【作者】 程振涛；

【导师】 李永明； 许乐仁；

【作者基本信息】 贵州大学 ， 动物学， 2009， 博士

【摘要】 山羊痘是由羊痘病毒属（Capripoxvirus）山羊痘病毒（Goat poxvirus,GPV）引起的一种高度接触性传染病,临床上以体温升高,全身皮肤、呼吸道和消化道黏膜出现痘疹为主要特征。本病被世界动物卫生组织（OIE）列为A类重大动物传染病,我国也将其列为一类动物疫病。2002年10月以来,贵州省羊群中首次暴发疑似山羊痘病例,并迅速波及到8个养羊较为集中的地区,对我省养羊业的健康发展构成了巨大的威胁。为了防制本病,本研究对我省两株山羊痘病毒分离株进行了某些生物学特性及其主要结构蛋白P32基因进行了详细研究。将贵州省山羊痘病毒分离株GPV-LD和GPV-QL接种Vero-E6、BHK-21细胞,3～7d感染细胞显现圆缩、聚集成簇等细胞病变效应;以GPV荧光抗体对感染细胞飞片染色,在细胞浆中检测到特异性的黄绿色荧光;取感染细胞超薄切片进行电子显微镜观察,细胞浆中发现大量成熟和未成熟的痘病毒颗粒;将感染细胞培养物提取总DNA样本,采用针对P32结构蛋白基因和ITR非编码区基因的两对引物进行PCR鉴定,分别扩增出969bp和289bp的DNA片段,对目的DNA片段的测序结果证实感染细胞培养物中存在GPV特异性的病原核酸;采用2000TCID₅₀的病毒感染细胞培养物通过皮下接种3月龄健康山羊,在14～45d成功复制出与山羊痘自然病例相类似的临床症状和剖检病变,并从感染组织材料中回收到接种的病毒。对GPV结构蛋白P32基因和非编码区ITR基因的两对引物进行比较,以期开展临床山羊痘病例的定性PCR检测。研究结果表明两对引物具有羊痘病毒属特异性,不与副痘病毒属羊传染性脓疱病毒、禽痘病毒属鸡痘病毒、健康山羊皮肤样本发生交叉反应。贵州分离毒P32基因和ITR基因与国内外参考毒株的核苷酸同源性分别达99.5%～100%和100%;PCR最小检出量分别为19.06ng和24.40ng。为此,ITR基因引物更适合于临床山羊痘病例的诊断,而P32基因则有利于病毒囊膜表面蛋白的深入研究。根据GPV参考毒株的全基因序列,针对gp064基因分别设计与合成了TaqMan-MGB探针和引物,应用实时荧光PCR方法开展对山羊痘病例的定量检测。一般PCR方法只能检测患羊出现痘疹病变的皮肤和黏膜材料,而FQ-PCR方法则能够从患羊鼻腔拭子、血液样本、皮肤、肺、胃、淋巴结等组织材料中检出GPV病原核酸。按照本试验构建的标准曲线,TaqMan-MGB-FQ-PCR方法的检测极限为0.1TCID₅₀的病毒量。检测结果表明在人工复制山羊痘病例中,不同组织的含毒量呈现皮肤＞瘤胃＞肺＞淋巴结的趋势;接种山羊从第9～15d形成病毒血症,持续时间较短;鼻拭子中从第7～22d均能检测到病毒的存在,表明患羊痘疹痂皮和鼻分泌物是山羊痘蔓延扩散的主要传染源。应用FQ-PCR方法在皮肤痘疹病变出现之前3～5d即可检测病毒的增殖动态,为山羊痘的早期诊断提供了实用的技术。对山羊痘现场自然病例和人工复制病例进行病理形态学观察,患病羊群的大体病变以皮肤、呼吸道和消化道黏膜出现痘疹为特征;病变部位上皮细胞增生、变性,同时出现巨噬细胞、淋巴细胞和嗜中性白细胞等炎性细胞浸润;受害细胞胞浆内观察到线粒体肿胀、内质网扩张、高尔基复合体扩张甚至破裂。在感染细胞的胞浆中观察到大量成熟和未成熟的痘病毒颗粒,包括形态较大,被膜完整或不完整,呈C形或花瓣状的初期病毒颗粒;在中央或偏中央部位开始形成致密类核体的中期病毒颗粒;和形态较小,有囊膜包裹,中央可见两面凹陷呈哑铃形核酸芯髓的成熟期痘病毒颗粒。将GPV-QL、GPV-LD、GPV-Y和GPV-B毒株的P32基因克隆至pMD18-T载体,重组质粒测序和序列分析显示,GPV贵州分离株之间核苷酸同源性高达99.9%,与国内其它GPV分离株的核苷酸同源性达到99.7%～100%,与国外GPV分离株核苷酸同源性达到99.5%～99.6%。系统发生树分析显示羊痘病毒属中SPV与LSDV之间亲缘关系较近,聚为一类,而它们与GPV亲缘关系较远,后者聚为一类,不同毒株之间表现出明显的种间差异和地域关系。P32蛋白的跨膜结构、亲水性、抗原指数、柔韧性、表面可及性和二级结构分析结果表明,GPV P32蛋白基因在肽段227-251区域存在一个潜在的优势抗原表位。以pMD18-T-P32/LD质粒为模板,扩增P32基因不同长度的片段并克隆至表达载体,构建原核重组表达载体和真核重组表达载体,转化至相应的宿主菌中进行诱导表达。结果表明,P32基因N端1/3片段和中间1/3片段未能通过原核表达载体pET-28a、毕赤酵母真核表达载体pPICZaA在各自宿主菌BL21（DE3）、X-33中获得表达;而P32全基因在两种表达系统中均获得了良好的表达,SDS-PAGE和Western-Blotting分析显示表达蛋白分子量分别为35.5KD和64KD。表达蛋白通过Ni柱亲和层析和Sephadex G-100层析获得纯化,该纯化蛋白在琼脂扩散试验中与山羊痘阳性血清出现特异性的沉淀线,而与阴性血清不发生反应;采用纯化蛋白接种家兔制备免疫血清,该免疫血清使山羊痘病毒感染力减少50%的中和指数达到1:123.07。采用纯化的P32蛋白建立了检测山羊痘血清抗体的间接ELISA方法。应用H.E染色法、凋亡试剂盒检测法、流式细胞仪检测法和DNA Ladder检测法证实GPV能够诱导BHK-21细胞发生凋亡,结果显示,GPV能引起BHK-21细胞凋亡。更多还原

【Abstract】 Goat pox, which is listed in Group A diseases of the OIE and in Group one diseases of China, is a highly contagious viral disease of goats, characterized by fever, ocular and nasal discharges. Pox lesions appear on the skin, the respiratory and gastro-intestinal mucosae. The disease is caused mainly by goat pox virus （GPV） which is one of pox viruses, classified in the genus Capripoxvirus of the family Poxviridae. Since October 2002, the disease firstly outbroke in Guizhou province and spreaded quickly. The disease inflicts the production of goat cultivation in Guizhou province. We have isolated and verified the pathogen of the doubtful goat pox disease in Guizhou province. Then we studied the major structural protein gene P32 of GPV and the pathomorphology of clinical goat pox disease cases and