柔嫩艾美耳球虫λMzp5-7基因在原核和真核细胞中的表达及其免疫保护性研究
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摘要
鸡球虫病是严重危害养禽业发展的重要疾病之一，而柔嫩艾美耳球虫(E.tenella)是
其中主要的病原，目前对该病的控制还存在许多问题。λMzp5-7的编码蛋白是艾美耳球虫
主要的表面抗原，是人们研究球虫新型疫苗和建立球虫血清学检测方法的侯选抗原之一。
本研究应用DNA重组技术将该基因分别克隆入原核和真核表达载体并表达，利用原核表
达的融合蛋白为包被抗原建立了检测柔嫩艾美耳球虫的间接ELISA方法，并用重组抗原和
真核重组DNA质粒分别免疫动物，观察其诱导产生的特异性免疫应答反应，以及对
E.tenella攻击的保护作用。
λMzp5-7抗原基因的克隆和序列分析 根据GENBANK上发表的序列设计上下游引
物，从E.tenella孢子化10h卵囊中提取RNA，通过RT-PCR技术扩增出编码该保护性抗原
基因的cDNA片段，插入到pMD18-T载体中，构建成克隆质粒：pMD-Mzp5-7，转化克隆
后进行核苷酸序列测定，并用DNAsis、PROsis软件对序列进行分析。结果为：λMzp5-7
基因长度为936bp，为一完整的开放阅读框，编码312个氨基酸，与国外报道的柔嫩艾美
耳球虫表面抗原基因λMzp5-7的同源性为98％，氨基酸的同源性为95.6％。
λMzp5-7基因原核表达载体的构建及其在大肠杆菌中的表达 将扩增出的基因片段
定向克隆到pET-28b(+)载体上构建成原核表达载体：pET-Mzp5-7，经酶切鉴定后在宿主菌
BI＿21(DE3)中表达。结果在1mmol／L IPTG诱导4h后，得到高效表达，产物主要以包涵体
形式存在，其表达量占菌体蛋白总量的18％。免疫印迹表明所表达的蛋白能与抗柔嫩艾美
耳球虫的多克隆抗体反应。
以λMzp5-7基因在E.coli中的表达产物为抗原的间接ELISA检测方法的建立 以在
E.coli中表达的融合蛋白为包被抗原，以辣根过氧化物酶(HRP)标记的鼠抗鸡IgG为二抗，
建立了检测柔嫩艾美耳球虫抗体的间接ELISA方法。最佳反应条件为：1μg／孔纯化的E.coli
表达的重组蛋白包被酶标板，用10％胎牛血清进行封闭，以正常E.coli裂解上清液稀释待
检血清。结果表明融合蛋白可以作为包被抗原检测柔嫩艾美耳球虫抗体的产生。该法快速，
抗原易纯化且成本低。
pET-Mzp5-7重组蛋白对鸡柔嫩艾美耳球虫攻击的免疫保护性 用柔嫩艾美耳球虫λ
MZps－7的重组融合蛋白经不同免疫途径接种免疫雏鸡，观察其诱导的免疫应答反应，以
及对球虫的攻击的保护作用。结果是口服工程活菌组可诱导机体的体液免疫和细胞免疫反
应，对鸡的增重、盲肠病变记分以及OPG值均有一定的改善（P＜0．05），保护率可达36％。
A MZps－7基因真核表达载体的构建及其在 Hela细胞中的表达 将入 Mzps－7基因亚克
隆到pVAXI中，构建成真核表达载体。pVAXI－Mzps－7。该重组质粒转染Hela细胞后，
RI”－PCR检测到M印5－7基因在Hela细胞内进行了转录，免疫印迹法检测到特异蛋白。
pVAX IMZps－7重组质粒对鸡柔嫩艾美耳球虫的免疫保护性作用 将 pVAX IMZps－7
质粒分别经滴鼻和肌注接种鸡并进行攻虫试验，首次对其诱导的系统免疫进行了检测。结
果表明重组质粒免疫雏鸡，均能诱导有效的细胞免疫和体液免疫反应。CD4＋／CDS十比率、
特异性抗体滴度等免疫指标均显著高于对照组（P＜0刀5）。且能够诱导鸡体对抗中等剂量的
球虫的攻击，试验组和对照组相比，攻虫后卵囊排出量和排出时间缩短，体重增长较快，
盲肠病变较小（P＜005），保护率均高达80％以上。其中以肌注100 p g组保护效果最好。
The λ Mzp5-7 on the sporozoites of Eimeria.tenella is major surface protein which is studied in the new pattern vaccine and developed a serologic detection method for E.tenella.In the experiment,prokaryotic and eukaryotic expression vectors of the gene was constructed by recombinant DNA technique and expressed in E.coli and Hela cells respectively.The nucleic acid vaccine could induce protective immune responses against E.tenella after chicken were inoculated .
Cloning and sequencing of λ. Mzp5-7 genes One cDNA fragment of λ Mzp5-7 genes was amplified by RT-PCR with primers according to GenBank.The recombinant plasmids pMD-Mzp5-7 was constructed .The ligation products were transformed to competent cells of E.coli host strain DH5 .The clone was cultured and the recombinant plasmids were prepared as template for automatic sequencing.The nucleotide sequence were analysed by DNAsis and PROsis sequence software.The results indicated that the gene fragment encoding λ. Mzp5-7 was 936bp in length.Compared with database in GenBank, λ Mzp5-7 shared 98%,DNA sequence homology and overall deduced amino acids identity of 95.6%.
Construction and expression of prokaryotic expression vector of λ. Mzp5-7 The recombinant plasmids pET-Mzp5-7 was constructed by cloning λ. Mzp5-7 gene into prokaryotic expression vector pET-28b(+) and expressed in E.coli host cells BL21(DE3).The expression levels were optimized by manipulating the host strain,the media,the time of harvest.The recombinant proteins were highly expressed in normal LB media,then induced by the Immol/L IPTG for 4h before harvesting cells.The recombinant proteins were inclusion bodies in the cytoplasm.The yield of recombinant λ Mzp5-7 were up to 18% of the total bacterial protein in the cell lysate.lt was specificity of E.t by Western blotting.
Development of indirect ELISA with recombiant proteins as antigen The indirect ELISA for the detection of serum antibodies against E.t was established with the recombinant fusion protein expressed in E.coli as antigen and mice anti-chicken IgG HRP conjugate as the second antibody .The best conditions were that coating antigen was 1 g per well for . Mzp5-7 recombiant protein.blocked with 10% serum of foetus Cattle and the sample diluted with the supernatant of normal E.coli lysate.The assay was characterized by simplicity.rapidity and economical cost.
