摘要
粒细胞-巨噬细胞集落刺激因子(granulocyte macrophage colony stimulatingfactor,GM-CSF)是一个具有多项潜能的造血生长因子,不仅能刺激造血祖细胞增殖分化和成熟,而且还能活化树突状细胞及其他单核细胞,在免疫反应中具有重要作用,许多研究证实GM-CSF与DNA疫苗联合免疫,能有效增强DNA疫苗所诱导的免疫应答。马立克病毒(Marek's disease virus,MDV)的VP22蛋白具有独特的蛋白转导特性,能通过非经典途径从原始表达细胞转导到邻近的非表达细胞中,而且VP22还能使与之融合的外源蛋白在细胞间转导,已被证实具有免疫增强作用。本试验成功克隆了四川山地乌骨鸡GM-CSF基因和MDV的VP22基因,研究了它们对IBV核酸疫苗的佐剂效应,具有重要的学术价值和应用前景,主要研究包括:
1.根据GenBank上注册的GM-CSF基因(登录号:AJ621740)序列设计一对引物,采用RT-PCR方法从ConA刺激的四川山地乌骨鸡外周血淋巴细胞总RNA中扩增出鸡GM-CSF基因,克隆入pGEM-T载体,进行序列测定与分析。结果表明,克隆的四川山地乌骨鸡GM-CSF基因全长为435bp,编码144个氨基酸残基。序列分析发现四川山地乌骨鸡GM-CSF(chGM-CSF)基因与其他品种鸡GM-CSF基因核苷酸同源性为99%左右,氨基酸同源率为98%左右,与哺乳动物GM-CSF基因的氨基酸同源性低于30%。蛋白二级预测发现山地乌骨鸡主要含4个α-螺旋,α-螺旋总含量高达55.2%,多处无规卷曲(coil),含量高达36.5%,含有2个β-折叠(strand),含量仅为8.3%,与哺乳动物的GM-CSF二级结构相似,也具有4个α-螺旋,2个β-折叠。四川山地乌骨鸡GM-CSF克隆及序列分析未见相关报道。
2.通过PCR方法扩增出不含信号肽的四川山地乌骨鸡GM-CSF成熟蛋白基因,克隆至原核表达载体pET-32a(+),经酶切和测序鉴定正确后,转化入BL21大肠杆菌,经IPTG诱导,SDS-PAGE电泳结果显示融合重组chGM-CSF蛋白主要以包涵体的形式表达,大小约为34 kD左右。通过镍离子金属螯合柱(Ni-NTA)进行重组蛋白纯化,采用纯化后重组蛋白免疫新西兰兔制备兔抗鸡GM-CSF蛋白的多克隆抗体。ELISA结果显示,制备的抗体效价达到了1:3200,Western Blot表明,制备的多克隆抗体能特异性与鸡GM-CSF重组蛋白结合,具有良好的反应原性。本研究获得的chGM-CSF重组蛋白及其多克隆抗体,为研究鸡GM-CSF蛋白生物学功能等提供关键的材料,国内外未见四川山地乌骨鸡GM-CSF基因的原核表达及抗体制备的相关报道。
3.通过酶切、连接等方法,将四川山地乌骨鸡GM-CSF基因亚克隆到真核表达载体pVAX1上以构建质粒pVAX1-chGM-CSF,利用菌落PCR和酶切对重组质粒进行鉴定,采用脂质体法转染哺乳动物COS-7细胞,采用间接免疫荧光试验(IFA)和RT-PCR检测质粒在体外的表达情况,收集质粒转染70 h后上清中表达蛋白,通过骨髓细胞增殖试验检测表达的chGM-CSF蛋白的生物学活性。RT-PCR结果显示转染了pVAX1-chGM-CSF质粒的COS-7细胞中能特异性扩增出GM-CSF基因片断,间接免疫荧光分析表明转染了pVAX1-chGM-CSF质粒的COS-7细胞显示出苹果绿荧光,而转染了空质粒pVAX1的细胞则没有显示出任何特异荧光。骨髓细胞增殖试验表明chGM-CSF蛋白能明显促进鸡骨髓细胞的增殖,这些结果为研究chGM-CSF对DNA疫苗佐剂效应提供了关键材料和奠定了理论基础,目前国内未见在COS-7细胞中表达四川山地乌骨鸡GM-CSF及表达蛋白活性分析的相关报道。
4.本研究根据发表的马立克病毒VP22的序列(登录号:L10283),设计了一对特异性引物,采用PCR方法从MDV弱毒株的基因组中扩增VP22基因。再根据IBV结构基因S1、M、N序列(登录号:DQ288927),设计引物通过PCR方法扩增出IBV结构基因S1、M、N,分别克隆入真核表达载体pVAX1,酶切鉴定正确后,再通过双酶切,电泳胶回收后与同样酶切处理的VP22片断相连,构建融合表达质粒pVAX-VP22/S1、pVAX-VP22/M、pVAX-VP22/N,酶切和测序鉴定后,通过脂质体转染COS-7细胞,转染40 h后,利用RT-PCR及间接免疫荧光检测重组蛋白在COS-7细胞中的表达。RT-PCR检测结果显示转染了融合表达质粒的COS-7细胞中分别扩增出S1、M、N基因片断,间接免疫荧光分析表明转染了融合表达质粒的COS-7细胞显示苹果绿荧光,而转染了空质粒pVAX1的细胞则没有显示出任何特异荧光。目前国内外未见马立克病毒VP22基因与IBV结构蛋白基因融合表达载体的构建及在COS-7细胞中表达的研究报道。
5.为研究chGM-CSF及VP22基因对IBV DNA疫苗的佐剂效应和比较chIL-2、chIL-8、chGM-CSF对DNA疫苗的佐剂效应差异,将大量提取的真核表达质粒,纯化后分别与脂质体溶液混合配制成DNA疫苗,通过腿部肌肉多点注射7日龄雏鸡,21日龄加强免疫一次。在免疫前和免疫后0、7、14、21、28 d采血检测ELISA抗体效价和外周血CD4+、CD8+T淋巴细胞亚群的数量,并在二免后第10d,采用MTT、法检测脾淋巴细胞增殖水平。二免两周后用IBV强毒进行攻毒,观察两周,记录发病及死亡情况,评价疫苗免疫保护力。抗体水平检测结果显示, VP22融合疫苗组pVAX-VP22/S1、pVAX-VP22/M、pVAX-VP22/N产生的抗体水平一直高于对照组pVAX-S1、pVAX-N、pVAX-M,从免疫后第14 d开始,差异显著(P＜0.05),chGM-CSF分子佐剂组pVAX-chGM-CSF+pVAX-S1、pVAX-chGM-CSF+pVAX-N、pVAX-chGM-CSF+pVAX-M抗体水平从免疫后第7 d开始明显高于对照组pVAX-S1、pVAX-N、pVAX-M,且差异显著(P＜0.05),chGM-CSF+pVAX-S1疫苗组产生的抗体水平高于chIL-2+pVAX-S1、chIL-18+pVAX-S1疫苗组,从免疫后第7 d开始差异显著(P＜0.05);脾淋巴细胞增殖实验显示,VP22融合疫苗组和chGM-CSF分子佐剂组都能有效刺激脾淋巴细胞的增殖,增殖指数高于对照组,且差异显著(P＜0.05),chGM-CSF+pVAX-S1疫苗组淋巴细胞增殖指数高于chIL-2+pVAX-S1、chIL-18+pVAX-S1组,且差异显著(P＜0.05),其中pVAX-chGM-CSF+pVAX-S1组增殖指数最高,平均值达2.83;外周血CD4+、CD8+T淋巴细胞亚群数量检测结果显示,VP22融合疫苗组淋巴细胞亚群数量免疫后高于对照组,在免疫后21 d开始,差异显著(P＜0.05),chGM-CSF分子佐剂组在免疫后明显高于对照组,从免疫后第7 d开始差异显著(P＜0.05),chGM-CSF+pVAX-S1疫苗组免疫后明显高于chIL-2+pVAX-S1、chIL-18+pVAX-S1组,从免疫后第7d开始差异显著(P＜0.05);攻毒结果表明,chGM-CSF分子佐剂组保护率介于80～90%,高于VP22融合表达的疫苗组的75～80%和对照组65～75%,其中pVAX-chGM-CSF+pVAX-S1为所有组最高,达到了90%,也高于chIL-2+pVAX-S1、chIL-18+pVAX-S1的85%和80%。这些结果表明,chGM-CSF、VP22作为分子免疫佐剂能提高IBV DNA疫苗体液和细胞免疫水平,提高疫苗的攻毒保护率,chGM-CSF对IBVDNA疫苗免疫增强效应要略优于chIL-2、chIL-18。
本研究开展的四川山地乌骨鸡GM-CSF和MDV的VP22基因的克隆、表达以及对IBV核酸疫苗佐剂效应的研究,为进一步研制新型、高效、安全禽类DNA疫苗的分子佐剂提供理论依据。
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that regulates proliferation and differentiation of hematopoietic progenitors. It has a potent effect on differentiation and maturation of dendritic cell (DC)as well as the expression of MHC and co-stimulatory molecules . Co-injection with plasmid DNA expressing GM-CSF has been reported to increase protective immunity induced by plasmid DNA.VP22, a tegument protein that has demonstrated the property of intercellular transport, is capable of distributing fused protein to many surrounding cells. It has been confirmed that it has adjuvant effect on vaccine.
In this study, chicken GM-CSF and herpervirus VP22 were cloned and expressed and further used to explore their adjuvant efects on on DNA vaccine (IBV DNA vaccine).The main content is as follows:
