【作者】 常华；

【导师】 程安春； 汪铭书；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2011， 博士

【摘要】 1.鸭瘟病毒gE基因的序列特征及密码子偏爱性的生物信息学分析本实验室注册的鸭瘟病毒(Duck plague virus, DPV) gE基因全长为1473bp (GenBank登录号为EU071044)。用生物信息学软件分析gE基因及该基因编码的蛋白,结果表明该基因编码490个氨基酸的多肽,含有信号肽切割位点,在整个多肽链中存在21个抗原决定簇、4个潜在的酰基化位点、29个磷酸化位点和6个N-糖基化位点；gE糖蛋白是典型的Ⅰ型膜蛋白,N端由396个氨基酸组成胞外区,中间为23个氨基酸组成跨膜区,C端为71个氨基酸组成胞内区；亚细胞定位分析表明gE糖蛋白主要位于细胞质；系统进化树分析显示DPV gE与α-疱疹病毒亚科中的马立克病毒属(Mardivirus)亲缘关系较近；同时分析了DPV gE基因密码子偏爱性,结果表明DPV gE基因在同义密码子中较偏爱第三位为A和T的密码子,如选择原核表达系统表达该蛋白则要选择宿主表达菌,才能利于DPV gE基因的体外表达。2.鸭瘟病毒gE基因的克隆、原核表达及其多克隆抗体的制备用Primer Premier5.0软件设计一对鸭瘟病毒(DPV)gE基因序列的特异性引物,采用PCR方法从鸭瘟病毒基因组DNA中扩增gE基因,将gE基因片段克隆至pMD18-T载体中,用双酶切(EcoR I和Xho I)方法和DNA测序鉴定正确后,命名为pMD18-T-gE。并采用酶切的方法将gE基因正向插入原核表达载体pET32a(+) (EcoR I和XhoI位点间),成功构建了重组表达质粒pET32a-gE。将该重组表达质粒转化到表达宿主菌BL21(DE3)、BL21(pLysS)和Rosseta中,用IPTG诱导,经过对表达宿主菌、诱导温度、诱导时间的优化,确定了该重组表达质粒的最佳诱导条件为在表达宿主菌Rosseta中用0.2mmol/L IPTG,30℃条件下诱导4.5h,表达出了大小约为74KD的重组蛋白pET32a/DPV-gE,且主要以包涵体形式存在。将表达产物用包涵体洗涤方法纯化后,高纯度的表达蛋白与等量弗氏佐剂混合作为免疫原,经过四次免疫家兔,获得了兔抗重组蛋白pET32a/DPV-gE的多克隆抗体。血清用饱和硫酸铵法粗提IgG,并经High Q阴离子交换柱层析纯化后,得到了特异性强的兔抗重组蛋白pET32a/DPV-gE的抗体IgG。3.鸭瘟病毒gE蛋白在病毒感染宿主细胞中的定位本研究将纯化的兔抗重组蛋白pET32a/DPV gE IgG作为一抗,用间接免疫荧光技术检测DPV gE蛋白在病毒感染鸭胚成纤维细胞内的定位,结果显示早在感染DPV 5.5 h后,细胞质中检测到特异性荧光点,在感染后9-24h荧光强度增强,在感染36h时绿色荧光广泛分布在细胞浆中,之后随着细胞病变在48 h时胞浆中的绿色荧光开始减弱,且一些绿色荧光聚集且靠近核区域,60h细胞病变脱落形成空斑,此时绿色荧光减少且减弱。4.鸭瘟病毒gE基因在感染宿主细胞中的转录和表达特征本研究应用实时荧光定量PCR方法和Western blotting检测DPV感染鸭胚成纤维细胞后DPV gE基因的转录和表达情况。结果表明gE基因在DPV感染后4h时开始转录,8h检测到表达,在感染36h后转录和表达量最高,随后逐渐降低。且DPV gE基因在鸭胚成纤维细胞中表达产物的分子量约为54KD。5.利用间接免疫酶组化法(IPA)检测DPV gE蛋白在感染鸭组织中的分布规律本研究将30日龄鸭人工感染DPV强毒,在感染病毒后于不同的时间点采集不同的器官或组织,并制备切片,用兔抗重组蛋白pET32a/DPV-gE IgG作为一抗,用间接免疫酶组化法(IPA)检测DPV gE蛋白在感染鸭组织中的分布规律。在感染后6h DPV gE蛋白在免疫器官(胸腺、法氏囊、脾)中被检测到,在感染后8h-12h检测到DPV gE蛋白分布在哈德氏腺、食道、腺胃、肝和肠道,且随感染时间增加阳性信号也增强,在感染后24h-48h在肾、肺、心、脑也检测到DPV gE蛋白。6.用间接免疫荧光方法(IFA)检测DPV gE蛋白在感染鸭组织中的分布将30日龄鸭人工感染DPV强毒,在感染病毒后于不同的时间点采集不同的器官或组织,用兔抗重组蛋白pET32a/DPV-gE IgG作为一抗,经建立优化的间接免疫荧光(IFA)方法检测DPV gE蛋白在鸭体组织中的分布。结果显示感染鸭瘟病毒后DPV gE蛋白分布在免疫器官(脾脏、法氏囊、胸腺、哈德氏腺)、消化器官(肝脏、肠道、食管、腺胃)及实质器官(肾、心、脑及肺)。在感染后4h, DPV gE蛋白首先在脾脏和法氏囊中检测到,随后在感染后8h在哈氏腺、胸腺、肝脏和肠道也检测gE蛋白,在感染后12h在肾、心、肺和脑中也开始检测到弱阳性信号,且随感染时间从12h到216h阳性信号逐渐增多。7.基于重组蛋白pET32a/DPV-gE的间接ELISA法检测鸭瘟病毒抗体的建立及应用本研究基于纯化的重组蛋白pET32a/DPV-gE建立了间接ELISA方法检测DPV血清抗体。重组蛋白pET32a/DPV-gE最佳包被稀释度为1:100(2ug/100ul),最佳酶标二抗的稀释度为1：1000,最佳血清稀释度为1：160。用建立的DPV-gE-ELISA方法检测鸭沙门氏菌(Salmonella anatum, S.anatum)、鸭大肠杆菌(E.coli)、鸭疫里默氏菌(RA)阳性血清和鸭病毒性肝炎病毒(DHV),结果均显示阴性；批内或批间重复试验的变异系数均小于10%；可以检测出经1：1280倍稀释的DPV阳性血清。用DPV-gE-ELISA与本实验室已建立的包被DPV全病毒ELISA法(DPV-ELISA)对55份地方鸭血清进行检测,结果显示,与DPV-ELISA的符合率为87.27%。更多还原

【Abstract】 1. Bioinformation analysis of sequence characteristics and codon bias of duck plague virus gE gene. DPV gE gene was 1473bp, and the GenBank accession number was EU071044. The characteristics of the protein encoding DPV gE gene were analyzed by the bioinformatics software, the results showed that DPV gE gene was encoded a protein comprising 490 amino acids, which contained an N-terminal signal peptide,21 antigenic determinants,4 potential palmitoylation sites,29 phosphorylation sites, and 6 glycosylation sites. DPV gE was a type-I membrane protein that could be resolved into 3 distinct functional domains:a 396-amino-acid extracellular domain, a 23-amino-acid hydrophobic transmembrane domain, and a 71-amino-acid cytoplasmic