【作者】 徐超；

【导师】 汪铭书；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2008， 博士

【摘要】 鸭瘟(Duck Plague,DP),又称,鸭病毒性肠炎(Duck Enteritis),是由α-疱疹病毒引起的鸭、鹅等雁行目动物的一种急性败血和高度接触性的传染病。相对其它疱疹病毒而言,DPV研究的总体水平较低,分子生物学所知甚少。本文对本实验室发现的鸭瘟病毒gC基因开展系列研究,获如下结果:1.鸭瘟病毒gC基因发现、克隆及分子特性分析对构建的鸭瘟病毒(DPV)DNA基因文库一个重组质粒测序,ORF Finder和BLASTN工具初步分析测序结果发现一个全长为1296bp的完整ORF片段(ORF1296)。大量生物信息学分析和斑点杂交证实该ORF与其它疱疹病毒gC基因同源,与禽疱疹病毒gC亲缘关系最近,为DPV的gC基因(GenBank登录号EU076811)。其大小为1296bp,编码432个氨基酸,编码的gC蛋白的理论分子量为45kD,理论等电点(PI)为5.292。推导的gC氨基酸序列的21～24,50～58,67～70,161～171,273～281,387～393位最有可能为抗原表位。2.鸭瘟病毒gC基因原核表达、产物纯化及其抗体制备根据发现的鸭瘟病毒的gC基因序列,设计一对引物扩增DPV gC基因并将其克隆至pMD18-T载体,经酶切和DNA测序鉴定正确后,将DPV gC基因正向插入原核表达载体pET-32a(+)的EcoRI和XhoI位点间,成功构建了重组表达质粒pET32a-gC。将重组表达质粒pET32a-gC转化表达宿主菌BL21(DE3),经IPTG诱导、SDS-PAGE电泳分析表达出了大小约为65kD的目的重组蛋白。对pET32a-gC表达的融合蛋白Western blot免疫印迹分析发现,该蛋白能与DPV阳性血清发生特异性反应。大量提取pET32a-gC表达产物,纯化后免疫家兔成功制备兔抗DPV gC蛋白的多克隆抗体。3.鸭瘟病毒gC基因原核表达产物免疫原性研究将经纯化、复性的鸭瘟病毒gC基因原核表达蛋白与等量弗氏佐剂混合制备gC重组蛋白疫苗,免疫雏鸭研究对鸭的免疫保护效果。结果表明:重组蛋白可以诱导机体产生中和抗体,其中和效价和弱毒组变化一致,在免疫后21d达到最高。ELISA检测的血清抗体消长规律亦同弱毒组一致。DPV gC重组蛋白对鸭瘟病毒强毒感染具有一定的保护能力。4.抗gC蛋白抗体介导的免疫组化和免疫荧光检测DPV建立和应用以饱和硫酸铵沉淀法结合High Q阴离子交换柱层析纯化的兔抗DPV gC原核表达蛋白的IgG为一抗,分别建立检测石蜡切片上DPV的SP免疫组化法和间接免疫荧光双染法。应用建立的两种方法研究DPV-CHv强毒株人工感染雏鸭肝、肺、肾、脑、十二指肠、空肠、回肠、直肠、法式囊、脾脏、腺胃、食道等组织中DPV病毒定殖、动态变化规律和gC蛋白在各组织中表达时相。结果表明:DPV是一种泛嗜性的病毒,SP法和免疫荧光法均可特异检测到DPV感染鸭肝、肺、肾、脑、十二指肠、空肠、回肠、直肠、法式囊、脾脏、腺胃、食道中DPV抗原的存在。人工感染8h即可在十二指肠、空肠、回肠和直肠中检测到DPV gC的表达;攻毒12h时,肝、食道、腺胃可检测到病毒gC表达蛋白;24h可在法式囊、肾和脾脏可检测到gC表达蛋白;2d在肺、大脑检测到gC表达蛋白;3d到死亡鸭各组织器官均可持续检测到gC表达蛋白存在。5.抗gC蛋白抗体介导的抗原捕获ELISA检测DPV建立和应用以兔抗DPV gC基因原核表达蛋白为第一抗体,以纯化的兔抗DPV IgG为夹心抗体,建立并优化了检测DPV的抗原捕获ELISA方法。结果表明:gC重组蛋白的最佳包被浓度为1.6μg/孔,检测血清的最佳稀释度为1:80,酶标二抗的最佳稀释度是1:1000。本方法敏感、特异、稳定,可应用于临床病料DPV病毒的快速、批量诊断。6.DPV gC蛋白作为包被抗原检测鸭瘟抗体间接ELISA建立和应用以纯化的DPV gC表达蛋白作为包被抗原初步建立了检测鸭瘟抗体水平的间接ELISA法(gC-ELISA),并对该方法进行了标准化研究。用本法(gC-ELISA)和本实验室研制DPV抗体检测试剂盒平行检测56份送检血清,符合率为94%,表明建立的gC ELISA检测方法具有很好的特异性和敏感性,可以用于DPV感染的诊断和疫苗免疫后的抗体监测。7.基于DPV gC基因的PCR检测方法建立和应用根据发现的DPV gC基因序列,建立检测DPV的PCR方法。特异性试验表明,设计引物对正常DEF细胞、鸭胚尿囊液及鸭肝炎病毒、鸭乙型肝炎病毒、小鹅瘟病毒、鸭巴氏杆菌、鸭大肠杆菌、鸭疫里默氏杆菌的核酸提取物进行的PCR的结果为阴性;而对DPV基因组DNA,其灵敏度可达到1pg;应用该PCR方法对DPV感染病料各组织进行检测,均能扩增出与预期大小一致的特异性条带。8.基于鸭瘟病毒gC基因建立的AC-ELISA、免疫组化、免疫荧光和PCR检测方法比较比较AC-ELISA、免疫组化、免疫荧光和PCR对DPV CHv强毒株感染鸭组织和临床病料检测结果的特异性、敏感性。结果发现,四种方法对鸭瘟阳性病料检测结果均为阳性,对正常鸭组织、鸭乙型肝炎病毒(DHBV)、鸭病毒性肝炎(DHV)、鹅细小病毒(GPV)、鸭疫里默氏杆菌(R.A.)、鸭源大肠杆菌(E.coli)、鸭源沙门氏菌(S.anatum)等病毒和细菌基因组的检测结果均为阴性。灵敏度试验显示:PCR方法灵敏度大于ELISA,ELISA的灵敏度大于SP免疫组化和间接免疫荧光。对临床鸭瘟病料检测显示四种方法检测结果基本一致,无显著性差异。更多还原

【Abstract】 Duck plague(DP),also called duck virus enteritis(DVE),is an acute,contagious and lethal septicemic herpesvirus infectionn of ducks,geese,and swans.It had causesed great economical loss in domestic ducks and wild waterfowls.Only few researches in the field of duck plague virus genomics had been performed.According to DPV gC gene sequence discovered by our laboratory,a series of scientific research were conducted and the results were obtained as followed:1.Discovery,Cloning and Molecular