, et al.Vaccine and skin testing properties of two avirulent Mycobacterium bovis mutants with and without an additional esat-6 mutation[J]. Tuberculosis (Edinb). 2003;83(6):361-6.
[14] Langermann S, Palaszynski SR, Burlein JE, et al. Protective humoral response against pheumococcal infection in mice elicited by recombinant Balille Calmette-Gurein vaccines expressing pneumococcal surface protein A[J]. Exp Med, 1994, 180 (6): 2277-2286.
[15] Honda M, Matsuo K, Nakasone T et al, Protective immune responses induced by secretion of a chimeric soluble protein from a recombinant Mycobacterium bovis BCG vector candidate vaccine for human immunodeficlency virus type 1 in small animals[J]. Proc Natl Acad Sci USA, 1995, 92 (23): 10693-10697.
[16] Lagranderie M, Winter N, Balazuc AM, et al. A cocktail of Mycobacterium bovis BCG recombinants expressing the SIV Nef, Env, and Gag antigens induces antibody and cytotoxic responses in mice vaccinated by different mucosal routes[J]. AIDS Res Hum Retroviruses, 1998, 14(18): 1625-1633.
[17] Wada N, Ohara N, Kameoka M, et al. Long-lasting immune response induced by recombinant bacillus Balille Calmette-Gurein(BCG) secretion system[J]. Scand J Immunol, 1996, 43(2): 202-209.
[18] Stover CK, Bansal GP, Hanson MS, et al. Protective immunity elicited by recombinant Bacille Calmette Guerin(BCG) expression outer surface protein A(OspA) lipoprotein:A candidate fume disease vaccine[J]. J Exp Med, 1993, 178: 197-209.
[19] Edelman R, Palmer K, Russ KG, et al. Safety and immunogenicity of recombinant Bacille Calmette-Guérin (rBCG) expressing Borrelia burgdorferi outer surface protein A (OspA) lipoprotein in adult volunteers: a candidate Lyme disease vaccine[J]. Vaccine, 1999, 17(7-8): 904-914.
[20] Lagranderie M, Murray A, Gicquel B, et a1. Oral immunization with recombinant BCG induces cellular and homoral immune responses against the foreign antigen[J]. Vaccine, 1993, 11(13): 1283-1290.
[21] Rauzier J, Moniz-Pereira J, Gicquel-Sanzey B. Complete nucleotide sequence of pAL5000, a plasmid from Mycobacterium fortuitum[J]. Gene, 1988, 30: 71(2):315-321.
[22] Stover CK, dela Cruz VF, Fuerst TR, et al. New use of BCG for recombinant vaccines[J]. Nature, 1991, 351(6326): 456-460.
[23] Winter N, Lagranderie M, Rauzier J, et al. Expression of heterologous genes in Mycobacterium bovis BCG: induction of a cellular response against HIV-1 Nef protein[J].Gene, 1991, 109(1): 47-54.
[24] Triccas JA, Britton WJ, Gicquel B. Isolation of strong expression signals of Mycobacterium tuberculosis[J]. Microbiology, 2001, 147(5): 1253-1258.
[25]程继忠,皇甫永穆,海涛.外源基因在耻垢分枝杆菌中表达效率的研究[J].生物化学与生物物理进展,1997, 24(3): 249-253.
[26]路艳燕,冯作化,皇甫永穆,等.新型大肠杆菌-分枝杆菌穿梭载体的构建及卡那霉素抗性基因表达的研究[J].同济医科大学学报,1998, 27(2): 89-93.
[27]程继忠,郑波,海涛,等.大肠杆菌-分枝杆菌穿梭表达质粒pBCG2100的构建及应用[J].生物工程学报,1999, 15(2): 225-229.
[28]程继忠,郑波,肖红,等.结核杆菌HSP70在耻垢分枝杆菌中的表达及其免疫原性研究[J].中华微生物学和免疫学杂志,1998, 18(5): 337-341.
[29] Matsuo K, yamaguchi R, Yamazaki A, et al. Establishment of foreign antigen secretion system Mycobacteria [J].Infect Immun, 1990, 58(12): 4049-4054.
[30] Stover CK, Marana DP, Carter JM, et al. The 56-kilodalton major protein antigen of Rickettsia tsutsugamushi: molecular cloning and sequence analysis of the sta56 gene and precise identification of a strain-specific epitope[J]. Infect Immun, 1990, 58(7): 2076-2084.
[31] Young RA. Stress proteins and immunology[J]. Annu Rev Immunol, 1990, 8: 401-420.