1.In this study, according to the gene sequence published in the GenBank (GenBank No: AJ621740), one pair of specific primer was designed. The Sichuan silky chicken GM-CSF cDNA was isolated from a concanavalin A (ConA)-stimulated peripheral blood lymphocytes using the reverse transcriptase-polymerase chain reaction (RT-PCR).The GM-CSF cDNA was ligated into the T vector and send to Sequence. Sequence analysis showed that the full-length cDNA is compose of 435 bp ,encoding an open reading frame of 144 amino acids with a predicted molecular mass of 16.15 kD . The deduced amino acid sequence of silky c hicken GM-CSF shares higher 98% and 15%-30% similarity to known GM-CSF in chicken species and other mammalian, respectively. The protein structure prediction showed that the GM-CSF protein has four a- helix and twoβ-strand and much coil. The percentage of a-helix andβ-strand and coil is 55.2%, 8.3%,36.5%,respectively. The c hGM-CSF structure is similar to that of mammalian GM-CSF.
2.The GM-CSF without the signal peptide was amplified by PCR and ligated into prokaryotic expression plasmid pET-32a(+) to construct plasmid p32GM-CSF.After identification with restriction enzyme digestion and DNA sequencing,the p32GM-CSF was transformed into BL21 E.coil.The recombinant chGM-CSF was produced with induction by IPTG at 37℃.SDS-PAGE demonstrated that the fusion protein expressed in form of inclusion body is approximately 34 kD in size. The acquired recombinant protein was purified by Nickel ion affinity chromatography and used to immunize rabbbit to prepare the Polyclonal antibodies (pAbs) specific for chicken GM-CSF .The ELISA assays showed that the antibody titre is very high and reach 1:3200. Western blotting analysis showed that it has good reactionogenicity and could bind with recombinant chGM-CSF protein specially expressed in E.coil.The acquired chGM-CSF protein and specific Polyclonal antibodies would offer critical material to the furt her research on biological activity of chGM-CSF.
3.The full-length chGM-CSF,which was obtained from the previous constructed plasmid pGM-CSF, was ligated into the plasmid pVAX1.After identification by restriction enzyme digestion and DNA sequencing,the plasmid pVAX1-chGM-CSF was transfected into COS-7 cell by lipofectamine. The expression of heterologous genes were detected by RT-PCR and indirect immunofluorescence assay. The recombinant chGM-CSF protein was prepared from COS-7 cell expressing supernatant 70 h after transfecting.The biological activity of chGM-CSF was measured by bone marrow cell proliferation assay. The results of restriction enzyme digestion and DNA sequencing showed that the plasmid pVAX1-chGM-CSF were constructed successfully. The mRNA transcription of chGM-CSF gene could be detected by RT-PCR in the COS-7 cells which were transfected with plasmids pVAX1-chGM-CSF. The expression of chGM-CSF protein could be detected by indirect immunofluorescent assay in the COS-7 cells which were transfected with plasmids.The bone marrow cell proliferation assay showed that chGM-CSF protein could enhanced bone marrow cell proliferation obviously. This study would provide materials for the further development of DNA vaccine against IBV.
4.According to VP22 gene sequence(GenBank No:L10283 ) already published in t he GenBank, one pair of specific primer was designed.T he VP22 gene was amplified from the MDV genome by PCR.Then, According to the sequence of IBV S1,M,N gene, the specific primer of S1,M,N gene were designed,respectively.The S1,M,N gene were amplified by PCR and ligated into the plasmid pVAX1 to construct recombinant plasmid ,respectively.After identification by restriction enzyme digestion and DNA sequencing,these recombinant plasmid were digested by two restriction enzyme,respectively and the big fragment of each recombinant plasmid was purified by agar electrophoresis. To construct fusion plasmid pVAX-VP2/S1 ,pVAX-VP22/M,pVAX -VP22/N,the each big fragment ligated with VP22 fragment dealed with same restriction enzyme,respectively.After identification by restriction enzyme digestion and DNA sequencing,the fusion plasmid pVAX-VP22/S1. pVAX-VP22/M, pVAX-VP22/N was transfected into COS-7 cell by lipofectamine,respectively. The expression of heterologous genes were detected by RT-PCR and indirect immunofluorescence assay. The results of restriction enzyme digestion and DNA sequencing showed that these fusion plasmid were constructed successfully. The mRNA transcription of S1,M,N gene could be detected respectively by RT-PCR in t he COS-7 cells which were transfected with these fusion plasmids. The expression of S1,M,N protein could be detected by indirect immunofluorescent assay in the COS-7 cells which were transfected with these fusion plasmids.This study would provide materials and foundation for the further evaluating the adjuvant effection of VP22 for IBV.DNA vaccine.
