新型鸭细小病毒DS-15株生物学特性研究及间接ELISA方法的建立

发布时间：2018-11-27 13:20

【摘要】：2014年以来,在我国许多樱桃谷肉鸭养殖地区广泛流行一种“鸭大舌病”,其临床症状主要表现为鸭生长发育迟缓,鸭喙萎缩舌头外露下垂,跛行瘫痪等,又称鸭短喙-侏儒综合征。已经鉴定出该病的病原是一种新型鸭细小病毒(Novel duck parvovirus,NDPV)。目前对此病毒的生物学特性暂不清楚,临床上还没有有效的疫苗或药物行防治,也没有检测该病毒的方法或技术。为此,本研究对NDPV DS-15株的生物学特性进行了研究,同时为了给该病的流行病学调查提供一个可靠的检测方法,我们还对NDPV的主要抗原蛋白VP3进行了表达和纯化,并在此基础上建立了检测NDPV抗体的ELISA方法。首先,我们对新型鸭细小病毒DS-15株在鸭胚上的增殖特性及对鸭胚的致病力进行研究。试验结果显示,鸭胚接种NDPV DS-15株以后,96~120 h出现死亡,胚体全身出血,病毒滴度(ELD50)为10-5.5/0.2 mL。以鸭胚增殖的病毒感染樱桃谷肉鸭,成功复制出典型的“鸭大舌病”临床症状,并从发病鸭脏器中分离到病毒。其次,根据NDPV DS-15株的基因序列设计PCR引物扩增VP3基因并将其序列与国内外报道的部分GPV及MDPV分离株的VP3基因进行比对和分析。结果表明,NDPV DS-15株VP3基因与鹅细小病毒(Goose parvovirus,GPV)之间的核苷酸和氨基酸同源性分别为91.4%~98.4%和96.6%~98.1%,而与番鸭细小病毒(Muscovy duck parvovirus,MDPV)核苷酸和氨基酸同源性则较低,分别为81.4%~91.5%和91%~97%;在亲缘关系方面,NDPV DS-15株与GPV较近,提示二者可能是由同一病毒进化而来。最后,我们将NDPV的VP3基因克隆并插入到pGEX-4T-1中构建重组表达质粒pGEX-4T-VP3,诱导表达重组GST-VP3蛋白,重组蛋白分子量为84kDa。Western blot方法检测结果显示重组VP3蛋白能与NDPV多克隆抗体发生反应,重组VP3蛋白具备用作诊断抗原的潜力。以表达的VP3蛋白作为抗原,包被ELISA板;然后,分别对包被抗原的浓度及条件、抗原封闭条件、一抗的孵育条件、酶标二抗的孵育条件和显色条件等进行优化,最终成功建立间接ELISA方法,确定方法的阳性阈值为0.35。所建立的方法有较好的重复性,运用该方法对临床样本进行了初步检测,结果表明该方法可用于NDPV疫苗的免疫效力评价,及NDPV感染鸭群的流行病学调查,同时也可用于鸭群GPV的检测。综上,本论文对NDPV的增殖特性及致病特性进行了初步研究,推测新型鸭细小病毒与鹅细小病毒可能是从同一病毒祖先进化而来。此外,我们建立了检测NDPV抗体的间接ELISA方法,为开展NDPV的疫苗研发和流行病学调查等提供了可靠的检测方法。
[Abstract]:Since 2014, a kind of "duck hyoid disease" has been prevalent in many cherry valley meat duck breeding areas in China. Its clinical symptoms mainly include duck growth retardation, duck beak atrophy tongue protruding, claudication paralysis and so on. Also known as duck short beak-dwarf syndrome. The pathogen of the disease has been identified as a new type of duck parvovirus (Novel duck parvovirus,NDPV). At present, the biological characteristics of the virus are not clear, there is no effective vaccine or drugs to prevent and cure the virus, and there is no method or technology to detect the virus. In order to provide a reliable method for the epidemiological investigation of NDPV DS-15, we also expressed and purified VP3, the main antigen protein of NDPV. On this basis, a ELISA method for detection of NDPV antibody was established. Firstly, we studied the proliferation and pathogenicity of a novel duck parvovirus DS-15 strain on duck embryos. The results showed that the duck embryo died at 96120 h after inoculation with NDPV DS-15 strain, the embryo body was bleeding and the virus titer (ELD50) was 10-5.5 / 0.2 mL.. The typical clinical symptoms of "duck hyoid disease" were successfully duplicated and the virus was isolated from the infected duck organs after infecting Cherry Valley Meat Duck with duck embryo proliferating virus. Secondly, according to the gene sequence of NDPV DS-15 strain, PCR primers were designed to amplify the VP3 gene, and the VP3 gene was compared and analyzed with the VP3 gene of some GPV and MDPV isolates reported at home and abroad. The results showed that the nucleotide and amino acid homology between VP3 gene of NDPV DS-15 strain and goose parvovirus (Goose parvovirus,GPV) was 91.4% and 96.698. 1%, respectively, and 91.4% and 96. 6% with muscovy duck parvovirus (Muscovy duck parvovirus,), respectively. The nucleotide and amino acid homology of MDPV was 81.4% and 91.5%, respectively. The close relationship between NDPV DS-15 strain and GPV suggests that the two strains may have evolved from the same virus. Finally, we cloned the VP3 gene of NDPV and inserted it into pGEX-4T-1 to construct the recombinant expression plasmid pGEX-4T-VP3, to induce the expression of recombinant GST-VP3 protein. The molecular weight of the recombinant protein was determined by 84kDa.Western blot method. The results showed that the recombinant VP3 protein could react with NDPV polyclonal antibody, and the recombinant VP3 protein had the potential to be used as diagnostic antigen. The expressed VP3 protein was used as antigen and coated with ELISA plate. Then, the concentration and condition of coated antigen, the condition of antigen sealing, the incubation condition of first antibody, the incubation condition of enzyme labeled second antibody and the condition of color development were optimized, and the indirect ELISA method was established successfully. The positive threshold of the method is 0.35. The results showed that the method could be used to evaluate the immune efficacy of NDPV vaccine and to investigate the epidemiology of NDPV infected ducks. It can also be used for the detection of GPV in ducks. In summary, the proliferation and pathogenicity of NDPV were studied preliminarily, and it was inferred that the new duck parvovirus and goose parvovirus might have evolved from the same virus ancestor. In addition, we established an indirect ELISA method for the detection of NDPV antibodies, which provided a reliable method for the development of NDPV vaccine and epidemiological investigation.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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