[32] Vodkin MH, Williams JC. A heat shock operon in Coxiella burnetti produces a major antigen homologous to a protein in both mycobacteria and Escherichia coli[J]. J Bacteriol, 1988 , 170(3): 1227-1234.
[33] Yi Y, Zhong G, Brunham RC. Continuous B-cell epitopes in Chlamydia trachomatis heat shock protein 60[J]. Infect Immun, 1993, 61(3): 1117-1120.
[34] Buchmeier NA, Heffron F. Induction of Salmonella stress proteins upon infection of macrophages[J]. Science, 1990, 248(4956):730-732.
[35] Ohara N, Yamada T. Recombinant BCG vaccines[J]. Vaccine, 2001, 19: 4089-4098.
[36] Silva CL, Lowrie DB. A single mycobacterial protein (HSP 65) expressed by a transgenic antigen-presenting cell vaccinates mice against tuberculosis[J]. Immunology, 1994, 82(2): 244-248.
[37] Kremer L, Riveau G, Baulard A, et al. Neutralizing antibody responses elicited in mice immunized with recombinant bacillus Calmette-Guerin producing the Schistosoma mansoni glutathione S-transferase[J]. J Immunol, 1996, 156(11): 4309-4317.
[38] Kremer L, Dupre L, Riveau G, et al. Systemic and Mucosal Immune Responses after Intranasal Administration of Recombinant Mycobacterium bovis Bacillus Calmette-Guerin Expressing Glutathiones-Transferase from Schistosoma haematobium[J]. Infect Immun, 1998, 66(12): 5669-5676.
[39] Varaldo PB, Leite LC, Dias WO, et al. Recombinant Mycobacterium bovis BCG Expressing the SmL4 Antigen of Schistosoma mansoni Protects Mice from Cercarial Challenge[J]. Infect Immun, 2004, 72(6): 3336–3343.
[40]肖红,郑波,祁学忠,皇甫永穆.日本血吸虫32kDa抗原基因的克隆及在耻垢分枝杆菌中的表达[J].同济医科大学学报,2000,29(1):4-7.
[41]程继忠,皇甫永穆,海涛,等.日本血吸虫26kDa抗原基因在BCG中的表达[J].中国生物化学与分子生物学报,1998,14(6):661-667.
[42]李文桂,石佑恩,汪燕鸣,等.血吸虫重组BCG-Sj26GST疫苗接种后保护力的观察[J].同济医科大学学报,1999,24(1):13-16.
[43]戴五星,郑波,皇甫永穆.日本血吸虫重组BCG-Sj26GST疫苗对小鼠免疫应答的影响[J].同济医科大学学报,2000,29(1):1-3.
[44] Matsumoto S, Yukitake H, Kanbara H, et al. Recombinant Mycobacterium bovis bacillus Calmette-Guérin secreting merozoite surface protein 1 (MSP1) induces protection against rodent malaria parasite infection depending on MSP1-stimulated interferon gamma and parasite-specific antibodies[J]. J Exp Med, 1998, 188(5): 845-854.
[45] Zheng C, Xie P, Chen Y. Molecular cloning and sequencing of the circumsporozoite protein gene from Plasmodium falciparum strain FCC-1/HN and expression of the gene in Mycobacteria[J]. J Clin Microbiol. 2001, 39(8): 2911-2915.
[46] Abdelhak S, Louzir H, Timm J, et al. Recombinant BCG expressing the leishmania surface antigen Gp63 induces protective immunity against Leishmania major infection in BALB/c mice[J]. Microbiology. 1995, 141(7): 1585-1592.
[47]王洪法.弓形虫ROP2基因在大肠杆菌和卡介苗中的表达及免疫原性的研究[D].长春:吉林大学,畜牧兽医学院,2005.
[48]李文桂,王鸿,朱佑明.多房棘球绦虫重组BCG-EmL4-3-3疫苗不同接种途径诱导小鼠细胞因子的研究[J].四川大学学报(医学版),2008,39(1):130-132.
[49]李文桂,朱佑明,王鸿.多房棘球绦虫重组BCG-EmⅡ/3疫苗免疫小鼠后脾细胞亚群的动态观察[J].免疫学杂志,2008,24(6),146-149.
[50]周宇.肝片吸虫保护性抗原基因在卡介苗中的表达[D].成都:四川大学,2003.