5.In order to evaluate the immune-modulatory of VP22 and GM-CSF as a genetic adjuvant, the plasmids used for vaccination were extracted from E.coil and purified by a C hinese invention patent method which was established previously in our lab. The diluted plasmids were encapsulated by liposome in equal volume, and administered to the 7-day-old chickens by intramuscularly injection. After two weeks later, the chickens were boosted by DNA vaccine. Indirect ELISA methods and the Fluorescence activated cell sorter were used to detect anti-IBV antibody and the number of CD3+, CD4+and CD8+ T lymp hoctyes on day 0, 7,14,21,28 after immunization. Ten days after boosting, MTT assay were used to detect the proliferation level of immunized chicken.Two weeks after boosting, all chicken were challenged by virulent IBV strain and observed for two week. The results of specific antibody show that there was a significant difference (P＜0.05) in ELISA antibody levels between VP22 fusion group pVAX-VP22/S1 ,pVAX-VP22/M, pVAX-VP22/N and control group p VAX-S1、pVAX-N、pVAX-M since the 14t h-day after first inoculation. There was also a significant difference (P＜0.05 ) in ELISA antibody levels between chGM-CSF as molecular adjuvant group pVAX-chGM-CSF+pVAX-S1,pVAX-chGM-CSF+pVAX-N, pVAX-chGM-CSF+pVAX -M and control group since the 7th-day after first inoculation.The antibody level of pVAX-chGM-CSF+pVAX-S1 was significant higher than pVAX-chIL-2+pVAX-S1, pVAX-chIL-18+pVAX-S1.The results of lymphocyte proliferation showed that there was a significant difference (P＜0.05) between VP22 fusion group and control group.There was also a significant difference (P＜0.05)between chGM-CSF as molecular adjuvant group and contol group. The SI index of lymphocyte proliferation of pVAX-chGM-CSF+pVAX-S1 is 2.83,significant higher than those of pVAX-chIL-2+pVAX-S1,pVAX-chIL-18 +pVAX-S1.The percentage of CD4+, CD8+ T-lymphocytes from chickens immunized with VP22 fusion group was significantly higher (P＜0.05) than t hat of control group since the 21d after the first vaccination. W hen compared to that of control group,the significantly higher percentage of CD4+, CD8+ T-lymphocytes was observed in group immunized with chGM-CSF as molecular adjuvant since the 7~(th) day after the first vaccination(P＜0.05).The percentage of CD4+, CD8+ T-lymphocytes of pVAX-chGM-CSF+pVAX-S1 was significant higher than pVAX-chIL-2+p VAX-S1, pVAX-chIL8+p VAX-S1. The result of virus-challenge assay showed that the protection rate of chGM-CSF as molecular adjuvant group was from 80% to 90%, higher than 75～80% of VP22 fusion group and 65～75% of control group.In all group, protection rate of the group immunized with pVAX-chGM-CSF+pVAX-S1 is highest and reach 90%,higher than 85%, 75% of pVAX-chIL-2+pVAX-S1, pVAX-chIL-18+pVAX-S1, respectively, far from 80% induced by inactivited vaccine. These results suggested that VP22 and chGM-CSF as a genetic adjuvant is feasible to enhance the immune response of IBV DNA vaccine .The chGM-CSF as molecular adjuvant is better than IL-2,IL-18 in the immune-modulatory of IBV DNA vaccine .These results provide a new way to develop effective avain vaccine.
引文
1.Cavanag h, D. Coronavirus avian infectious bronc hitis virus[J]. Vet. Res. 2007 38,281-297.
2.Ignjatovic J, Galli L.The S1 glycoprotein but not t he N or M proteins of avian infectious bronc hitis virus induces protection in vaccinated chickens[J]. Arc h.Virol,1994,138:117-134.
3.陈洪岩,江国托,杨奇伟等.鸡传染性支气管炎病毒S1基因免疫对鸡的保护作用[J].中国预防兽医学报,1999,21(4):250-252.
4.步志高,江国托,王秀荣等.传染性支气管炎病毒S1基因DNA免疫质粒的构建及其免疫原性[J].中国兽医学报,1998,18(6):527-530.
5.D. Metcalf .The Molecular Biology and Functions of the Granulocyte- Macrophage Colony-Stimulating Factors [J] .Blood,1986,67(2):257-267.
6.Tazi A, Bouchonnet F, Grandsaigne M, et al. Evidence that granulocyte macrop hage-colony-stimulating factor regulates the distribution and differentiated state of dendritic cells/Langerhans cells in human lung and lung cancers[J]. J. Clin. Invest, 1993 91:566-576.
7.Inaba K, Inaba M, Romani N, et al. Generation of Large Numbers of Dendritic Cells from Mouse Bone Marrow Cultures Supplemented with Granulocyte /Macrophage Colony- stimulating Factor[J].J. Exp. Med,1992,176 :1693-1702.
8.Kim J J, Ayyavoo V, Bagarass M L, et al. In vivo engineering of a cellular immune response by coadministration of IL-12 expression vetor with a DNA immunogen [ J]. J Immunol, 1997, 158(2):816-826.
9.Chow Y H,Chiang B L,Lee YL, et al. Development of T h1 and T h2 populations and t he nature of immune responses to hepatitis B virus DNA vaccines can be modulated by codelivery of various cytokine genes [J].J Immunol,1998,160(3):1320-1329.
10.Sedegah M, Weiss W, Sacci J B,et al. Improving Protective Immunity Induced by DNA-Based Immunization: Priming wit h Antigen and GM-CSF-Encoding Plasmid DNA and Boosting with Antigen-Expressing Recombinant Poxvirus[J]. J.Immunol,2000,164:5905- 5912.
11.Du YJ, Jiang P, Li YF, et al. Immune responses of two recombinant adenoviruses expressing VP1 antigens of FMDV fused wit h porcine granulocyte macrop hage colony-stimulating factor[J].Vaccine ,2007,25:8209-8219.
12.Zhang X Z, Divangahi M, Ngai P. Intramuscular immunization with a monogenic plasmid DNA tuberculosis vaccine: Enhanced immunogenicity by electroporation and co-expression of GM-CSF transgene[J].Vaccine,2007,25:1342-1352.
13.Schwarze S R, Dowdy S F. In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA [J ]. Trends Pharmacol Sci, 2000, 21 (2) : 45248.
14.Elliot G, Hare P. Intercellular trafficking and protein delivery by a herpesvirus structural protein [J].Cell ,1997,88(2):223-233.
15.Dorange F, Mehdaoui S, Pichon C, et al. Mareks disease virus (MDV) homologues of herpes simplex virus type1 UL49 (VP22) and UL48 (VP16) genes : high-level expression and characterization of MDV21 VP22 and VP16[J]. J Gen Virol,2000,81(9):2219-2230.
16.Harms JS, Ren X, Oliveira SC, et al. Distinctions between bovine herpesvirus-1 and herpes simplex virus type-1 VP22 tegument protein subcellular associations [J]. J Virol, 2000, 74:3301-3312.
17.Cheng W F, Hung C F, Hsu K F, et al. Cancer immunot herapy using Sindbis virus replicon particles encoding a VP22-antigen fusion [J]. Hum Gene Ther, 2002,13:553-568.