The Immunoprotection induced by recombinant antigen in chicken The chicken were inoculated with recombinant antigen three times,then were challenged with E.t oocysts.The results showed the inoculation with live E.coli expressing E.t recombinant antigen can protect against E.t infection partial.Oocyst number and time shedding of experiment groups were shorter,and the increase of body weight (BW) were swifer,and the cecum Lesion were slighter than those of control groups.
Construction and expression of eukaryotic expression vectors The recombiant plasmids pVAXl-Mzp5-7 were contructed by cloning X Mzp5-7 genes into eukaryotic expression vector pVAXl and expressed in Hela cell strain.After transfected them into Hela cells.the specific recombiant proteins were detected in supernatant of Hela cell by Wester blot. The immunoprotection response induced by nucleic acid vaccine in chicken All the chicken were immunized with the vaccine plasmids by different inoculation pathway.dosage three times,then were challenged with E.t oocysts.The responses of specific humoral and cell immunity were elevated by the immunological methods such as the specific antibodies responses,the ratio of CD4+/CD8+. The results showed the nucleic acid vaccines can induce the immune response The specific immune responses were strengthened with the increase of immunization times.The immunity indexes among experiment groups were not significant,while those between experiment and control groups were significant.At the same time, the results showed the nucleic acid vaccines can protect against E.t infection.Oocyst number and time
shedding of experiment groups were significantly shorter
其中主要的病原，目前对该病的控制还存在许多问题。λMzp5-7的编码蛋白是艾美耳球虫
主要的表面抗原，是人们研究球虫新型疫苗和建立球虫血清学检测方法的侯选抗原之一。
本研究应用DNA重组技术将该基因分别克隆入原核和真核表达载体并表达，利用原核表
达的融合蛋白为包被抗原建立了检测柔嫩艾美耳球虫的间接ELISA方法，并用重组抗原和
真核重组DNA质粒分别免疫动物，观察其诱导产生的特异性免疫应答反应，以及对
E.tenella攻击的保护作用。
λMzp5-7抗原基因的克隆和序列分析 根据GENBANK上发表的序列设计上下游引
物，从E.tenella孢子化10h卵囊中提取RNA，通过RT-PCR技术扩增出编码该保护性抗原
基因的cDNA片段，插入到pMD18-T载体中，构建成克隆质粒：pMD-Mzp5-7，转化克隆
后进行核苷酸序列测定，并用DNAsis、PROsis软件对序列进行分析。结果为：λMzp5-7
基因长度为936bp，为一完整的开放阅读框，编码312个氨基酸，与国外报道的柔嫩艾美
耳球虫表面抗原基因λMzp5-7的同源性为98％，氨基酸的同源性为95.6％。
λMzp5-7基因原核表达载体的构建及其在大肠杆菌中的表达 将扩增出的基因片段
定向克隆到pET-28b(+)载体上构建成原核表达载体：pET-Mzp5-7，经酶切鉴定后在宿主菌
BI＿21(DE3)中表达。结果在1mmol／L IPTG诱导4h后，得到高效表达，产物主要以包涵体
形式存在，其表达量占菌体蛋白总量的18％。免疫印迹表明所表达的蛋白能与抗柔嫩艾美
耳球虫的多克隆抗体反应。
以λMzp5-7基因在E.coli中的表达产物为抗原的间接ELISA检测方法的建立 以在
E.coli中表达的融合蛋白为包被抗原，以辣根过氧化物酶(HRP)标记的鼠抗鸡IgG为二抗，
建立了检测柔嫩艾美耳球虫抗体的间接ELISA方法。最佳反应条件为：1μg／孔纯化的E.coli
表达的重组蛋白包被酶标板，用10％胎牛血清进行封闭，以正常E.coli裂解上清液稀释待
检血清。结果表明融合蛋白可以作为包被抗原检测柔嫩艾美耳球虫抗体的产生。该法快速，
抗原易纯化且成本低。
pET-Mzp5-7重组蛋白对鸡柔嫩艾美耳球虫攻击的免疫保护性 用柔嫩艾美耳球虫λ
MZps－7的重组融合蛋白经不同免疫途径接种免疫雏鸡，观察其诱导的免疫应答反应，以
及对球虫的攻击的保护作用。结果是口服工程活菌组可诱导机体的体液免疫和细胞免疫反
应，对鸡的增重、盲肠病变记分以及OPG值均有一定的改善（P＜0．05），保护率可达36％。
A MZps－7基因真核表达载体的构建及其在 Hela细胞中的表达 将入 Mzps－7基因亚克
隆到pVAXI中，构建成真核表达载体。pVAXI－Mzps－7。该重组质粒转染Hela细胞后，
RI”－PCR检测到M印5－7基因在Hela细胞内进行了转录，免疫印迹法检测到特异蛋白。
pVAX IMZps－7重组质粒对鸡柔嫩艾美耳球虫的免疫保护性作用 将 pVAX IMZps－7
质粒分别经滴鼻和肌注接种鸡并进行攻虫试验，首次对其诱导的系统免疫进行了检测。结
果表明重组质粒免疫雏鸡，均能诱导有效的细胞免疫和体液免疫反应。CD4＋／CDS十比率、
特异性抗体滴度等免疫指标均显著高于对照组（P＜0刀5）。且能够诱导鸡体对抗中等剂量的
球虫的攻击，试验组和对照组相比，攻虫后卵囊排出量和排出时间缩短，体重增长较快，
盲肠病变较小（P＜005），保护率均高达80％以上。其中以肌注100 p g组保护效果最好。
The λ Mzp5-7 on the sporozoites of Eimeria.tenella is major surface protein which is studied in the new pattern vaccine and developed a serologic detection method for E.tenella.In the experiment,prokaryotic and eukaryotic expression vectors of the gene was constructed by recombinant DNA technique and expressed in E.coli and Hela cells respectively.The nucleic acid vaccine could induce protective immune responses against E.tenella after chicken were inoculated .