[51] O'Donnell MA, Aldovini A, Duda RB, et al. Recombinant Mycobacterium bovis BCG secreting functional interleukin-2 enhances gamma interferon production by splenocytes[J]. Infect Immun, 1994, 62(6): 2508-2514.
[52] Murray PJ, Aldovini A, Young RA. Manipulation and potentiation of antimycobacterial immunity using recombinant bacille Calmette-Guerin strains that secrete cytokines[J]. Proc Natl Acad Sci USA, 1996, 93(2): 934-939.
[53] Kong DK, Danimoto DY. Secretion of human interleukin 2 by recombinant Mycobacterium bovis BCG[J]. Infect Immun, 1995, 63(3): 799~803.
[54] Wangoo A, Brown IN, Marshall BQ, et al. Bacille Calmette-Guerin (BCG) associated inflammation and fibrosis: modulation by recombinant BCG expressing interferon-gamma (IFN-gamma)[J]. Clin Exp Immunol, 2000, 119 (1): 92-98.
[55] Miller LA, Johns BE, Elias DJ, et al. Oral vaccination of white- tailed deer using a recombinant Bacillus Calmette–Guerin vaccine expression the prorrelia burgdorferi outer surface protein A: prospects for immunocontraleption[J]. vaccine, 1999, 17 (7-8): 904-914.
[56] Langermann S, Palaszynski S, Sadziene A, et al. Systemic and mucosal immunity induced by BCG vector expressing outer-surface protein A of Borrelia burgdorferi[J]. Nature, 1994, 6506: 552–555.
[57] Hayward CM, O’Gaora P, Young DB, et al. Construction and murine immunogenicity of recombinant Bacille Calmette Guerin vaccines expressing the B subunit of Escherichia coli heat labile enterotoxin[J]. Vaccine, 1999,17 (9/10): 1272-1281.
[58] Power CA, Wei G, Bretscher PA. Mycobacterial dose defines the Th1/Th2 nature of the immune response independently of whether immunization is administered by the intravenous, subcutaneous, or intradermal route[J]. Infect Immun. 1998, 66(12): 5743–5750.
[59] Griffin JF, Mackintosh CG, Slobbe L, et al. Vaccine protocols to optimise the protective efficacy of BCG[J]. Tuberc Lung Dis, 1999, 79(3): 135-143.
[60] YANG Wen-chong. Study on live vehicle of recombinant BCG[J]. Chi Microbiology immunology progress, 2003, 31(2): 79-82.
[61] De Bruyn J, Soetaert K, Buyssens P, et al. Evidence for specific and non-covalent binding of lipids to natural and recombinant Mycobacterium bovis BCG HSP60 proteins, and to the Escherichia coli homologue GroEL[J]. Microbiology. 2000, 146(7): 1513-1524.
[62] Chapman HD, Cherry TE, Danforth HD, et al. Sustainable coccidiosis contorl in poultry production: the role of live vaccines[J]. Int J Parasitol, 2002, 32: 617-629.
[63] Shirley MW, Blake D, White SE, et al. Integrating genetics and genomics to identify new leads for the control of Eimeria spp[J].Parasitology, 2004, 128: 33-42.
[64] Shirley MW, Smith AL, Blake DP. Challenges in the successful control of the avian coccidia[J]. Vaccine, 2007, 25(30): 5540-5547.
[65]韩春来,汪明,王茂荣.鸡球虫苗的研制与应用[J].山东家禽,2004,11:38-41.
[66] Shirley MW. The genome of Eimeria spp.with special reference to Eimeria tenella-a coccidium from the chicken[J]. Int J Parasitol, 2000, 30(4):485-493.
[67] McDonald V, Rose ME, Jeffers TK. Eimeria tenella: immunogenicity of the first generation of schizogony[J]. Parasitology, 1986, 93 (1): 1-7.
[68] Shirley MW, Smith AL, TomLey FM. The biology of avian Eimeria with an emphasis on their control by vaccination[J]. Adv Parasitol, 2005, 60:285-330.
[69] Bhogal BS, Miller GA, Anderson AC, et al. Potential of a recombinant antigen as a prophylactic vaccine for day-old broiler chickens against Eimeria acervulina and Eimeria tenella infections[J]. Vet Immunol Immunopathol, 1992, 31(3-4): 323-335.
[70] Jenkins MC, Lillehoj HS, Dame JB. Eimeria acervulina: DNA cloning and characterization of recombinant sporozoite and merozoite antigens[J]. Exp Parasitol, 1988, 66(1):96-107.