18.Saha S, Yoshida S, Ohba K, et al.A fused gene of nucleoprotein (NP) and herpes simplex virus genes (VP22) induces highly protective immunity against different subtypes of influenza virus [J].Virology,2006,354:48-57.
19.宋云峰,肖少波,金梅林等.猪2型圆环病毒核酸疫苗免疫效应研究[J].畜牧兽医学报.2005,36(10):1049-54
20.肖少波.伪狂犬病毒gC基因“自杀性”DNA疫苗及VP22蛋白转导的免疫增强效应研究[D].博士学位论文.武汉:华中农业大学.2004.
21.赵武,肖少波,方六荣等.牛疱疹病毒Ⅰ型VP22和猪繁殖与呼吸综合征病毒GP5融合基因DNA疫苗的免疫效应[J].生物工程学报,2005,21(5):725-28.
22.陈鸿军.MDVCVI988弱毒疫苗株VP22蛋白转导功能研究[D].博士学位论文.扬州:扬州大学,2006.
23.汪洋,王红宁等.高密度培养重组菌生产鸡传染性支气管炎DNA疫苗[J].安徽农业科学,2006,34(9):1894-1897.
24.胡慧琼.四川山地乌骨鸡IL-2、15、18、IFN-γ基因克隆测序及分子佐剂效应研究[D].博士学位论文.雅安:四川农业大学,2006.
25.余协中.山地乌骨鸡IL-16基因克隆测序及对IBV DNA疫苗免疫佐剂效应研究[D].硕士学位论文.雅安:四川农业大学,2007.
26.徐永莉.山地乌骨鸡IL-1β基因克隆测序及对IBV DNA疫苗免疫佐剂效应研究[D].硕士学位论文.雅安:四川农业大学,2007.
27.周生,王红宁,唐梦君等.禽传染性支气管炎冠状病毒SAIBK株全基因组cDNA文库的构建与序列测定[J].畜牧兽医学报,2007,38(1):72-77.
28.Gurunathan S, Klinman D M, Seder R A. DNA VACCINES: Immunology, Application, and Optimization. Annu. Rev. Immunol.,2000,18:927-974.
29.Maliszewski C R, Schoenborn M A, Cerretti D P,et al.Bovine GM-CSF: molecular cloning and biological activity of the recombinant protein[J].Molecular Immunology ,1988,25(9): 843-850.
30.Mclnnes C J,Haig D M.Cloning and expression of a cDNA encoding ovine granulocyte -macrop hage colony-stimulating factor[J]. Gene,1991,105: 275-279.
31.Inumaru S,Sakamatsu H. CDNA cloning of porcine granulocyte-macrophage-colony -stimulating factor[J]. Immunology and Cell Biology,1995,73:474-476.
32.Wu H L, Chen P J, Lin H K, et al.Molecular Cloning and Expression of Woodchuck Granulocyte-Macrophage Colony Stimulating Factor[J]. Journal of Medical Virology, 2001, 65:567-575.
33.Avery S, Rothwell L,Degen W.D.J,et al. Characterization of the first nonmammalian T2 cytokine gene cluster: the cluster contains functional single-copy genes for IL-3, IL-4, IL-13, and GM-CSF, a gene for IL-5 t hat appears to be a pseudogene, and a gene encoding anot her cytokine like transcript, KK34[J].Journal of Interferon & Cytokine Research, 2004,24: 600-610.
34.Dunham S P, Bruce J. Isolation, expression and bioactivity of feline granulocyte-macrophage colony-stimulating factor[J].Gene,2004,332: 97-106.
35.Gaucher D, Chadee K. Molecular cloning and expression of gerbil granulocyte/macrophage colony-stimulating factor[J]. Gene,2002, 294 :233-238.
36.Burgess AW, Camakaris J, Metcalf D. Purification and properties of colony-stimulating factor from mouse lung - conditioned medium [J]. J Biol Chem, 1977, 252 (6): 1 998 - 2 003.
37.Caux C,Dezutter-Dambuyant C,Schmitt D, et al.GM-CSF and TNF-a cooperate in the generation of dendritic Langer hans cell[J].Natrue.,1992,360:258-259.
38.Nicola N A, Burgess A W, Metcalf D.Similar Molecular Properties of Granulocyte -Macrophage Colonystimulating Factors Produced by Different Mouse Organs in Vitro andin Vivo[J]. T he journal of biological chemistry, 1979,254(12): 5290-5299.
39.DeLamarter F, Mermod J J, Liang C.-M,et al. Recombinant murine GM-CSF from E. coli has biological activity and is neutralized by a specific antiserum[J] .The EMBO Journal,1985,4 (10):2575-2581.
40.Greenberg R, Lundell D, Alroy Y, et al. Expression of Biologically Active, Mature Human Granulocyte-Macrophage Colony Stimulating Factor with an E. coli Secretory Expression System[ J ] .Current microbiology ,1988,17 :321-332.
41.舒邓群,茆达干,曹少先等.猪粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因的克隆和序列分析及其基因表达[J].江西农业大学学报2007,29(2):234-240.
42.林少华,罗红霞,郭慧媛等.重组牛乳铁蛋白肽包含体的复性与纯化[J].中国乳品工业,2006,34(1):23-27.
43.Dranoff G,Jaffee E ,Lazenby A ,et al. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity [J]. Proc Natl Acad Sci USA ,1993 ,90 (8) :3539-3543.
44.FDA.Considerations for Plasmid DNA Vaccines for Infectious Disease Indications [J]. Biotec hnology Law Report, 2005,24(3):304-311
45.卢圣栋.现代分子生物学实验技术(第2版)[M].北京:中国协和医科大学出版社,2004
46.Harvey T J, Macnaug hton T. BGowans E. J. The development and characterisation of a SV40 T-antigen positive cell line of human hepatic origin[J]. Journal of Virological Methods,1997, 65(1):67-74
47.Sabbioni S, Negrini M., Rimessi P.et al. A BK virus episomal vector for constitutive high expression of exogenous cDNAs in human cells[J]. Arch Virol ,1995,140:335-339.
48.Feigner PL, Gadek TR, Holm M, et al. Lipofection: a highly efficient lipid-mediated DNA transfection procedure. Proc Natl Acad Sci USA, 1987,84(21):7413-7417.
49.Connor J, Yatvin MB, Huang L. PH-sensitive liposomes:acid-induced liposome fu-Sion[J].Ptoc Natl Acad Sci USA, 1984,81:1715-1718.
50.Koshizaka T,Hayashi Y, Yagi K. Novel liposomes for efficient transfection of beta-alctosidase gene into COS -lcells[J].J Chin Biochem Nutr,1989,7:185-192.
51.Kapcynski D R. Protection of c hickens from infectious bronc hitis by in ovo and intramuscular vaccination wit h a DNA vaccine expressing t he S1 glycoprotein[J]. Avian Dis, 2003,47(2):272-285.
52.刘兆球,姜世金,常维山等.传染性支气管炎病毒S1基因的真核表达及免疫原性检测[J].中国兽医学报,2005,25(3):241-243.
