【作者】 宋德光；

【导师】 高丰；

【作者基本信息】 吉林大学 ， 基础兽医学， 2008， 博士

【摘要】 水泡性口炎病毒(Vesicular Stomatitis Virus, VSV)是引起多种动物水泡性口炎的一种重要人兽共患病病原,属于弹状病毒科的成员。VSV对马、牛、猪特别易感,羊、山羊、多种野生动物和人均有易感性。水泡性口炎与口蹄疫、猪水泡病、猪水泡疹的临床症状十分相似,以口腔黏膜、舌、唇、乳头和蹄冠部上皮出现水泡为主要特征。VSV对不同动物致病性的共同特征为具有嗜上皮性,根据这一特性开展该病毒的致病机制研究,对于水泡性口炎疾病的防治意义重大。本研究以犊牛口腔黏膜上皮为材料,分离培养犊牛原代口腔黏膜上皮细胞,经形态学观察、细胞贴壁率和生长曲线计算、流式细胞仪检测等系统鉴定,确定已成功建立了一种原代犊牛口腔黏膜上皮细胞培养方法,并获得高纯度的原代上皮细胞,为研究具有嗜上皮性病毒的侵入机制和细胞cDNA文库构建提供了良好的实验体系。将VSV接种于犊牛口腔黏膜上皮细胞,应用超薄切片技术进行病毒的形态发生和细胞超微结构变化特征的观察,通过接毒细胞出现病变和电镜观察到弹状病毒样粒子,证实VSV可感染原代培养的犊牛口腔黏膜上皮细胞,并能繁殖扩增病毒。通过超微结构观察,初步明确VSV对该细胞的侵袭过程,且病毒通过细胞膜和胞浆内的空泡膜上出芽增殖后获得囊膜而成熟。将原代细胞大量培养增殖后,提取细胞的总RNA后分离其mRNA,利用噬菌体表面展示技术,构建了对VSV具有高反应性的CPMBCs高质量cDNA表达文库。未扩增文库的滴度为1.3×107 pfu/mL,文库重组率为95.8%,扩增后文库滴度为2.4×1010 pfu/mL。为高通量地从库中筛选出与VSV相互作用的噬菌体重组子,即寻找该病毒相互作用受体蛋白奠定了坚实的基础。用纯化的VSV与构建的T7噬菌体展示文库进行文库筛选,获得一个VSV可能的受体蛋白基因功能区片段。该基因片段长度为801bp,含有一大小为375bp的开放阅读框,编码124个氨基酸残基,将其命名为CPMBCsCP1。测序结果经登陆NCBI BLAST核酸同源性检索为与人类的剪接因子3b的同源性较高。DNAStar软件和ScanProsite分析结果表明是CPMBCsCP1蛋白亲水性较好,具有免疫原性和良好的抗原性,其生物学活性有可能在细胞信号传导通路中接受多种信号的调控。该项研究不仅为VSV由其受体介导的分子致病机制研究奠定坚实的基础,而且将为我国针对VSV引发疾病的防治研究提供参考依据。更多还原

【Abstract】 Vesicular stomatitis(VS) is an acute, febrile and highly contagious infectious disease in cattle and in human which caused by vesicular stomatitis virus(VSV). The disease is characterized by vesic in the epithelium of oral mucosa, tongue, lip, papillae and coronet. The disease first occurred in the middle of United States and North America in 1821, then spreading to some countries and areas of South America, Africa, Europe and Asia. Since 1995, outbreaks of VSV had been reported in the western of United States one after another. VSV infection is frequently occurred in cattle, horses, pigs, deer and so on, but human could be infected occasionally. The mainly clinical sign of VSV infection is acute fever which is similar to Flu and Aden fever. In the summer of 2002, outbreaks of VSV were reported in Jilin province and Altay area in Xinjiang province of China. Although the infection spectrum of VSV is broad, the tropism epithelium is a common characteristics for different infected animals. However, up to now, the mechanism of tropism epithelial of VSV is not clear.In this study, we first cultured calf primary oral mucosal epithelial cells in vitro. The result indicated that we had successfully established a high-grade cultural method of calf primary oral mucosal epithelial cells (CPMBCs) by the observation of morphology, optical microscopy, ultrastructure using scanning electron microscopy and transmission electron microscopy, the identification of cytokeratin by immunohistochemistry and the examination of flow cytometry. This would establish foundation for subsequent research. We observed the morphogenesis of VSV and the changed characteristic of cellular ultrastructure using ultrathin section by collecting cells in different time after infection. The result indicated that the CPMBCs appeared cytopathic effect(CPE) after infection of VSV. We observed virions like bullet by negative staining. The result confirmed that VSV could infect primary CPMBCs. We had initially identified the process of VSV infecting CPMBCs by the observation of cellular ultrastructure. We considered that the maturity of VSV was by pullulation proliferation in cellular membrane and cytoplasm.The CPMBCs produced typical CPE after infection of VSV, so we supposed that the surface of CPMBCs should exist receptors of VSV. In order to more overall sieve the receptors of VSV, we first established T7 phage display library of CPMBCs in the research. The data showed that the library contained 1.3×107 clones, and approximately 95.8% of the library were recombinant. The titer of the amplied library was 2.4×1010 pfu/mL. The cDNA fragments longer than 300bp in length were 93% by using the PCR identification. The VSV virions after purification by sucrose gradient centrifugation were used to sieve receptors. We coated 96 ELISA plate with purificated virions, then added amplified library to carry out sieving receptors. We carried out five series sieves. At last we spread on the plate using the supernatant of the last serie. We picked out negative colonies, then extracted genome DNA from them. The cDNA inserts in these plaques were amplified by PCR using the primers. The phage DNA were sequenced , and the nucleotides were compared with the Genbank database by BLAST searches. Amino acid sequences were analyzed through ExPASy Proteomics Server and DNAStar software. About 100 clones were randomly picked from individual plaques, and their DNA sequences were amplified by PCR and analysed on agarose gel to determine the size of the inserts. The result of PCR analysis showed that 34% of the phage clones had an insert size of 400bp or longer. The sequence showed that the insert was 801bp in length and contained a 375bp open reading frame(ORF), which was predicted to encode 124 amino acids. The protein was designated CPMBCsCP1 and high homology appears between CPMBCsCP1 and the human splicing factor of 3b. The analytic result of CPMBCsCP1 protein had the characteristic of hydrophilicity, immunogenicity and good antigenicity, and biologic activity of the protein might be regulation by a variety of signals in the transmission pathway of cellular signals.This study will not only establish a foundation for the study of VSV infectious mechanism mediated by receptors, but also provide certain theory basis for the research of prevention and cure of VSV infection in our country.更多还原