artificial infection cases were observed. The mainly study content are followed.The Vero-E6, BHK-21 cells were infected by GPV-LD and GPV-QL strains , then they showed some cytopathic effects after 3⁷ days, such as getting to round, gathering to clustering. The fluorescent antibody test showed some specific flavovirens fluorescence in the cytoplasm. There were lots of mature and immature particles in the cytoplasm of infected cells under the transmission electron microscope（TEM）. The genus special fragments P32 gene（969bp） and ITR gene（289） were amplified from the DNA samples which were extracted from the infected cell cultures by PCR, the results of sequencing showed the fragments were the special fragments in the GPV genome. The 2000TCID₅₀ GPV-LD strain infected cell cultures were used to infect the 3 months old healthy goats through subcutaneous inoculation. The infected goats showed the same clinical symptom and pathological changes as the clinical cases of goat pox and the goat pox viruses were retrieved from the infected goats.Two specific primers were designed according to the P32 gene and ITR gene sequences of goat poxvirus, the sensitivity and specificity were compared with each other. The results showed the primers have the specificity of Capripoxvirus and do not have consensual reaction with the Orf virus of parapoxvirus, Fowl pox virus of Avipoxvirus and the skin samples of healthy goats. The homologies of nucleotide were above 99.5% and 100% between P32 gene or ITR gene sequences of goat pox viruses. The minimum DNA detection amount of P32 gene primers and ITR gene primers were 19.06ng and 24.40pg. On the whole, the ITR primers are more fit used to diagnose the clinical goat pox disease, and the P32 primers are more fit used to research the virus membrane protein.According to the goat pox virus complete gene sequence, a size of 64 bp gene fragment which locates in gp064 region of goat pox virus （GPV） genome was selected and a pair of primers and a TaqMan-MGB probe against the gene fragment were designed with Primer Express 2.0 software. Then, the fluorescence quantitative PCR （FQ-PCR） assay was developed for quantitative detection of goat pox cases. The FQ-PCR can detect the goat pox virus from the nose swabs, blood samples, skin samples, lung samples, stomach samples, et al., which were selected from GPV infected goats, while the common PCR can detect the GPV only from the skin samples and mucous membrane samples with exanthema variolosum. The minimum DNA detection amount of the FQ-PCR is 0.1 TCID₅₀ according to the standard curve of this test. And the FQ-PCR can detect the exist of GPV before 3～5 days of the skin exanthema variolosum appearance. So the FQ-PCR is a useful technology for the technology of goat pox.The pathomorphology of clinical goat pox cases and artificial infection cases in Guizhou province were observed. The major pathological changes were pox lesions on the skin, respiratory tract and gastro-intestinal tract. And there were hyperplasia and degeneration in epithelial cell and inflammatory cell in diseased regions, such as macrophages, leukomonocyte, leucocyte neutrophils, et