Cloning and sequencing of λ. Mzp5-7 genes One cDNA fragment of λ Mzp5-7 genes was amplified by RT-PCR with primers according to GenBank.The recombinant plasmids pMD-Mzp5-7 was constructed .The ligation products were transformed to competent cells of E.coli host strain DH5 .The clone was cultured and the recombinant plasmids were prepared as template for automatic sequencing.The nucleotide sequence were analysed by DNAsis and PROsis sequence software.The results indicated that the gene fragment encoding λ. Mzp5-7 was 936bp in length.Compared with database in GenBank, λ Mzp5-7 shared 98%,DNA sequence homology and overall deduced amino acids identity of 95.6%.
Construction and expression of prokaryotic expression vector of λ. Mzp5-7 The recombinant plasmids pET-Mzp5-7 was constructed by cloning λ. Mzp5-7 gene into prokaryotic expression vector pET-28b(+) and expressed in E.coli host cells BL21(DE3).The expression levels were optimized by manipulating the host strain,the media,the time of harvest.The recombinant proteins were highly expressed in normal LB media,then induced by the Immol/L IPTG for 4h before harvesting cells.The recombinant proteins were inclusion bodies in the cytoplasm.The yield of recombinant λ Mzp5-7 were up to 18% of the total bacterial protein in the cell lysate.lt was specificity of E.t by Western blotting.
Development of indirect ELISA with recombiant proteins as antigen The indirect ELISA for the detection of serum antibodies against E.t was established with the recombinant fusion protein expressed in E.coli as antigen and mice anti-chicken IgG HRP conjugate as the second antibody .The best conditions were that coating antigen was 1 g per well for . Mzp5-7 recombiant protein.blocked with 10% serum of foetus Cattle and the sample diluted with the supernatant of normal E.coli lysate.The assay was characterized by simplicity.rapidity and economical cost.
The Immunoprotection induced by recombinant antigen in chicken The chicken were inoculated with recombinant antigen three times,then were challenged with E.t oocysts.The results showed the inoculation with live E.coli expressing E.t recombinant antigen can protect against E.t infection partial.Oocyst number and time shedding of experiment groups were shorter,and the increase of body weight (BW) were swifer,and the cecum Lesion were slighter than those of control groups.
Construction and expression of eukaryotic expression vectors The recombiant plasmids pVAXl-Mzp5-7 were contructed by cloning X Mzp5-7 genes into eukaryotic expression vector pVAXl and expressed in Hela cell strain.After transfected them into Hela cells.the specific recombiant proteins were detected in supernatant of Hela cell by Wester blot. The immunoprotection response induced by nucleic acid vaccine in chicken All the chicken were immunized with the vaccine plasmids by different inoculation pathway.dosage three times,then were challenged with E.t oocysts.The responses of specific humoral and cell immunity were elevated by the immunological methods such as the specific antibodies responses,the ratio of CD4+/CD8+. The results showed the nucleic acid vaccines can induce the immune response The specific immune responses were strengthened with the increase of immunization times.The immunity indexes among experiment groups were not significant,while those between experiment and control groups were significant.At the same time, the results showed the nucleic acid vaccines can protect against E.t infection.Oocyst number and time
shedding of experiment groups were significantly shorter
引文
[1] Bhogal B S,Miller G A, et al..Potential of a recombinant antigen as a prophylactic vaccine for one-day-old broiler chickens against E.acervulina and E.tenella infections. Veterinary Immunology and Immunopatholog, 1992,31:323-335.
[2] McDougald L R.Anti-coccidia drug resistance in the southeastern United States:polyether drugs.Avian Dis, 1981,25:606～609.
[3] Chapman Hd. Drug resistance in avian coccdia (review).Vet Parasital,1984,15:11～27.
[4] 索勋，李国清：《鸡球虫病学》[M]，北京：中国农业大学出版社，1998。
[5] Jeffers, T.K. Genetics of coccidia and the host response. In:P. L.Long,K.N.Boorman, and B.M.Freeman(eds.),Avian Coccidiosis, 1978, 57～125.
[6] Jenkins,M.C.,Castle,M.D,and Danforth,HD. 1991.Protective immunization against the intestinal parasite Eirneria acervulina with recombinant coccidial antigen. Poultry Science,70:539～547
[7] E.L.Long. The biology of coccidia. U.S.A.: University Park Press, 1982.329～371
[8] 谢明权，吴惠贤，张键驯：柔嫩艾美尔球虫裂殖子免疫原性的研究[J]，《畜牧兽医学报》，1994，25(4)，340～345
[9] E.J,LSoulbsy. Immune responses in parasitic infections. Florida:Boca Raton,CRC Press, 1989.276～312
[10] Stotish RL et al. Studies of a glycoprotein in the oocystsof Eimeria tenella. J.Biol. Chem, 1976,25(2):302～307.
[11] Karkhanis YD et al.Purification and characterization of a protective antigen from Eimeria tenel-la. Infect.Immun, 1991,59:983～989.
[12] Dantbrth HD et al. Hybridoma antibody characterization of stage-specific and stage-cross-reativeanti-gens of Eimeria tenella. J.Parasitol, 1987,73:985～992
[13] Wisher MH. Identification of the sporozoites antigens of Eimeria tenella. Mol. Biochem. Parasitol, 1986,21:7～15.
[14] Jenkins MC et al. Identification of immunodominat surface antigens ofEimeria acervulina sporozoites and merozoites. Mol. Biochem. Parasitoi, 1987,25:155～164.
[15] Xie MQ et al. Electrophoretic and immunologic characterization of proteins of merozoites of Eimeria acervulina, Eimeria maxima, Eimeria necatrix and Eimeria tenella. J.Parasitol, 1992,78(1):82～86
[16] 蒋建林，蒋金书：柔嫩艾美耳球虫的抗原分析，《畜牧兽医学报》，1997，28(2)，146-151
[17] Jenkins. Eimeria acervulina DNA Cloning and Characterization of Recombinant Sporozoite and Mewzoite[J].ANTIGENS Experimental Parasitology, 1988,66:96～107
[18] Jenkins. Eimeria acervulina:Cloning of a cDNA encoding an immunogenic of related merozoite surface amgrhoptry proteins[J].Experimental parasitology, 1990,70:353～362.