53.焦红梅,焦新安,殷月兰等.鸡传染性支气管炎病毒S1基因的真核表达及其产物的免疫原性[J].扬州大学学报(农业与生命科学版),2006,27(2):44-47.
54.Elliott G, O'Hare P.Intercellular Trafficking and Protein Delivery by a Herpesvirus Structural Protein[J] .Cell, 88:223-233.
55.Sciortino MT, Taddeo B, Poon AP, et al. Of the three tegument proteins that package mRNA in herpes simplex virions, one (VP22) transports the mRNA to uninfected cells for expression prior to viral infection[J]. Proc Natl Acad Sci USA, 2002, 99:8318-8323.
56.Liu CS, Kong B, Xia H, et al. VP22 enhanced intercellular trafficking of HSV thymidine kinase reduced the level of ganciclovir needed to cause suicide cell death[J]. J Gene Med, 2004, 3: 145-152.
57.Normand N, van Leeuwen H, O' Hare P. Particle formation by a conserved domain of the herpes simplex virus protein VP22 facilitating protein and nucleic acid delivery[J]. J Biol Chem,2001,276:15042-15050.
58.Dorange F, EMehdaoui S, Pichon C, et al.Marek's disease virus (MDV) homologues of herpes simplex virus type 1 VP22 (VP22) and ULA8 (VP16) genes: high-level expression and characterization of MDV-1VP22 and VP16[J]. J Gen Virol, 2000,81:2219-2230.
59.Harms J S, Ren X, Oliveira S C,et al. Distinctions between bovine herpesvirus 1 and herpes simplex virus type 1VP22 tegument protein subcellular associations[J]. J Virol. 2000,74(7): 3301-3312.
60.Hung CF, Cheng WF, Chai CY, et al. Improving vaccine potency through intercellular spreading and enhanced MHC class I presentation of antigen[J]. J Immunol, 2001,166:5733-5740.
61.Becker-Hapak M, Mcallister SS, Dowdy SF. TAT mediated protein transduction into mammalian cells[J]. Met hods, 2001,24:247-256
62.Saha S, Yoshida S, Ohb K,et al. A fused gene of nucleoprotein (NP) and herpes simplex virus genes (VP22) induces highly protective immunity against different subtypes of influenza virus[J].Virology, 2006,354,48-57
63.Oliveira S C, Harms J S, Afonso R R ,et al. A genetic immunization adjuvant system based on BVP22-antigen fusion [J]. Hum Gene Ther,2001,12(10): 1353-9.
64.瞿素,胡凝珠,施海晶等.HSV-1 VP22和hIL-18增强HBV DNA微球疫苗诱导的小鼠体液免疫应答[J].现代免疫学,2005,25(4):300-304.
65.马道新,于修平,张晓梅等.VP22增强人巨细胞病毒pp65核酸疫苗在小鼠体内免疫活性的实验研究[J].中华医学杂志,2005,85(15):1049-1052
66.Saskia A R, Bosma PJ ,Rohn J L, et al. Induction of insolubility by herpes simplex virus VP22 precludes intercellular trafficking of N-terminal Apoptin-VP22 fusion proteins [J]. J. Mol. Med.2003, 81:558-565
67.Saha S, Yoshida S , Ohba K, et al.A fused gene of nucleoprotein (NP) and herpes simplex virus genes (VP22) induces highly protective immunity against different subtypes of influenza virus [J].Virology,2006.,354:48-57
68.Cook J K, Epidemiology of infectious bronc hitis virus.The Coronaviridae[M]. Plenum Press,1995,317-336.
69.Yu L, Wang Z, Jiang Y, et al.Molecular epidemiology of infectious bronc hitis virus isolates from China and Southeast Asia[J]. Avian Dis,2001,45(l):201-219.
70.田占成,王云峰,童光志.传染性支气竹炎病毒疫苗研究进展[J].动物医学进展,2005,26(6):21-24.
71.Wang L,Jun her D,Collisson E W. Evidence of natural recombination wit hin the S1 gene of infectious bronc hitis virus[J].Virology, 1993,192: 710-716.
72.Xiang ZQ, Erd H C J. Manipulation of the immune response to a plasmid encoded viral atigen by coinocultion with plasmids expressing cytokines [J]. Immuntiy, 1995, 2:129-135.
73.廖国阳,姜述德,孙明波等.双表达载体研究GM-CSF对HCV C基因免疫应答的影响[J].免疫学杂志,2001,17(4):289-292
74.Michel N ,Osen W, Gissmann L, et al. Enhanced immunogenicity of HPV 16 E7 fusion proteins in DNA vaccination[J ] . Virology ,2002 ,294 (1) :47-59.
75.Krishnan L,Dicaire C J, Patel G B , et al. Archaeosome Vaccine Adjuvants Induce Strong Humoral, Cell-Mediated, and Memory Responses: Comparison to Conventional Liposomes and Alum[J]. Infect Immun, 2000; 68 (1): 54-63
76.Wong H T,Cheng S C,Sin F W.et al.A DNA vaccine against foot-and mouth disease elicits an immune response in swine which is enhance by co-administration with interleukin-2[J].Vaccine,2002,20(21-22):2641-2647.
77.Kim J J, Ayyavoo V, Bagarass M L, et al. In vivo engineering of a cellular immune response by coadministration of IL-12 expression vetor wit h a DNA immunogen[J]. J Immunol, 1997, 158(2):816-826
78.Okada H,Giezeman-Smits K M,Ta hara H,et al. Effective cytokine gene therapy against an intracranial glioma using a retrovirally transduced IL-4 plus HSV tk tumor vaccine [J]. Gene Ther,1999,6(2): 219-226.
79.Caux C,Dezutter-Dambuyant C,Schmitt D, et al. GM-CSF and TNF-a cooperate in t he generation of dendritic Langer hans cell[J].Natrue,1992,360:258
80.Collisson E W, Pei J, Dzielawa J, et al. Cytotoxic T lymphocytes are critical in the control of infectious bronc hitis virus in poultry[J]. Dev Comp Immunol, 2000,24(2):187-200.
81.Seo S H, Wang L, Smit h R, et al. The carboxyl-terminal 120-residue polypeptide of infectious bronc hitis virus nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection[J]. J Virol, 1997,71(10):7889-7894.
82.Williams A E, Davison T F. Enhanced immunopat hology induced by very virulent infectious bursal disease virus[J]. Avian Path. 2005,34(11): 4-14.
83.Nathalie S. Canine Distemper Virus DNA Vaccination Induces Humoral and Cellular Immunity and Protects against a Lethal Intracerebral Challenge[J]. J. Virol. 1998,72(11): 8472-76.
84.Feltquate D M, Heaney S, Webster RG, et al. Different T helper cell types and antibody isotypes generated by saline and gene gun DNA immunization[J]. J. Immunol. 1997,158: 2278-84.
85.Hung C F, He L M,Juang J,et al. Improving DNA vaccine potency by linking Marek's disease virus type 1VP22 to an antigen[J]. J Virol. 2002,76(6): 2676-82.
