犬细小病毒YBYJ株的分离鉴定及VP2基因克隆与多克隆抗体的制备

发布时间：2018-04-13 09:57

本文选题：犬细小病毒 + 分离鉴定　；参考：《延边大学》2015年硕士论文

【摘要】：犬细小病毒病(canine parvovirus infection, CP)是由犬细小病毒(canine parvovirus, CPV)感染而引起犬科及其肉食性经济型动物患病的一种急性接触性传染病。CP临床患病犬主要有两种表现型,即出血性肠炎型和非化脓性心肌炎型,且发病率及致死率均较高,是危害我国犬科类动物健康的主要疫病之一,对犬科类动物的生存构成极大威胁,同时也严重影响我国经济动物养殖业的健康发展。我国专家学者在CP流行病学、CP病原学、CP诊断及CP预防等多方面进行了大量研究,且已经取得阶段性研究进展。为丰富我国CP流行病学调查资料,了解延边地区CPV的遗传进化趋势,进一步深入研究CPV的生物学特性,制备更有针对性的地区性疫苗,本研究采集延边地区疑似感染CPV犬组织病料,将病料预处理后接种F81细胞进行病毒分离试验,并对分离株病毒进行形态学及血清学鉴定，，克隆CPV-VP2基因,分析其遗传进化规律,并构建pGEX-CPV-VP2重组表达质粒,对重组融合表达蛋白进行优化诱导表达及纯化,且经Western-blot分析纯化蛋白反应原性后,将纯化的重组CPV-VP2蛋白免疫BALB/c小鼠,制备鼠抗CPV-VP2蛋白多克隆抗体,采用ELISA方法监测其抗体效价。结果显示,病毒接种F81细胞后,可致细胞变圆、肿胀、脱落等明显细胞病变(CPE)；经IFA鉴定,可观察特异性绿色荧光；回归动物试验可致试验幼犬出现典型CP临床特征,且剖检可见与自然感染相似的病理变化；CPV-VP2基因测序结果经分析后进一步证明,本试验已成功分离到延边地区本地株犬细小病毒,命名为YBYJ株CPV,YBYJ株CPV-VP2基因全长约为1755bp,编码584氨基酸；该分离株病毒与GenBank中收录的国内外47株CPV-VP2基因核苷酸同源性为98.7～99.9%,氨基酸同源性为96.9～99.8%,且与参考株比对后有氨基酸位点变异；经鉴定已成功构建原核表达重组质粒pGEX-CPV-VP2,经IPTG诱导表达,SDS-PAGE分析鉴定表达蛋白约为118 Ku,且以包涵体形式存在；纯化蛋白经Western-blot分析鉴定,具有较好的反应原性；免疫BALB/c小鼠,能够诱导其产生抗CPV-VP2抗体,表明该重组表达蛋白同时具有较好的免疫原性。本研究成果将为下一步CPV基因工程疫苗研制、单克隆抗体制备,以及后期开展CPV相关研究奠定基础。
[Abstract]:Canine parvovirus infection (CPV) is an acute contact infectious disease caused by canine parvovirus (CPV) infection in canine canine parvovirus.CP has two main phenotypes.Hemorrhagic enteritis type and non-suppurative myocarditis type, with high morbidity and mortality, are one of the major diseases that endanger the health of canines in China, and pose a great threat to the survival of canines.At the same time, it also seriously affects the healthy development of China's economic animal husbandry industry.Chinese experts and scholars have made a great deal of research on CP epidemiology, CP etiology, CP diagnosis and CP prevention, and have made progress in stages.In order to enrich the epidemiological investigation data of CP in China, to understand the genetic and evolutionary trend of CPV in Yanbian area, to further study the biological characteristics of CPV, and to prepare a more targeted regional vaccine.In this study, samples of suspected infected CPV canine tissue in Yanbian area were collected and inoculated with F81 cells for virus isolation test after pretreatment. The virus was identified by morphology and serology, CPV-VP2 gene was cloned and its genetic evolution was analyzed.The recombinant pGEX-CPV-VP2 expression plasmid was constructed, and the recombinant fusion protein was optimized for induction and purification. After Western-blot analysis, the purified recombinant CPV-VP2 protein was immunized with BALB/c mice to prepare anti- polyclonal antibody.The antibody titers were detected by ELISA method.The results showed that after inoculation with F81 cells, the cells became round, swelling, exfoliation and other obvious cytopathic changes. The specific green fluorescence could be observed by IFA, and the typical clinical features of CP could be observed in the experimental puppies.The results of CPV-VP2 gene sequencing showed that the local canine parvovirus strains in Yanbian area had been isolated successfully.The total length of CPV-VP2 gene of YBYJ strain was about 1755 BP, encoding 584 amino acids, and the nucleotide homology of CPV-VP2 gene was 98.799. 9, amino acid homology was 96. 9%, and there was amino acid mutation after comparison with reference strain.The prokaryotic expression plasmid pGEX-CPV-VP2was successfully constructed, and the expressed protein was identified to be about 118Kuand existed as inclusion body by IPTG induced SDS-PAGE analysis. The purified protein was identified by Western-blot analysis and had good reactivity. BALB/c mice were immunized with pGEX-CPV-VP2.It can induce the production of anti CPV-VP2 antibody, which indicates that the recombinant protein has good immunogenicity at the same time.The results of this study will lay a foundation for the further development of CPV gene engineering vaccine, the preparation of monoclonal antibody, and the later research on CPV.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.655

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