domain. Subcellular location analysis demonstrated that gE mainly located in the cytoplasm, and the phylogenetic tree analysis showed that DPV gE was evolutionarily closer to the mardivirus genus of the Alphaherpesvirinae subfamily. Meanwhile, the codons usage bias analysis revealed that DPV gE was strong bias towards the synonymous codons with A and T at the third codon position. And we should choose the host bacteria, which should impove the expression of the exogenous genes, if gE was expressed by using the prokaryotic expression system.2. Cloning, prokaryotic expression and polyclonal antibody preparation of DPV gE gene. The primers were designed based on the sequence of DPV gE by Primer Premier 5.0. DPV gE gene was amplified from the genome of DPV by PCR, and cloned into pMD18-T vector, which was identified by restriction enzymes digestion (EcoR I and Xho I) and sequenced. The correct recombinant vector was named pMD18-T-gE. Then gE gene from the pMD18-T-gE vector with two restriction enzymes digestion was subcloned into the prokaryotic expression vector pET-32a (+) to generate the recombinant plasmid pET32-gE. And the recombinant plasmid pET32-gE was transformed into E. coli BL21(DE3), BL21(pLysS) and Rosseta strain, expressed by IPTG (isopropylβ-D-thiogalactopyranoside) induction, and the analysis of the expression conditions was optimized, which contained the expressed host strans、the different induction time and temperature. The result showed that the optimal condition was 0.2mmol/L IPTG as inductor, duration of 4.5 hours at 30℃, and SDS-PAGE analysis showed that the induced expressed protein is about 74 KD, and the recombinant protein was mostly existed in inclusion examined by soluble analysis, and purified by washing inclusion body, and used to immunize rabbits for the preparation of polyclonal antibody, which was subsequently obtained by using ammonium sulfate precipitation and High-Q anion-exchange chromatography, and the pET32a/DPV-gE antiserum had a high level of specificity.3. Cellular localization of DPV gE protein in DPV-infected cells. The cellular localization of DPV gE protein was tested with the anti-DPV polyclonal IgG as the first antibody by indirect immunofluorescence analysis. The result showed that specific fluorescence was first appeared in cytoplasm at 5.5h PI (post infection), and the fluorescence was stronger from 9 to 24h PI gradually. At 36h PI, these fluorescence granules was detected widely distributed in the cytoplasm, and became more bigger and brighter. The gE-specific fluorescence was gradually diminished at 48h PI with the cytopathic effect (CPE), and a deal of the specific fluorescence was concentrated in the juxtanuclear region. Then at 60h PI, the gE-specific fluorescence was sparser and weaker following the cytoplasm disintegration in infected cells, when the DEFs cells were exfoliated and the plaques size become bigger.4. The transcription and expression characteristics of DPV gE gene in DPV-infected host cells. The transcription and expression characteristics of DPV gE gene were determined by real-time quantitative PCR and western blotting. These results revealed that the transcripts of DPV gE were appeared at 4 h post infection (PI), and its expression products were detected