Characterization of duck plague virus gC gene According to the duck plague virus(DPV) DNA gene library constructed in our laboratory,A complete open reading frame(ORF1296) discovered was homology to other herpesvirus glycoprotein C gene.Sequence anylysis and Dot-blot hybridization confirmed that the ORF1296 is DPV gC gene(GenBank accession number is EU076811).Bioinformatics analysis showed that DPV gC gene is 1296bp in length,encoded a 432 amino acid.Its molecular weight was 45kD and the isoelectric point(PI) was 5.292.Duck plague virus gC gene is more close genetic relationship to gallid herpesvirus than to other’s gC gene.Amino acids sequcence at positions 21～24,50～58,67～70,161～171,273～281,387～393 maybe the most possible antigenic epitopes.2.Prokaryotic expression,purification and antibody preparation of Duck plague virus gC gene According to the Duck plague virus gC gene sequence found by our laboratory,DPV gC gene was amplified and cloned into pMD18-T vector.It was identified by PCR,restriction endonuclease digestion analysis and DNA sequencing.The gC gene was subcloned into EcoRI and XhoI site of prokaryotic expression vector pET-32a(+) and recombinant expression plasmid pET-32a-gC was constructed successfully.The plasmid was transformed into host bacterium of BL21(DE3) induced with IPTG and analyzed by SDS-PAGE electrophoresis.the expected 65kD fusion protein was expressed.Western Blot test showed that the expressed fusion protein reacted to DPV serum was positive.Then rabbits were immuned with the extracted,purified pET32a-gC expressed products.The rabbit-anti DPV gC protein polyclonal antibodies were purified and proved to have good immunogenicity.3.Imunogenicity of Duck plague virus gC gene prokaryotie products The DPV gC prokaryotic expression products were purified,renatured and prepared as vaccine with Freund’S adjuvant to ducklings.The results showed that recombinant protein can induce neutralization antibody anti DPV,its titer reached the maximum 21 days after immuned in accordance with attenuated vaccine group and its variation law of antibody titers detected by ELISA was coincided with attenuated vaccine group.There was no difference in the immune protective efficiency between the recombinant protein groups and attenuated vaccine group.All shows that DPV gC gene recombinant protein has certain protective capacity to virulent DPV challage.4.Development and aplication a SP assay and an indirect immunofluorescence assay to detect Duck Plague Virus gC protein Rabbit anti-DPV gC IgG purified by ammonium sulfate precipitation method and High Q anion exchange column chromatography were used as the first antibody,a streptavidin-peroxidase conjugated method and an indirect immunofluorescence assay was established respectively.DPV antigens distribute,dynamic distribution in spleen,thymus, brain,bursa of Fabricius,liver,lung,kidney and intestine