[71] Belli SI, Witcombe D, Wallach MG, et al. Functional genomics of gam56: characterisation of the role of a 56 kilodalton sexual stage antigen in oocyst wall formation in Eimeria maxima[J]. Int J Parasitol, 2002, 32(14):1727-1737.
[72] Lillehoj HS, Ding X, Dalloul RA, et al. Embryo vaccination against Eimeria tenella and E.acervulina infections using recombinant proteins and cytokine adjuvants[J]. J Parasitol, 2005, 91(3): 666-673.
[73] Talebi A, Mulcahy G. Eimeria tenella: B-cell epitope mapping following primaryand secondary infections[J]. Exp Parasitol, 2006, 113(4):235-238.
[74] Danforth HD, Augustine PC. Use of hybridoma antibodies and recombinant DNA technology in protozoan vaccine development[J]. Avian Dis, 1986, 30(1):37-42
[75] Du A, Hu S, Wang S. Eimeria tenella: ginsenosides-enhanced immune response to the immunization with recombinant 5401 antigen in chickens[J]. Exp Parasitol, 2005, 111(3):191-197.
[76] Du A, Wang S. Efficacy of a DNA vaccine delivered in attenuated Salmonella typhimurium against Eimeria tenella infection in chickens[J]. Int J Parasitol, 2005, 35(7): 777-785.
[77] Binger MH, Hug D, Weber G, et al. Cloning and characterization of a surface antigen of Eimeria tenella merozoites and expression using a recombinant vaccinia virus[J]. Mol Biochem Parasitol, 1993, 61(2):179-187.
[78]秦睿玲.柔嫩艾美耳球虫λMzp5-7基因在原核和真核细胞中的表达及免疫保护性研究[D].长春:中国人民解放军军需大学,2003.
[79]格日勒图.鸡柔嫩艾美耳球虫Mzp5-7基因免疫调节型DNA疫苗的研究[D].南京:南京农业大学,2005.
[80] Brothers VM, Kdahn I, Paul IS, et a1. Characterization of a surface antigen of Eimeria tenella sporozoites and synthesis from a cloned cDNA in E.scherichia coli[J]. Mo1 Biochem Parasitol, 1988, 28(3):235-247.
[81]潘晓亮.TA4和EtMIC-2表达产物免疫后对感染柔嫩艾美耳球虫(Eimeria tenella)鸡增重和盲肠卵囊数的影响[J].黑龙江畜牧与兽医,2002, 8: 6-7.
[82]吴绍强,蒋金书,刘群,等.柔嫩艾美耳球虫BJ株核酸疫苗的构建及其免疫保护效果研究[J].中国兽医杂志,2004,40(9):76-82.
[83] Miller GA, Bhogal BS, McCandliss R, et a1.Characteriatiori and vaccine potential of a novel recombinant coccidial[J]. Infection and Immunity, 1989, 57(7): 2014-2020.
[84] Klotz C, Gehre F, Lucius R, et al.Identification of Eimeria tenella genes encoding for secretory proteins and evaluation of candidates by DNA immunisation studies in chickens[J]. Vaccine, 2007, 25(36): 6625-6634.
[85] Crane MS, Goggin B, Pellegrino RM, et al. Cross-protection against four species of chicken coccidia with a single recombinant antigen[J].Infect Immun, 1991, 59(4): 1271-1277.
[86] TomLey FM, Billington KJ, Bumstead JM, et al. EtMIC4: a microneme protein from Eimeria tenella that contains tandem arrays of epidermal growth factor-like repeats and thrombospondin type-I repeats[J].Int J Parasitol, 2001, 31(12): 1303-1310.
[87] Periz J, Gill AC, Knott V, et al. Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4[J]. Mol Biochem Parasitol, 2005, 110: 311-321.
[88] Labbe M, Venevelles P, Girard-Misguich F, et al. Eimeria tenella microneme protein EtMIC3: identification, localization and role in host cell infection[J]. Mol Biochem Parasitol, 2005, 140: 43-53.
[89] TomLey FM, Bumstead JM, Billington KJ, et al. Molecular cloning and characterization of a novel acidic microneme protein (Etmic-2) from the apicomplexan protozoan parasite, Eimeria tenella[J]. Mol Biochem Parasitol, 1996, 79 (2): 195-206.
[90] Urban S. Rhomboid proteins: conserved membrane proteases with divergent biological functions[J]. Genes, 2006, 20(22): 3054-3068.