at 8h PI, and the transcripts and expression products were up to a peak at 36h PI, thereafter both of them were reduced. And the molecular weight of expression products was approximate 54 KD in lysates of DPV-infected cells.5. The distribution of DPV gE protein in experimentally DPV-infected ducks detected by indirect immunoperoxidase assay (IPA).30-day-old ducks were intramuscularly inoculated with DPV CHv strain, and the different tissues were collected from DPV-infected ducks at sequential time points. And the tissues were tested with the polyclonal pET32a/DPV-gE IgG as the first antibody by indirect immunoperoxidase assay (IPA). The result showed that DPV gE was first detected in the immunological organs (Bursa of Fabricius, thymus and spleen) at 6h PI (post infection), then it appeared in the Harders glands, macrophages, glandularis ventriculus, liver and intestine at 8h-12h PI, and the intensity of positive staining in various tissues increased steadily, finally, in the kidney, lung, myocardium and cerebrum at 24h-48h PI.6. The distribution of DPV gE protein in experimentally DPV-infected ducks detected by indirect immunofluorescence assay (IFA).30-day-old ducks were intramuscularly inoculated with DPV CHv strain, and the different tissues were collected from DPV-infected ducks at sequential time points. And the distribution of DPV gE on DPV-infected ducks was detected with the polyclonal pET32a/DPV-gE IgG as the first antibody by indirect immunofluorescence assay (IFA). DPV gE was distributed in the immunological organs (spleen, Bursa of Fabricius, thymus, Harders glands), digestive organs (liver, intestine, esophagus, glandularis ventriculus) and other parenchymatous organ (kidney, myocardium, cerebrum, lung). And DPV gE was first seen in the BF and spleen at 4h PI (post infection), then it was detected in Harderian gland, thymus, liver and the intestine at 8h PI, and it was shown in the kidney, lung, myocardium, and cerebrum 12h PI. And the positive fluorescent signals increased from 12h to 216h PI.7. Development and application of an indirect ELISA based on the recombinant protein pET32a/DPV-gE for detecting the antibody against duck plague virus. Based on the purified recombinant protein pET32a/DPV-gE, DPV-gE-ELISA was developed for detecting the DPV serum antibodies. And the optimum conditions for DPV-gE-ELISA were determined, and the results showed that the optimized concentration of the recombinant protein pET32a/DPV-gE is l:100(2μ.g/100μl), the enzyme linked antibody dilution is 1:1000, and the dilution of the examined serum is 1:160. Duck S.anatum, duck E.coli, duck riemirella anatipestifer (RA) and duck hepatitis virus (DHV) were employed as negative controls, and detected by DPV-gE-ELISA, and the result was negative. The coefficients of intra-assay and inter-assay variation were less than 10%, and DPV-gE-ELISA could detect DPV positive antiserum with a dilution of 1:1280. To evaluate the effect of the DPV-gE-ELISA,55 duck serum samples collected from several duck flocks were simultaneously tested by the DPV-gE-ELISA and the whole DPV antigen as coated antigen ELISA method (DPV-ELISA). The results revealed that DPV-gE-ELISA have higher coincidence compared with the DPV-ELISA (about 87.27%).更多还原