[19] Binger Mary Helen,Hug D,Weber G,et al Cloning and characterization of a surface antigen of Eimeria tenella merozoites and expression using a recombinant vaccinia virus. Molecular and Biochemical Parasitology, 1993,61:179～188
[20] H.S.Lillehoj.Infection and Immunity, 1987,55:1616～1621
[21] 谢庆阁，翟中和主编，畜禽重大疫病防治研究进展，北京：中国农业科技出版，1996，181～190
[22] 程金科，段嘉树：球虫免疫机制进展[J]，《寄生虫与昆虫学报》，1994，1(1)：61～67
[23] Rose,M.E.,and Long, P.L. 1976.Immunity to coccidiosis:Interactions in vitro between Eimeria tenella and chicken phagocytic cells.In:H.Van den Bossche(ed.),Biochemistry of Parasites and Host-Parasite Relationships, PP. 449～455.Elsevier North Holland
[24] P O Lorcain Mapping for Ball epitopes in the GX3262 antigenic sequence derived from Eimefia tenella sporulated oocysts[J].Vet Para, 1996,66:159～169
[25] Grane M S J,Goggin B,Pellegrino R M, et al.Vaccination against coccidiosis with recombinant antigens[A].Yvore P. Coccidia and intestinal coccidiomorphs. Proceedings of the 5th International Coccidiosis Conference[C].Tours(France)INRA Paris, 1989.649～654
[26] 杨林。鸡球虫Etp28基因的克隆及其在杆状病毒系统中的表达[J]，生物化学与生物物理学报，1998，3019：293～298
[27] Danforth HD,Augustine PC,Ruff MD.Genetically engineered antigen confers partial protection agains tavian coccidial parasites. Poultry Science, 1989,68:1643～1652
[28] 王世若．分子免疫学[M]，中国人民解放军军需大学，1998，79～85．
[29] Wolff, JA, Malone R, Williams P, et al.Direct gene transfer into mouse muscle in vivo[J].Science, 1990,247:1465～1468.
[30] Ulmer JB, Donnelly JJ, Parker SE, et al, Heterologous protection against influenza by injection of DNA encoding a viral protein, Science, 1993, 259:1745～1749.
[31]. Cox G. JM, Lamb ML, Babuuk LA, Bovine herpesvirus 1: immune response in mice and cattle injected with plasmid DNA, J Virol, 1993, 67:5664～5667.
[32] Montgomery DL, Shiver, JW, Leadner, KR, et al, Heterologous and homologous protection against influenza A by DNA vaccination optimization of DNA vector, DNA Cell Biol, 1993, 12:777～783.
[33]. Shiver JW, Perry HC, Davies ME, et al, Immune responses to HIV gp-120 elicited by DNA vaccination, in Vaccines 95, Brown F, Chanock RM, ginsberg HS, and Norrby E, Eds, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1995:95～98.
[34]. Kurth R, Risk potential of the chromosomal insertion of foreign DNA, Ann Y Acad Sci, 1995, 772:140～151
[35]. Wang B, Boyer J, Srikautan V, et al, DNA inoculation induces neutralizing immune responses against human immunodeficiency virus type I in mice and non-human primates, DNA Cell Biol, 1993, 12:799～805.
[36]. Xiang ZQ, Spitalnick S, Tran M, et al, Vaccination with a plasmid vector carrying the rabeis virus glycoprotein gene induce protective immunity against rabies virus, Virology, 1994, 199:132～140.
[37]. Davis HL, Michel ML, Whalen RG, DNA based immunization induced continuous secretion of hepatitis B surface antigen and high levels of circulating antibody, Hum Mol Genet, 1993, 1847～1851.
[38]. Lagging LM, Meyer K, Holt D, et al, Immune responses to plasmid DNA encoding the hepatitis C virus core protein, J Virol, 1995, 69:5859～5863.
[39]. Yokoyama M, Zhang J, Whitton JL, DNA immunization confers protection against lethal lymphocytic choriomeningitis virus infection, J Virol, 1995, 69:2684～2688.
[40]. Kriesel JD, Spruance SL, Daynes RA, et al, Nucleic acid vaccine encoding D1 protects mice from Herps simplex virus type 2 disease, J Infect Dis, 1996, 173:536～541.
[41] Fu TM, Ulmer JB,Caulfield MJ,et al. Priming of cytotoxic T lymphocytes by DNA vaccines: requirement for professional antigen presenting cells and evidence for antigen transfer from myocytes[J].Mol Med, 1997,3:362～371.
[42] Sedegah M,Hedstrom R, Hobart P. Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein[J].Proc Natl And Sci USA, 1994,91(21):9866～9870.
[43] Xu D,Liew FY. Genetic vaccination against leishmaniasis[J].Vaccine, 1994:12(16):1534～1536.
[44] Ho TY, Hsiang CY, Hsiang CH, et al.DNA vaccination induces a long-term antibody response and protective immunity against pseudorabies virus in mice[J].Arch Viroi,1998,143:115～125.
[45] Serge S,Dominique BG, Sophil I.Protection against of kids Cryptosporidium parvum infection after immunization of dams with CP15-DNA[J].Vaccine, 1999,17:2346～2355.
[46] Kopko SH, Martin DS,et al. Responses of chickens to a recombinant refractile body antigen of Eimeria tenella administered using various immunizing strategies.2001,12;20(1-2):267～74
[47] Song KD, Lillehoj HS,等, A DNA vaccine encoding a conserved Eimeria protein induces protective immunity against live Eimeria acervulina challenge. ,Vaccine 2000 Sep 15; 19(2-3):243-248
[48] Min W, Lillehoj HS,等.Adjuvant effects of IL-1beta, IL-2, IL-8, IL-15, IFN-alpha, IFN-gamma TGF-beta4 and lymphotactin on DNA vaccination against Eimeria acervulina., Vaccine 2001 Oct 12;20(1-2):267-74
[49] Binger Mary Helen,Hug D,Weber G,et al Cloning and characterization of a surface antigen of Eimeria tenella merozoites and expression using a recombinant vaccinia virus. Molecular and Biochemical Parasitology, 1993,61:179～188
[50] Qiu P, Ziegelhoffer P, Sun J,et al. Genegunde livery of mRNA insitu results inefficient transgene expression and genetic immunization. Gene Ther 1996,3:262-268
[51] Dalemans W, DelersA, Delmellec,et al. Protectiona-gains thomologous influenza challenge by genetic immu-nization with SFV-RNA encoding FHI-HA.AnnNYAcad Sci 1995, 772:255-256
[52] JohnstonSA, BarryMA.Genetictogenomic vaccination. Vaccine199715(8):808-809 UlmerJB,liuMA.ELI'coming:expression library immunization and vaccine antigen discovery. Trends Microbiol 1996,4(5): 169-170
[53] BarryMA,LaiWC.JohnstonSA.Protection againstmy-coplama infection using expression library immunization.Nature i 995,377(6550):632-635
[54] 蒋建林，蒋金书，柔嫩艾美耳球虫各阶段虫体纯化方法的改进。中国农业大学学报，1996，1(5)：99～102
[55] Wisher M H,Rose M E.The large-scale preparation of purified sporozoites of Eimeria spp by metriamide density-gradient centrifugation.Parasitology, 1984,88:515～519
[56] Stotish R L,Wang c c. Preparation and purification of merozoites of Eimeria tenelIa. J Parasitol, 1975,61(l):700～703
[57] Stotish R Let al. Separation of sporozoites,sporocysts and oocysts of Eimeria tenella by centrifugal elutriation.J Parasitol, 1997,63(6): 1124～1126
[58] Xie M Q,et aI.A new method for purification of Eimeria tenella merozoites. Parasitol Res, 1990,76:5664～569
[59] Vetterling J M.Continuous-fiow differential fensity flotation of coccidial oocysts and a comparision with other methods. J Parasitol, 1969,55(2):412～417.