86.Kusters J G, Niesters H G M. Phytogeny of antigenic variants of avian coronavirus IBV[J].Virology,1989,169:217-221
87.王红宁主编,禽传染性支气管炎综合防治[M].北京:中国农业出版社,2000
88.Endo-munoz L B, Faraghe J T. Avian infectious bronchitis: cross-protection studies using different Australian subtypes[J]. Australian Veterinary Journal, 1989,66:345-348
89.Avellaneda G E, Villeges P, Jackwood M W, et al. In vivo evaluation of t he pathogenecity of isolates of infectious bronc hitis virus[J]. Avian Dis, 1994,38:589-597
90.Albassam M A, Winterfield R W, Thacker H L. Comparision of the nephropath-ogenecity of four strains of infectious bronchitis virus[J].Avian Dis,1986,30:468-476
91.King D J, Cavang h D. Infectious bronchitis. In: Diseases of poultry, 9t h ed[M]USA, Iowa State University Press, 1991
92.郭文和.鸡传染性支气管炎病毒的免疫研究进展[J].中国兽医杂志,1992,10:38-39
93.江国托,刘思国,康丽娟等.鸡传染性支气管炎病毒的遗传与变异(一)[J].养禽与禽病防治,1999,(4):6-9
94.Finney P M, Box P G, Holmes H C. Studies with a bivalent infectious bronchitis killed virus vaccine[J].Avian Pathology, 1990,19: 435-450
95.Binns M M, Tomley F M, Mockett A P, et al. Expression of the infectious bronc hitis virus spike protein by reeombinant vaccinia virus and induction of neutralizing antibodies in vaccinated mice[J].J Gen Virol, 1987, 68(9):2291-2298.
96.Tomley F M, Mockett A P, Boursnell M E, et al. Expression of the infectious bronchitis virus spike protein by recombinant vaccinia virus and induction of neutralizing antibodies in vaccinated mice[J].J Gen Virol, 1987, 68(9):2291-2298.
97.Evans S, Cavanagh D. Utilizing fowlpox virus recombinants to generate defective RNAs of the coronavirus infectious bronchitis virus[J]. J Gem Virol, 2000, 81(12): 2855-2865.
98.Wang X, Schnitzlein W M, Tripat hy D N, et al. Construction and immunogenicity studies of recombinant fowl poxvirus containing the S1 gene of Massachusetts 41 strain of infectious bronc hitis virus[J]. Avian Dis, 2002,46(4):831-838.
99.Yu L, Liu W, Schnitzlein W M, et al. Study of protection by recombinant fowl poxvirus expressing C-terminal nucleocapsid protein of infectious bronchitis virus against challenge[J]. Avian Dis, 2001,45(2):340-348.
100.Cavanagh D. Severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus [J]. Avian Pathology,2003,32:6,567 - 582
101.Johnson M A, Pooley C, Ignjatovic J, et al. A recombinant fowl adenovirus expressing the S1 gene of infectious bronchitis virus protects against challenge with infectious bronc hitis virus[J]. Vaccine,2003,21(21):2730-2736.
102.焦红梅,焦新安,田华荣等.减毒鼠伤寒沙门氏菌运送的鸡传染性支气管炎病毒S1基因DNA疫苗及其免疫效力研究[J].病毒学报,2007,23(3):212-217.
103.Stirrups K, S haw K. Expression of reporter genes from the defective RNA CD-61 of the coronavirus infection bronchitis virus[J]. J Gen Virol, 2000, 81(7): 1687-1698.
104.孙永科,川战成,土云峰,等.鸡传染性支气竹炎病毒S1基因和鸡且型十扰素基因在重组鸡痘病毒中的共表达fJl.畜牧兽医学报,2005,36(8):800-806.
105.黄亚东,王林川,郑青等.鸡传染性支气管炎病毒H株纤突蛋白S1基因的克隆及其在毕赤酵母中表达[J].病毒学报,2003,19(2):144-148.
106.刘胜旺,杜恩歧,孔宪刚等.鸡传染性支气管炎病毒核蛋白基因的体外表达和纯化[J].中国实验动物学杂志,2002,12(5):302-302.
107.周继勇,JimmyKwang.传染性支气管炎病毒核衣壳蛋白基因变异及在大肠杆菌中的表达[J].浙江大学学报:农业与生命科学版,2003,29(1):1-9.
108.陈萍,王红宁.间接ELISA检测IBV DNA疫苗免疫抗体的研究[J].安徽农业科学,2006.34(16):3881-3884.
109.廖明,辛朝安.鸡传染性支气管炎病毒s1基因在昆虫细胞中的表达[J].中国兽医学报,1997,17(6):539-543.
110.宋延华、刘福安.含有IBV S1基因的昆虫杆状病毒表达载体的构建[J].华南农业大学学报,1998,14(4):1-4.
111.戴亚斌,陈德胜,丁铲等.表达鸡传染性支气管炎病毒SD/97/01株S1蛋白的重组杆状病毒的构建[J].厦门大学学报:自然科学版,2002,41(4):487-492.
112.余祖华,王红宁,周生等.禽传染性支气管炎病毒S1基因毕赤酵母表达载体的构建[J].中国家禽,2006,28(21):14-16.
113.Song C S, Lee Y J, Lee C W, et al. Induction of protective immunity in chickens vaccinated with infectious bronchitis virus S1 glycoprotein expressed by a recombinant baculovirus [J]. Journal of General Virology ,1998, 79:719-723.
114.Zhou J Y , Wu J X, Cheng L Q,et al. Expression of Immunogenic S1 Glycoprotein of Infectious Bronchitis Virus in Transgenic Potatoes [J]. Journal of Virology,2003, 77 (16):9090-9093
115.Kapcynski D R, Hilt D A,shapiro D,et al.Protection of chickens from infectious bronchitis by in ovo and intramuscular vaccination with a DNA vaccine expressing the S1 glycoprotein[J]. Avian Dis,2003, 47(2): 272-285.
116.刘思国,康丽娟,江国托等.鸡传染性支气管炎病毒核蛋白基因重组真核表达载体的构建[J].中国兽医学报,2001,21(1):14-16.
117.杨帆,王红宁,李春等.禽传染性支气管炎病毒四川分离株M基因的克隆及真核表达载体构建[J].四川畜牧兽医,2003,30(B09):19-19.
118.李春,王红宁,周生等.禽传染性支气管炎病毒S1基因玉米表达载体的构建[J].四川畜牧兽医,2003,30(B09):18.