[91] Dowse TJ, Soldati D. Rhomboid-like proteins in Apicomplexa: phylogeny and nomenclature[J]. Trends Parasitol, 2005, 21(6): 254-258.
[92] Brossier F, Jewett TJ, Sibley LD, et al. A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma[J]. Proc Natl Acad Sci U S A, 2005, 102(11): 4146-4151.
[93] O'Donnell RA, Blackman MJ. The role of malaria merozoite proteases in red blood cell invasion[J]. Curr Opin Microbiol, 2005, 8(4): 422-427.
[94] O'Donnell RA, Hackett F, Howell SA, et al. Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite[J]. J Cell Biol, 2006, 174(7): 1023-1033.
[95] Li J, Zhang X, Liu Q, et al. Eimeria tenella: Cloning of a novel Eimeria tenella cDNA encoding a protein related to rhomboid family from F2 hybrid strain[J]. Exp Parasitol, 2006, 113: 215-220.
[96]陈超.柔嫩艾美耳球虫ET-Rho-2基因在原核和真核细胞中的表达及免疫保护性研究[D].长春:吉林大学,2005.
[97] Yang G, Li J, Zhang X, et al. Eimeria tenella: construction of a recombinant fowlpox virus expressing rhomboid gene and its protective efficacy against homologous infection[J]. Exp Parasitol, 2008, 119: 30-36.
[98] Kinnaird JH, Bumstead JM, Mann DJ. EtCRK2, a cyclin-dependent kinase gene expressed during the sexual and asexual phases of the Eimeria tenella life cycle[J]. Int J Parasitol, 2004, 34(6): 683-692.
[99] Wiersma HI, Galuska SE, TomLey FM. A role for coccidian cGMP-dependent protein kinase in motility and invasion[J]. Int J Parasitol, 2004, 34(3): 369-380.
[100] Péroval M, Péry P, LabbéM, et al. The heat shock protein 90 of Eimeria tenella is essential for invasion of host cell and schizont growth[J]. Int J Parasitol, 2006, 36(10-11): 1205-1215.
[101] Haygreen L, Davison F, Kaiser P. DNA vaccines for poultry: the jump from theory to practice[J]. Expert Rev Vaccines, 2005, 4(1): 51-62.
[102] Kopko SH, Martin DS, Barta JR. Responses of chickens to a recombinant refractile body antigen of Eimeria tenella administered using various immunizing strategies[J].Poult Sci, 2000, 79(3): 336-342.
[103] Songa KD, Lillehoja HS, Choia KD, et al. A DNA vaccine encoding aconserved Eimeria protein induces protective immunity gainst live Eimeria acervulina challenge [J]. Vaccine, 2001, 9: 243-252.
[104]秦睿玲,张西臣,李建华,等.柔嫩艾美球虫重组质粒pVAX1-Mzp5-7的免疫保护性试验[J].中国寄生虫学与寄生虫病杂志,2004,22(6):334-337.
[105] Krieg AM. From A to Z on CpG[J]. Trends Immunol, 2002, 23(2): 64-65.
[106] Dalloul RA, Lillehoj HS, Klinman DM, et al. In ovo administration of CpG oligodeoxynucleotides and the recombinant microneme protein MIC2 protects against Eimeria infections[J].Vaccine, 2005, 23(24): 3108-3113.
[107] Lillehoj HS, Ding X, Quiroz MA, et al. Resistance to intestinal coccidiosis following DNA immunization with the cloned 3-1E Eimeria gene plus IL-2, IL-15, and IFN-gamma[J]. Avian Dis, 2005, 49(1): 112-117.
[108] Geneva WHO. Nucleic Acid Vaccines[J]. Vaccine, 1994, 12: 1491-1550.
[109] McCluskie MJ, Brazolot-MiUan CL, Gramzinski RA. Route and method of delivery of DNA vaccine influence immune Responses in mice and non-human primates [J].Mol Med, 1999, 5(50): 287-300.
[110] Fomsgaard A, Nielsen HV, Nielsen C, et al. Comparisons of DNA-mediated immunization procedures directed against surface glycoproteins of human immunodefi-ciency virus type-1 and hepatitis B virus[J]. APMIS, 1998, 106(6): 636-646.
[111] 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 (24): 11478-11482.
[112] Fried Michal, Sar-Shalom O, et al. Developmental gene expression of a 230-klodalton in of the avian coccididal parasite, Eimeria maxima[J]. Molecular and Biochemical Parasitology, 1992, 51: 251-262.