[60] 金冬雁，黎孟枫 等译．分子克隆实验指南，第二版 科学出版社 1989
[61] MARK S,J.CRANE, et al.Cross-Protection against Four Species of Chicken Coccidia with a Single Recombinant Antigen, INFECTIONAND IMMUNITY, Apr. 1991,p. 1271～1277.
[62] 陈名勇等，应用ELISA检测鸡柔嫩艾美耳球虫免疫血清抗体水平，中国兽医杂志，1991(第17卷)第2期，4-6，49．
[63] 余兴龙．猪瘟病毒E2基因的原核和真核细胞表达及基因疫苗的研究．博士论文，1999．
[64] 索勋，李国清等．《鸡球虫病学》．北京：中国农业大学出版社，1998，254～257
[65] 索勋，李国清等，《鸡球虫病学》．北京：中国农业大学出版社，1998，
[66] 宋长绪，刘福安等．用PCR检测鸡新城疫病毒的初步实验[J]，中国兽医科技，1995，25：20-24。
[67] 宋长绪，刘福安，杜伟贤，新城疫病毒融合蛋白裂解位点基因的分离克隆及在原核细胞中的表达[J]．中国兽医学报，1996，16(6)：527-533．
[68] Kim K S,Jenkins M C,Lillehoj H S.Immunization of chickens with live Escherichia coli expressing Eimeria acervulina merozoite recombinant antigen induces partial protectionagainst coccidiosis[J].Infect Immun, 1989,57(8):2434～2440.
[69] 丁熙成，蒋金书．EtMIC-2表达产物对鸡三种艾美耳球虫的保护试验，畜牧兽医学报，2002，33(2)，205～208
[70] Miller G A, Bhogal B S,McCandliss R, et al. Characterization and vaccine potential of a novel recombinant coccidial antigen[J].Infect Immun, 1989,57(7)2014～2020
[71] 李安兴，蒋金书．柔嫩艾美耳球虫BJ株SO7重组抗原的免疫试验．中国兽医学报2000，3，20(2)167～170。
[72] Shiley M W. Control of eoccidiosis with live vaccines. World Poultry. 1993,8,24-27.
[73] 刘华忠等，基因免疫的研究现状，畜禽业，2001年第4期，10～11
[74] ZelpHati O, Nguyen C, Ferrari M, et al.Stable and monodisperse lipoplex formulations for gene delivery[J].GeneTher, 1998, 5: 1272.
[75] Simoes RA, Slepushkin V, Pretzer E, et al.Transfection of human maeropHages by lipoplexes via the combined use oftransferrin and PH-sensitive peptides[J].J Leukoc Biol, 1999, 65: 270.
[76] 吴乃虎．基因工程原理[M]，第二版．北京：科学出版社，1998．
[77] Calarola S,Brall G, et al. Cellolar cytotoxic response induced by DNA vaccine infected patientients. Lancel, 1998,351:1320
[78] Restifo N,et al.The development of recombinant and synthetic cancer vaccines. CurrOpinOncol, 1999, 11:50
[79] Wang R,et al.Induction of antigen specific cytotoxic Tlymph ocytesin humans by malaria DNA vaccine.
Science, 1998,282:476
[80] 鲁风良，庄辉．核酸疫苗研究进展．中华预防医学杂志，1995；29(5)：303～304．
[81] Fynan FF. DNA vaccines; protective immunizations by parenteral,mucoral and gene-gun inoculations[J].PNAS. 1993,90:11478～11482.
[82] 石宁，董敏，殷彬．免疫接种方法对恶性疟DNA疫苗诱导产生ISG亚类的影响[J]．中国动物学会第七次全国寄生虫学学述会议论文集，1999，14～15．
[83] Sedegah M,Hedstrom R, Hobart P. et al. Protetion against Malaria by Immunization with Plasmid DNA Encoding Circum sprozoite Protein. ProcNatl Acad Sci USA, 1994;91:9866～9870
[84] FURTHpa, Shasnay A, Wall RJ.et al.Gene Transfer into Somatic Tissues by Jet Injection. Analy Biochem, 1992;205:365～368
[85] Xiang ZQ, Spitainik SL,Cheng et al. Immune Response to Nucleic Acid Vaccine to Rabies Virus. Virology, 1995 ;209:569～579
[86] Davis HL,Whalen RG,Demeneix BA.Direct gene transfer into skeletal muscle in vivo:factors affecting efficiency of transfer and stability of expression[J]. Human Gene Therapy, 1993,4:151～159.
[87] Vitadello M,Schiaffino MV, Picard A, et al. Gene transfer in regenerating muscle[J].Hum Gene Ther, 1994,5(1): 11～18.
[88] Manickan E,Karem KL,Rouse BT. DNA vaccines:A modem gimmick or a boon to vaccinology[J].Crit Rev Immunol, 1997,17,139～154.
[89] Kriesel JD,Spruance SL,Daynes RA.Nucleic acid vaccine encoding gD2 protects mice from herpes simplex virus type 2 disease[J].J Infect Dis, 1996,173,536～541.
[90] 冯志华，周永兴，等，脂质转染剂增强基因疫苗诱导的免疫应答效力，第四军医大学学报，2000，21(7)718～723
[91] Wang B,Boyer J,Strikantan V. DNA inoculation induces neutralizing immune responses against human immunodeficiency virus type I in mice and nonprimates[J].DNA ceil biol, 1993,12:799.