al. The cytopathic effects such as cytochondriome tumentia, endocytoplasmic reticulum expansion and Golgi’s complex expansion were also observed under the TEM. A lots of mature and immature particles in the cytoplasm of affected cells were observed under TEM, including initial stage particles, intermediate stage particles and mature particles. The phenomena that GPV could damage the vascular endothelial cells and medullated nerve fibers were finded under TEM. The test results remain that mature particles got the peplos through the cell organ membrane perhaps was one of the way for particles getting the peplos.The P32 genes of GPV-QL, GPV-LD, GPV-Y and GPV-B were inserted into the pMD18-T vector. The sequence analysis of recombinant plasmids showed that the P32 gene of GPV strains isolated from Guizhou shared 99.9% nucleotides with each other, 99.5%～100% with the GPV strains of Chinese and 99.5%-99.6% with the GPV strains of foreign country. Amino acids homology is similar to the nucleotide homology. The Phylogenetic analysis showed the relationship of Guizhou strains were nearer with Chinese other strains than that of foreign strains. The research indicated the P32 gene of capripoxvirus is very conservative and the heredity of capripoxvirus has some relation with the geographic setting. The bioinformatics analysis showed that P32 protein was a kind of membrane protein which had a larvaceous dominant epitope in the 227-251 segment of the amino acid sequence.The different P32 gene fragments were amplified by PCR method from pMD18-T-P32/LD recombinant plasmids. The different P32 gene fragments were cloned into prokaryotic expression vector and eukaryotic expression vector. The proteins were induced to express in their host bacterium and their antigenicity were researched. Then the ELISA for detecting the serum antibody of goat pox were established against the pure P32 protein. The results showed the P32/N_1/3 and P32/M_1/3 gene fragments did not express in both eukaryotic expression system and prokaryotic expression system, while the complete P32 gene expressed well in both eukaryotic expression system and prokaryotic expression system. The results of SDS-PAGE and Western-Blotting showed a specific protein band about 35.5KD in eukaryotic expression products and a specific protein band about 64KD in prokaryotic expression products were detected. The expression protein can be purified by Ni⁺ affinity chromatograph and G-100 chromotography techniques. The agar diffusion reaction and animal experiment identified that the pure P32 protein had good antigenicity. The clinical experiment verified the indirect ELISA assay which made use of the pure P32 protein was useful for detecting the goat pox serum antibody.For detecting the BHK-21 cell apoptosis caused by goat pox virus, the H.E staining method, apoptosis detection kit, flow cytometry and DNA Ladder test were used to detect the BHK-21 cell cultures which infected by the goat pox virus. The results showed that the goat pox virus could cause the BHK-21 cell apoptosis. The research consequences provide some evidences to explain some pathological changes of goat pox.更多还原