[113] Jeurissen SH, Janse EM, Vermeulen AN, et al. Eimeria tenella infections in chickens: aspects of host-parasite: interaction[J]. Vet Immunol Immunopathol, 1996, 54(1-4): 231-238.
[114] Yun CH, Lillehoj HS, Lillehoj EP, et al. Intestinal immune responses to coccidiosis[J]. Dev Comp Immunol, 2000, 24(2-3): 303-324.
[115] Lillehoj HS, Kim CH, Keeler CL Jr, et al. Immunogenomic approaches to study host immunity to enteric pathogens[J].Poult Sci, 2007, 86(7): 1491-1500.
[116] Vermeulen AN, Schaap DC, Schetters TP. Control of coccidiosis in chickens by vaccination[J]. Vet Parasitol, 2001, 100(1-2): 13-20.
[117] Williams RB. Anticoccidial vaccines for broiler chickens: path-way to success [J]. Avian Path, 2002, 31: 317-353.
[118] Dalloul RA, Lillehoj HS. Recent advances in immunomodulation and vaccination strategies against coccidiosis [J]. Avian Dis, 2005, 49: 1-8.
[119] Lillehoj HS. Effects of immunosuppression on avian coccidiosis: cyclosporin A but not hormonal bursectomy abrogates host protective immunity[J].Infect Immun, 1987, 55(7): 1616-1621.
[120] Choi KD, Lillehoj HS. Role of chicken IL-2 onγδT-cells and Eimeria acervulina-induced changes in intestinalIL-2 mRNA expression andγδT-cells[J]. Vet Immunol Immunopathol, 2000, 73: 309–321.
[121] Digby MR, Lowenthal JW. Cloning and expression of the chicken interferon-γgene[J]. Interferon Cytokine Res, 1995, 15: 939-949.
[122] Min W, Lillehoj HS, Burnside J, et a1. Adjuvant effects of Ilrlbeta, IL-2, IL-8, IL-15, IFN alpha, IFN gamma, TGF beta4 and lymphotactin on DNA vaccination against Eimeria acervulina[J].Vaccine, 2001, 20(122): 267-274.
[123] Dimier-Poisson IH, Soundouss Z, Naciri M, et a1. Mechanism of the Eimeria tenalla growth inhibitory activity induced by concanavalian A andrecyiculoendo theliosis virus supernatants with interferon gamma activity in chicken macrophages and fibroblasts[J]. Avian Dis, 1999, 43: 65-74.
[124] Talebi A, Torgerson PR, Mulcahy. Optimal conditions for mearsurement of blastogenic response of chickens to concanavalin-A in whole blood assays[J]. Vet Immunol Immunol, 1995, 46: 293-301.
[125]叶秀华,蔡建平,吴志光,等.重组鸡γ干扰素对E.tenella卵囊免疫后肠上皮组织结构的影响[J].中国兽医学报,2005, 25 (5): 484-486.
[126] Schauenstein K, Globerson A, Wick G. Avian lymphonkinesahymic cell growth factor in supernatants of mitogen stimulated chicken spleen cells[J]. Dev Comp Immunol, 1982, 6: 533-540.
[127] Sundick RS, Gill-Dixon C. A cloned chicken lymphokine homologous to both mammalian IL-2 and IL-15[J]. J.Immunol, 1997, 159: 720–725.
[128]李祥瑞,金红,卢景良.鸡白细胞介素-2 cDNA克隆[J]南京农业大学学报,1999,(02):45-49.
[129]谢昆,李祥瑞.重组鸡IL-2的抗球虫作用[J].中国兽医杂志,2006,42(8): 7-9.
[130]徐前明,李祥瑞.重组鸡IL-2对禽流感灭活苗免疫增强作用试验[J].动物医学进展,2006,10:22-26.
[131]李宏梅.重组鸡白细胞介素-2作为免疫增强剂的研究[D].南京:南京农业大学, 2000.
[132] Zhang S, Lillehoj HS, Ruff MD. In vivo role of tumor necrosis-like factor in Eimeria tenella infection[J]. Avian Dis, 1997, 39: 859–866.
[133] Byrnes S, Eaton R, Kogut M. In vitro interleukin-1 and tumor necrosis factor-αproduction by macrophages from chicken infected with Eimeria maxima or Eimeria tenella[J]. Int J Parasitol, 1993, 23: 639–645.
[134] Smith NC, Ovington KS. The effect of BCG, zymosan and Coxiella burnetti extract on Eimeria infections[J]. Immunol Cell Biol, 1996, 74: 346–348.