[92] Hoffman SL,Sedagah M,Hedtrom RC.Protcction against malaria by immunization with a Plasmodium Yoelii circumsporoite pritein nucleic acid vaccine[J].Vaccine, 1994,12:1259.
[93] Coney L,Wang B,Ugen KE.Facilitated DNA inoculation induces anti-HIV-1 immunity in vivo[J].Vaccine, 1994,12:1545.
[94] Chow YH,Huang WL,Chi WK[J].Improvement of hepatitis B virus DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2[J].J Virol, 1997,71:169～178.
[95] Larsen DL, Sissoko DN,McGregor MW, et aI.Coadministration of DNA encoding interleukin-6 and hemagglutinin cofers protection from influenza virus challenge in mice[J].J Virol, 1998,72:1704～1708.
[96] 吴丹，郭瀛军，孙树汉．猪囊尾蚴抗原与猪白介素-4基因融合DNA疫苗载体的构建[J]．中国寄生虫学与寄生虫病杂志，1999：17(3)：172～174．
[97] Horspool JH,Perrin PJ, Woodcock JB,et al.Nucleic acid vaccine-induced immune responses require CD28 costimulation and are regulated by CTLA4[J].J Immunol, 1998,160:2706～2714.
[98] Min W, Lillehoj HS,et,al. Adjuvant effects of IL-1beta, IL-2,IL-8,IL-15,IFN-alpha, IFN-gamma TGF-beta4 and lymphotactin on DNA vaccination against Eimeria acervulina.;Vaccine 2001 Oct 12;20(1-2):267-74
[2] McDougald L R.Anti-coccidia drug resistance in the southeastern United States:polyether drugs.Avian Dis, 1981,25:606～609.
[3] Chapman Hd. Drug resistance in avian coccdia (review).Vet Parasital,1984,15:11～27.
[4] 索勋，李国清：《鸡球虫病学》[M]，北京：中国农业大学出版社，1998。
[5] Jeffers, T.K. Genetics of coccidia and the host response. In:P. L.Long,K.N.Boorman, and B.M.Freeman(eds.),Avian Coccidiosis, 1978, 57～125.
[6] Jenkins,M.C.,Castle,M.D,and Danforth,HD. 1991.Protective immunization against the intestinal parasite Eirneria acervulina with recombinant coccidial antigen. Poultry Science,70:539～547
[7] E.L.Long. The biology of coccidia. U.S.A.: University Park Press, 1982.329～371
[8] 谢明权，吴惠贤，张键驯：柔嫩艾美尔球虫裂殖子免疫原性的研究[J]，《畜牧兽医学报》，1994，25(4)，340～345
[9] E.J,LSoulbsy. Immune responses in parasitic infections. Florida:Boca Raton,CRC Press, 1989.276～312
[10] Stotish RL et al. Studies of a glycoprotein in the oocystsof Eimeria tenella. J.Biol. Chem, 1976,25(2):302～307.
[11] Karkhanis YD et al.Purification and characterization of a protective antigen from Eimeria tenel-la. Infect.Immun, 1991,59:983～989.
[12] Dantbrth HD et al. Hybridoma antibody characterization of stage-specific and stage-cross-reativeanti-gens of Eimeria tenella. J.Parasitol, 1987,73:985～992
[13] Wisher MH. Identification of the sporozoites antigens of Eimeria tenella. Mol. Biochem. Parasitol, 1986,21:7～15.
[14] Jenkins MC et al. Identification of immunodominat surface antigens ofEimeria acervulina sporozoites and merozoites. Mol. Biochem. Parasitoi, 1987,25:155～164.
[15] Xie MQ et al. Electrophoretic and immunologic characterization of proteins of merozoites of Eimeria acervulina, Eimeria maxima, Eimeria necatrix and Eimeria tenella. J.Parasitol, 1992,78(1):82～86
[16] 蒋建林，蒋金书：柔嫩艾美耳球虫的抗原分析，《畜牧兽医学报》，1997，28(2)，146-151
[17] Jenkins. Eimeria acervulina DNA Cloning and Characterization of Recombinant Sporozoite and Mewzoite[J].ANTIGENS Experimental Parasitology, 1988,66:96～107
[18] Jenkins. Eimeria acervulina:Cloning of a cDNA encoding an immunogenic of related merozoite surface amgrhoptry proteins[J].Experimental parasitology, 1990,70:353～362.
[19] Binger Mary Helen,Hug D,Weber G,et al Cloning and characterization of a surface antigen of Eimeria tenella merozoites and expression using a recombinant vaccinia virus. Molecular and Biochemical Parasitology, 1993,61:179～188
[20] H.S.Lillehoj.Infection and Immunity, 1987,55:1616～1621
[21] 谢庆阁，翟中和主编，畜禽重大疫病防治研究进展，北京：中国农业科技出版，1996，181～190
[22] 程金科，段嘉树：球虫免疫机制进展[J]，《寄生虫与昆虫学报》，1994，1(1)：61～67
[23] Rose,M.E.,and Long, P.L. 1976.Immunity to coccidiosis:Interactions in vitro between Eimeria tenella and chicken phagocytic cells.In:H.Van den Bossche(ed.),Biochemistry of Parasites and Host-Parasite Relationships, PP. 449～455.Elsevier North Holland
[24] P O Lorcain Mapping for Ball epitopes in the GX3262 antigenic sequence derived from Eimefia tenella sporulated oocysts[J].Vet Para, 1996,66:159～169
[25] Grane M S J,Goggin B,Pellegrino R M, et al.Vaccination against coccidiosis with recombinant antigens[A].Yvore P. Coccidia and intestinal coccidiomorphs. Proceedings of the 5th International Coccidiosis Conference[C].Tours(France)INRA Paris, 1989.649～654
[26] 杨林。鸡球虫Etp28基因的克隆及其在杆状病毒系统中的表达[J]，生物化学与生物物理学报，1998，3019：293～298
[27] Danforth HD,Augustine PC,Ruff MD.Genetically engineered antigen confers partial protection agains tavian coccidial parasites. Poultry Science, 1989,68:1643～1652
[28] 王世若．分子免疫学[M]，中国人民解放军军需大学，1998，79～85．
[29] Wolff, JA, Malone R, Williams P, et al.Direct gene transfer into mouse muscle in vivo[J].Science, 1990,247:1465～1468.