119.程丽琴.传染性支气管炎病毒S基因马铃薯生物反应器的建立[D].博士学位论文.杭州:浙江大学,2003
120.Wills K N, Atencio I A, Avanzini J B, et al. Intratumoral spread and increased efficacy of a p53-VP22 fusion protein expressed by a recombinant adenovirus [J]. J Virol, 2001, 75:8733-8741
121.Dilber MS, P helan A, Aints A, et al. Intercellular delivery of thymidine kinase prodrug activating enzyme by the herpes simplex virus protein, VP22[J] .Gene Ther,1999, 6:12-21
122.Lai Z and Brady RO. Gene transfer into the central nervous system in vivo using a recombinanat lentivirus vector [J]. J Neurosci Res, 2002, 67:363-371
123.Kretz A, Wybranietz W A, Hermening S,et al. HSV-I VP22 augments Adenoviral gene transfer to CNS neurons in the retina and striatum in vivo [J]. Mol Ther,2003,7: 659-669
124.Lundberg M, Johansson M. Is VP22 nuclear homing an artifact? [J]. Nat Biotec hnol, 2001, 19:713-714
125.Boenicke L, Chu K, Pauls R,et al. Efficient dose-dependent and time-dependent protein transduction of pancreatic carcinoma cells in vitro and in vivo using purified VP22-EGFP fusion protein [J]. J Mol Med, 2003,81:205-213
126.Donnell L A,Clemmer J A,Czymmek K,et al.Marek's Disease Virus VP22: Subcellular Localization and Characterization of Carboxyl Terminal Deletion Mutations [J]. Virology,2002,292(2): 235-240
127.Elliott G, O'Reilly D, O' Hare P. Phosphorylation of the herpes simplex Virus type 1 tegument protein VP22[J]. Virology,1996,226:140-145
128.Morrison EE, Wang YF, Meredit h DM. Phosphorylation of structural components promotes dissociation of the herpes simplex virus type I tegument [J]. J Virol, 1998,72: 7108-7114
129.Brignati M J, Loomis J S, Wills J W, et al.Membrane association of VP22, a herpes simplex virus type 1 tegument protein[J]. J Virol, 2003,77:4888-4898.
130.Zender L, Kock R, Eckhard M, et al. Gene therapy by intrahepatic and intratumoral trafficking of p53-VP22 induces regression of liver tumors[J]. Gastroenterology, 2002a, 123: 608-618.
131.Aints A, Guven H, Gahrton G,'et al. Mapping of herpes simplex virus-1 VP22 functional domains for inter and subcellular protein targeting [J]. Gene Ther. 2001, 8(14): 1051-6.
132.Mitchell D J, Steinman L, Kim D T. Fathman Polyarginine enters cells more efficiently than other polycationic homopolymers [J]. Pept Res.2000: 318-325
133.Schwarze SR, Ho A, Vocero-A kbani A, et al. In Vivo Protein Transduction: Delivery of a Biologically Active Protein into the Mouse [J]. Science, 1999, 285: 1569-1572.
134.Normand N, Leeuwen H, O' Hare P.Particle Formation by a Conserved Domain of t he Herpes Simplex Virus Protein VP22 Facilitating Protein and Nucleic Acid Delivery [J] J. Biol. C hem., 2001,276:15042-15050.
135.Fang B, Xu B, Koch P, et al. Intercellular traffcking of VP22-GFP fusion proteins is not observed in cultured mammalian cells [J]. Gene Ther, 1998, 5: 1420-1424.
136.Derer W, Easwaran HP, Knopf CW,et al. Direct protein transfer to terminally differentiated muscle cells [J]. J Mol Med, 1999,77: 609-613.
137.Wybranietz WA, Gross CD, Phelan A,et al. Enhanced suicide gene effect by adenoviral transduction of a VP22-cytosine deaminase (CD) fusion gene [J]. Gene Ther, 2001, 8:1654-1664.
138.Brewis N ,Phelan A, Webb J, et al. Evaluation of VP22 spread in tissue culture [J]. J Virol, 2000,74:1051-1056.
139.Elliott G , O' Hare P. Cytoplasm-to-nucleus translocation of a herpesvirus tegument protein during cell division [J] J.Virol, 2000,74: 2131-2141.
140.Phelan A ,Elliott G,O' Hare P. Intercellular delivery of functional p53 by the herpesvirus protein VP22[J]. Nat Biotec hnol ,1998,16 (5) :440-443.
141.Dilber M S ,P helan A ,Aints A ,et al. Intercellular delivery of thymidine kinase prodrug activating enzyme by the herpes simplex virus protein VP22 [ J]. Gene Ther, 1999, 6(1) :12-21.
142.Wills K N,Atencio I A, Avanzini J B,et al. Intratumoral spread and increased efficacy of a p53-VP22 fusion protein expressed by a recombinant adenovirus [J]. J Virol. 2001,75(18): 8733-41.
143.Roy I, Holle L ,Song W ,et al. Efficient translocation and apoptosis induction by adenovirus encoded VP22-p53 fusion protein in human tumor cells in vitro [J]. Anticancer Res. 2002, 22(6A):3185-9.
144.Qiu Z H, Harms J S., Zhu J,et al. Bovine Herpesvirus Tegument Protein VP22 Enhances Thymidine Kinase/Ganciclovir Suicide Gene Therapy for Neuroblastomas Compared to Herpes Simplex Virus VP22[J].J. Virol. 2004 78:4224-4233.
145.孔北华,王文霞,刘春生等.病毒蛋白VP22增强单纯疱疹病毒胸苷激酶/更昔洛韦基因治疗系统杀伤卵巢癌细胞的体内研究[J].中华妇产科杂志,2003,38(4):192-198.
146. Perkins S D, Hartley M G, Lukaszewski R A,et al. VP22 enhances antibody responses from DNA vaccines but not by intercellular spread [J] .Vaccine. 2005,23(16): 1931-40.
147.瞿素,胡凝珠,施海晶等.HSV-1 VP22和hIL-18增强HBV DNA微球疫苗诱导的小鼠体液免疫应答[J].现代免疫学,2005,25(4):300-304.
148.马道新,于修平,张晓梅等.单纯疱疹病毒壳膜蛋白22增强人巨细胞病毒糖蛋白B核酸疫苗免疫效果的体内生物学活性[J].中国医学科学院学报,2005,27(6):793
149.Nunberg J H, Doyle M V, York S M, et al.Interleukin 2 acts as an adjuvant to increase the potency of inactivated rabies virus vaccine [J] .PNAS , 1989 ,86 (11) 4240-4243
150.Chow Y H,Huang W L,Cli W K,et al. Improvement of hepatitis B virus DNA vaccine by plasmids coexpressing hepatitis B surface antigen and interleukin-2 [J]. J Virol, 1997,71(1): 169-178.
151.Xin K Q, Hamajima K, Sasar S, et al. Intranasal administration of human immunodeficiency virus type 1 ( HIV1) DNA vaccine with interleukin2 expression plasmid enhance cell mediated immunity against HIV[J]. Immunology, 1998, 94(3): 438-444.
152.Wong H T,Cheng S C,Sin F W.et al.A DNA vaccine against foot-and mouth disease elicits an immune response in swine which is enhance by co-administration with interleukin-2 [J]. Vaccine,2002,20(21-22):2641-2647.
153.Li J,Liang X, Huang Y,et al.Enhancement of the immunogenicity of DNA vaccine against infectious bursal disease virus by co-delivery with plasmid encoding chicken interleukin 2 [J] .Virology,2004,329(1):89-100
154.Wassef N M, Plaeger S F. Cytokinesas adjuvantsf or HIV DNA vaccines[J].Clinical and Applied Immunology reviews.2002,2:229-234
155.Toka F N, Pack C D, Rouse B T. Molecular adjuvants for mucosal immunity [J] .Immunological Reviews,2004,199:100-11
156. Kim J J, Ayyavoo V, Bagarass M L, et al. In vivo engineering of a cellular immune response by coadministration of IL-12 expression vetor with a DNA immunogen [J]. J Immunol, 1997, 158(2):816-826
157. Okada H,Giezeman-Smits K M,Tahara H,et al. Effective cytokine gene therapy against an intracranial glioma using a retrovirally transduced IL-4 plus HSV tk tumor vaccine [J]. Gene Ther,1999,6(2): 219-226.