challenged DPV CHv strain and expression kintics of DPV gC gene were researched by the two methods.The results showed that DPV was one kind of pantropic virus,spreaded through blood to all kinds of tissues.The expression kintics was that 8 hour after challenge 12h,live,esophagus,glandular stomach were dectected gC protein expression.24h after infected,bursa of Fabricius,kidney and spleen were dectected gC protein;2d 1,gC proteins were dectected in lung and brain and 3d later,expressed protein were dectected in almost infect tiusses.5.Development and application of an AC-ELISA with Anti-Prokaryotie Expression Protein of DPV gC By using recombinant gC expressed protein as the primary antibody and purified Rabbit anti-DPV IgG as the second antibody,an antigen capture ELISA(AC-ELISA) to dectect DPV was developed and optimized.The results showed that the optimal concentration of the recombinant gC protein was dilution of 1:40,Rabbit anti-DPV IgG was dilution of 1:80 and HRP-goat anti rabbit IgG dilution of 1:2000.No cross reaction was observed with DI-IBV、DHV、GPV、R.A.,E.coli,S.anatum and the minimum DPV content detection is 30 ng DPV.In the intro-batch duplicativity test and in the inter-batch duplicativity test,the variation coefficient is less than 10%.6.Establishment and Aplication of an Indirect ELISA Diagnose with the DPV gC expresssion protein An indirect ELISA for detecting antibody of duck plague was developed using recombinant gC expressed in E.coli as coating antigen.The optimum conditions for the gC-ELISA were determined.The results showed that the concentration of the recombinant gC protein was 1.6μg for coating per well;the optimal dilution of the examined serum is 1:80 and the enzyme linked antibody dilution is 1:1000.The indirect gC-ELISA was specific and sensitive and used in diagnosis of DPV in clinic.7.A PCR method based on DPV gc gene was established to Detect DPV According to the DPV gC gene,a PCR asssy was developed.The specific DNA fragment(about 1112bp) was amplified from DPV DNA,but not from nucleic acid samples of DHV,DHBV,GPV,E.coli, S.anatum,R.A.The specific DNA product was also amplified from the infected livers and brain from 19 samples of DPV.These results suggested that the PCR was a specific and sensitive method which could be used for detection and diagnosis of clinical DPV infection.8.Comparison of the four assays to detect Duck Plague Virus In order to select more rapid,sensitive and specific method in detection of DPV directly from the clinical specimens, tissues infected DPV were detected with AC-ELISA,SP immunohistochemistry,indirect immunofluroscence and PCR.The results showed that the sensitivity of the PCR much higher than the AC-ELISA,and the AC-ELISA is higher than the SP and IF method.The remits to detect clinical samples showed that there were no significant difference in the positive rates among the four methods.更多还原