[30] Ulmer JB, Donnelly JJ, Parker SE, et al, Heterologous protection against influenza by injection of DNA encoding a viral protein, Science, 1993, 259:1745～1749.
[31]. Cox G. JM, Lamb ML, Babuuk LA, Bovine herpesvirus 1: immune response in mice and cattle injected with plasmid DNA, J Virol, 1993, 67:5664～5667.
[32] Montgomery DL, Shiver, JW, Leadner, KR, et al, Heterologous and homologous protection against influenza A by DNA vaccination optimization of DNA vector, DNA Cell Biol, 1993, 12:777～783.
[33]. Shiver JW, Perry HC, Davies ME, et al, Immune responses to HIV gp-120 elicited by DNA vaccination, in Vaccines 95, Brown F, Chanock RM, ginsberg HS, and Norrby E, Eds, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1995:95～98.
[34]. Kurth R, Risk potential of the chromosomal insertion of foreign DNA, Ann Y Acad Sci, 1995, 772:140～151
[35]. Wang B, Boyer J, Srikautan V, et al, DNA inoculation induces neutralizing immune responses against human immunodeficiency virus type I in mice and non-human primates, DNA Cell Biol, 1993, 12:799～805.
[36]. Xiang ZQ, Spitalnick S, Tran M, et al, Vaccination with a plasmid vector carrying the rabeis virus glycoprotein gene induce protective immunity against rabies virus, Virology, 1994, 199:132～140.
[37]. Davis HL, Michel ML, Whalen RG, DNA based immunization induced continuous secretion of hepatitis B surface antigen and high levels of circulating antibody, Hum Mol Genet, 1993, 1847～1851.
[38]. Lagging LM, Meyer K, Holt D, et al, Immune responses to plasmid DNA encoding the hepatitis C virus core protein, J Virol, 1995, 69:5859～5863.
[39]. Yokoyama M, Zhang J, Whitton JL, DNA immunization confers protection against lethal lymphocytic choriomeningitis virus infection, J Virol, 1995, 69:2684～2688.
[40]. Kriesel JD, Spruance SL, Daynes RA, et al, Nucleic acid vaccine encoding D1 protects mice from Herps simplex virus type 2 disease, J Infect Dis, 1996, 173:536～541.
[41] Fu TM, Ulmer JB,Caulfield MJ,et al. Priming of cytotoxic T lymphocytes by DNA vaccines: requirement for professional antigen presenting cells and evidence for antigen transfer from myocytes[J].Mol Med, 1997,3:362～371.
[42] Sedegah M,Hedstrom R, Hobart P. Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein[J].Proc Natl And Sci USA, 1994,91(21):9866～9870.
[43] Xu D,Liew FY. Genetic vaccination against leishmaniasis[J].Vaccine, 1994:12(16):1534～1536.
[44] Ho TY, Hsiang CY, Hsiang CH, et al.DNA vaccination induces a long-term antibody response and protective immunity against pseudorabies virus in mice[J].Arch Viroi,1998,143:115～125.
[45] Serge S,Dominique BG, Sophil I.Protection against of kids Cryptosporidium parvum infection after immunization of dams with CP15-DNA[J].Vaccine, 1999,17:2346～2355.
[46] Kopko SH, Martin DS,et al. Responses of chickens to a recombinant refractile body antigen of Eimeria tenella administered using various immunizing strategies.2001,12;20(1-2):267～74
[47] Song KD, Lillehoj HS,等, A DNA vaccine encoding a conserved Eimeria protein induces protective immunity against live Eimeria acervulina challenge. ,Vaccine 2000 Sep 15; 19(2-3):243-248
[48] Min W, Lillehoj HS,等.Adjuvant effects of IL-1beta, IL-2, IL-8, IL-15, IFN-alpha, IFN-gamma TGF-beta4 and lymphotactin on DNA vaccination against Eimeria acervulina., Vaccine 2001 Oct 12;20(1-2):267-74
[49] Binger Mary Helen,Hug D,Weber G,et al Cloning and characterization of a surface antigen of Eimeria tenella merozoites and expression using a recombinant vaccinia virus. Molecular and Biochemical Parasitology, 1993,61:179～188
[50] Qiu P, Ziegelhoffer P, Sun J,et al. Genegunde livery of mRNA insitu results inefficient transgene expression and genetic immunization. Gene Ther 1996,3:262-268
[51] Dalemans W, DelersA, Delmellec,et al. Protectiona-gains thomologous influenza challenge by genetic immu-nization with SFV-RNA encoding FHI-HA.AnnNYAcad Sci 1995, 772:255-256
[52] JohnstonSA, BarryMA.Genetictogenomic vaccination. Vaccine199715(8):808-809 UlmerJB,liuMA.ELI'coming:expression library immunization and vaccine antigen discovery. Trends Microbiol 1996,4(5): 169-170
[53] BarryMA,LaiWC.JohnstonSA.Protection againstmy-coplama infection using expression library immunization.Nature i 995,377(6550):632-635
[54] 蒋建林，蒋金书，柔嫩艾美耳球虫各阶段虫体纯化方法的改进。中国农业大学学报，1996，1(5)：99～102
[55] Wisher M H,Rose M E.The large-scale preparation of purified sporozoites of Eimeria spp by metriamide density-gradient centrifugation.Parasitology, 1984,88:515～519
[56] Stotish R L,Wang c c. Preparation and purification of merozoites of Eimeria tenelIa. J Parasitol, 1975,61(l):700～703
[57] Stotish R Let al. Separation of sporozoites,sporocysts and oocysts of Eimeria tenella by centrifugal elutriation.J Parasitol, 1997,63(6): 1124～1126
[58] Xie M Q,et aI.A new method for purification of Eimeria tenella merozoites. Parasitol Res, 1990,76:5664～569
[59] Vetterling J M.Continuous-fiow differential fensity flotation of coccidial oocysts and a comparision with other methods. J Parasitol, 1969,55(2):412～417.
[60] 金冬雁，黎孟枫 等译．分子克隆实验指南，第二版 科学出版社 1989
[61] MARK S,J.CRANE, et al.Cross-Protection against Four Species of Chicken Coccidia with a Single Recombinant Antigen, INFECTIONAND IMMUNITY, Apr. 1991,p. 1271～1277.