158. Gherardi M M, Ramirez J C. Esteban M. Towards a new generation of vaccine: the cytokine IL-12 as an adjuvant to enhance cellular inmmune responses to pathogens during prime-booster vaccination regimens [J]. Histol Histopathol, 2001,16 (2): 655-667
159. Chen H W,Pan C H, Huan H W,et aLSuppression immune response and protective im munity to a Japanesee ncephalitis virus DNA vaccine by coadministration an IL-12 Expressing plasmid [ J]. J Immunol ,2001,166 :7419-7426
160. 朱晓华,石佑恩,宁长修等.白细胞介素IL-12在日本血吸虫脂肪酸结合蛋白诱导小鼠保护性免疫力中的佐剂作用[J].中国寄生虫学与寄生虫病杂志,2005,23(3):150-154.
161. Geissler M, Gesien A, Tokus hige K, et al. Enhancement of cellular and hunoral immune responses to hepatitis C virus core protein using DNA-based vaccinges augmented with cytokine-expressing plasmids[J]. J Immunol,1997,158(3): 1231-1237.
162. Barouch D H, Santra, S.,Steenbeke, T. D..et al. Augmentation and suppression of immune responses to an HIV-1 DNA vaccine by plasmid cytokine/Ig administration[J]. Journal of Immunology (Baltimore), 1998,161:1875-1882.
163. Kusakabe K, Xin KQ, Katoh H et al. The timing of GM-CSF expression plasmid administration influences the Th1/T h2 response induced by an HIV-1-specific DNA vaccine[J]. J Immunol.2000,15;164(6):3102-3111.
164. GobelT W , Sc hneider K, Sc haerer B ,et al. IL-18 stimulates t he proliferation and IFN-Y release of CD4+T cells in t he c hicken:Conservation of a T h1-like system in a nonmammalian species[J].Journal of immunology,2003,171:1809-1815.
165. Lee S ,Gierynska M, Eo S K,et al .Influence of DNA encoding cytokines on systemic and mucosal immunity following genetic vaccination against herpes · simplex virus[J].Microbes Infect,2003,5:571-578.
166. Kim S H, Cho D, Hwang S Y, et al. Efficient induction of antigenspecific T helper type I mediated immune responses by intramuscular injection with ovalbumin /interleukin-18 fusion DNA[J].Vaccine, 2001,19(30): 4107-4114.
167. Billaut-Mulot O, Idziorek T, Loyens M, et al. Modulation of cellular and humoral immune responses to a multiepitopic HIV-1 DNA vaccine by interleukin-18 DNA immunization/viral protein boost[J].Vaccine, 2001,19(20): 2803-2811.
168. Hanlon L, Argyle D,Bain D,et al .Feline leukemia virus DNA vaccine Eficacy is enhanced by coadministration with interleukin-12(IL-12)and IL-18 expression vectors[J].J Virol, 2001,75( 18):8424-8433.
169. Kim J J,Yang J S,Dang K,et al .Engineering enhancement of immune responses to DNA-based vaccines in a prostate cancer model in rhesus macaques through the use of cytokine gene adjuvants[J].Clin Cancer Res,2001,7 :88 2s-889s.
170. Sin J I,Kim J J, Boyer I D ,et al .In vivo modulation of vaccine-induced immune responses toward a Th1 phenotype increases potency and vaccine effectiveness in a herpes simplex virus type 2 mouse model[J]. J Virol,l 999,73(1):5 01-509.
171. Bossu P,Neumann D,Giudice E D,et al. IL-18 cDNA vacciantion protects mice from spontaneous lupus-like autoinunune disease. PNAS ,2003,100 :14181 -14186.
172. Egan M A, Israel Z R. The use of cytokines and chemokines as genetic adjuvants for plasmid D NAv accines[J]. Clinical and Applied Immunology Reviews,2002,2:255-287
173. Anderson K P et al.En hancement of a secondary antibody response to vesicular atomatatis virus "G" protein by IFN-γ treatment at primary immunization[J].J Immanol ,1989,140:3599-3614
174. Xiang Z Q, He Z, Wang Y,et al. The effect of interferon-gamma on genetic immunization [J].Vaccine.,1997,15(8):896-898.
175. Kim J J, Yang J S, Manson K H, et al. Coimmunization with IFN-gamma or IL-2, but Not IL-13 or IL-4 cDNA Can En hance Th1-Type DNA Vaccine-Induced[J]. Journal of Interferon & CytokineResearch,2000,20(3): 311-319.
176. Pertmera T M, Orana A E, Madorinb C A. et al.Th1 genetic adjuvants modulate immune responses in neonates[J].Vaccine. 2001,19(13-14): 1764-1771
177. 孙永科,田占成,王云峰,等。畜牧兽医学报,鸡传染性支气管炎病毒S1基因和鸡Ⅱ型干扰素基因在重组鸡痘病毒中的共表达[J].2005,36(8):800-806
178. Boulay, J.L., Paul, W.E. 1992. The interleukin-4 family of lymphokines[J]. Curr.Opin. Immunol. 4,294-298.
179. Stanley, E., Metcalf, D., Sobieszczuk, P., Gough, N.M., DunnAR.1985.The structure and expression of the murine gene encoding granulocyte-macrophage colony-stimulating factor:Evidence for utilization of alternative promoters. The EMBO Journal 4,2569-2578
180. Billaut-Mulot O, Id2iorek T, Loyens M, et al. Modulation of cellular and humoral immune responses to a multiepitopic HIV 1 DNA vaccine by interleukin-18 protein boost [J].Vaccine,2001,19(20-22):2803-281.
181. O'Hagan DT, MacKichan ML, Singh M. Recent developments in adjuvants for vaccines against infectious diseases [J].Biomol Eng,2001,18(3):69-85
182. Okada E, Sasaki S, Ishii N, et al. Intranasal immunization of a DNA vaccine with IL-12- and granulocyte macrophage colony-stimulating factor (GM-CSF)-expressing plasmids in liposomesinduces strong mucosal and cell-mediated immune responses against HIV-1 antigens[J].J Immunol,1997,159(7):3638-3647
183. Kusakabe K, Xin KQ, Katoh H, et al. The timing of GM-CSF expression plasmid administration influences the ThllTh2 response induced by an HIV 1-specific DNA [J]. J Immunol,2000,164(6):3102-3111
184. Borrello I, Pardoll D. GM-CSF-based cellular vaccines: a review of the clinical experience[J].Cytokine Growth Factor Rev, 2002,3(2):185-193
185. Gerber D, Trescol-Biemont MC, Ettouati L, et al. An accessory peptide binding site with allosteric effect on the formation ofpeptide-MHC-Ⅱ complexes [J]. C RAcad Sci Ⅲ,1998,321(l):19-24
186. Heath A W, Playfair J H. Cytokines as immunological adjuvants.Vaccine, 1992,10(7): 427-434