[62] 陈名勇等，应用ELISA检测鸡柔嫩艾美耳球虫免疫血清抗体水平，中国兽医杂志，1991(第17卷)第2期，4-6，49．
[63] 余兴龙．猪瘟病毒E2基因的原核和真核细胞表达及基因疫苗的研究．博士论文，1999．
[64] 索勋，李国清等．《鸡球虫病学》．北京：中国农业大学出版社，1998，254～257
[65] 索勋，李国清等，《鸡球虫病学》．北京：中国农业大学出版社，1998，
[66] 宋长绪，刘福安等．用PCR检测鸡新城疫病毒的初步实验[J]，中国兽医科技，1995，25：20-24。
[67] 宋长绪，刘福安，杜伟贤，新城疫病毒融合蛋白裂解位点基因的分离克隆及在原核细胞中的表达[J]．中国兽医学报，1996，16(6)：527-533．
[68] Kim K S,Jenkins M C,Lillehoj H S.Immunization of chickens with live Escherichia coli expressing Eimeria acervulina merozoite recombinant antigen induces partial protectionagainst coccidiosis[J].Infect Immun, 1989,57(8):2434～2440.
[69] 丁熙成，蒋金书．EtMIC-2表达产物对鸡三种艾美耳球虫的保护试验，畜牧兽医学报，2002，33(2)，205～208
[70] Miller G A, Bhogal B S,McCandliss R, et al. Characterization and vaccine potential of a novel recombinant coccidial antigen[J].Infect Immun, 1989,57(7)2014～2020
[71] 李安兴，蒋金书．柔嫩艾美耳球虫BJ株SO7重组抗原的免疫试验．中国兽医学报2000，3，20(2)167～170。
[72] Shiley M W. Control of eoccidiosis with live vaccines. World Poultry. 1993,8,24-27.
[73] 刘华忠等，基因免疫的研究现状，畜禽业，2001年第4期，10～11
[74] ZelpHati O, Nguyen C, Ferrari M, et al.Stable and monodisperse lipoplex formulations for gene delivery[J].GeneTher, 1998, 5: 1272.
[75] Simoes RA, Slepushkin V, Pretzer E, et al.Transfection of human maeropHages by lipoplexes via the combined use oftransferrin and PH-sensitive peptides[J].J Leukoc Biol, 1999, 65: 270.
[76] 吴乃虎．基因工程原理[M]，第二版．北京：科学出版社，1998．
[77] Calarola S,Brall G, et al. Cellolar cytotoxic response induced by DNA vaccine infected patientients. Lancel, 1998,351:1320
[78] Restifo N,et al.The development of recombinant and synthetic cancer vaccines. CurrOpinOncol, 1999, 11:50
[79] Wang R,et al.Induction of antigen specific cytotoxic Tlymph ocytesin humans by malaria DNA vaccine.
Science, 1998,282:476
[80] 鲁风良，庄辉．核酸疫苗研究进展．中华预防医学杂志，1995；29(5)：303～304．
[81] Fynan FF. DNA vaccines; protective immunizations by parenteral,mucoral and gene-gun inoculations[J].PNAS. 1993,90:11478～11482.
[82] 石宁，董敏，殷彬．免疫接种方法对恶性疟DNA疫苗诱导产生ISG亚类的影响[J]．中国动物学会第七次全国寄生虫学学述会议论文集，1999，14～15．
[83] Sedegah M,Hedstrom R, Hobart P. et al. Protetion against Malaria by Immunization with Plasmid DNA Encoding Circum sprozoite Protein. ProcNatl Acad Sci USA, 1994;91:9866～9870
[84] FURTHpa, Shasnay A, Wall RJ.et al.Gene Transfer into Somatic Tissues by Jet Injection. Analy Biochem, 1992;205:365～368
[85] Xiang ZQ, Spitainik SL,Cheng et al. Immune Response to Nucleic Acid Vaccine to Rabies Virus. Virology, 1995 ;209:569～579
[86] Davis HL,Whalen RG,Demeneix BA.Direct gene transfer into skeletal muscle in vivo:factors affecting efficiency of transfer and stability of expression[J]. Human Gene Therapy, 1993,4:151～159.
[87] Vitadello M,Schiaffino MV, Picard A, et al. Gene transfer in regenerating muscle[J].Hum Gene Ther, 1994,5(1): 11～18.
[88] Manickan E,Karem KL,Rouse BT. DNA vaccines:A modem gimmick or a boon to vaccinology[J].Crit Rev Immunol, 1997,17,139～154.
[89] Kriesel JD,Spruance SL,Daynes RA.Nucleic acid vaccine encoding gD2 protects mice from herpes simplex virus type 2 disease[J].J Infect Dis, 1996,173,536～541.
[90] 冯志华，周永兴，等，脂质转染剂增强基因疫苗诱导的免疫应答效力，第四军医大学学报，2000，21(7)718～723
[91] Wang B,Boyer J,Strikantan V. DNA inoculation induces neutralizing immune responses against human immunodeficiency virus type I in mice and nonprimates[J].DNA ceil biol, 1993,12:799.
[92] Hoffman SL,Sedagah M,Hedtrom RC.Protcction against malaria by immunization with a Plasmodium Yoelii circumsporoite pritein nucleic acid vaccine[J].Vaccine, 1994,12:1259.
[93] Coney L,Wang B,Ugen KE.Facilitated DNA inoculation induces anti-HIV-1 immunity in vivo[J].Vaccine, 1994,12:1545.
[94] Chow YH,Huang WL,Chi WK[J].Improvement of hepatitis B virus DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2[J].J Virol, 1997,71:169～178.
[95] Larsen DL, Sissoko DN,McGregor MW, et aI.Coadministration of DNA encoding interleukin-6 and hemagglutinin cofers protection from influenza virus challenge in mice[J].J Virol, 1998,72:1704～1708.
[96] 吴丹，郭瀛军，孙树汉．猪囊尾蚴抗原与猪白介素-4基因融合DNA疫苗载体的构建[J]．中国寄生虫学与寄生虫病杂志，1999：17(3)：172～174．
[97] Horspool JH,Perrin PJ, Woodcock JB,et al.Nucleic acid vaccine-induced immune responses require CD28 costimulation and are regulated by CTLA4[J].J Immunol, 1998,160:2706～2714.
[98] Min W, Lillehoj HS,et,al. Adjuvant effects of IL-1beta, IL-2,IL-8,IL-15,IFN-alpha, IFN-gamma TGF-beta4 and lymphotactin on DNA vaccination against Eimeria acervulina.;Vaccine 2001 Oct 12;20(1-2):267-74