山羊副流感病毒3型分离、鉴定及其核衣壳蛋白的原核表达

发布时间：2018-02-10 16:35

本文关键词： 山羊副流感病毒3型分离鉴定原核表达豚鼠致病性　出处：《南京农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：副流感病毒3型(Parainfluenza virus type 3,PIV3)是引起人和动物呼吸道疾病的重要病原。主要有牛PIV3(BPIV3)和人PIV3(HPIV3),BPIV3可引起牛严重的呼吸道疾病,并有较高的发病率和致死率,对养牛业造成巨大的损失。但PIV3在羊群中的感染情况在国内外都鲜有研究和报道。2013年下半年起江苏、安徽多地山羊养殖场育肥山羊陆续出现以呼吸道症状为主的疾病,临床表现较以往严重,传统药物治疗的治愈率低,向我们提示了病原的特殊性。本研究将采集自表现为严重呼吸道症状的山羊临床病料处理后接种MDBK细胞,连续传代分离到病毒,进一步通过病毒蚀斑纯化实验纯化病毒,经RT-PCR和血凝性鉴定证明病毒分离成功。通过对其基因测序及病原学特性研究证实其不同于其他PIV3,是PIV3新成员,将该毒株命名为山羊副流感病毒 3 型(Caprine parainfluenza virus 3,CPIV3)JS2013 株。为了进一步探究新分离得到的CPIV3JS2013株的致病性,本试验采用PIV3易感动物豚鼠研究此病毒在动物体内的复制及机体组织病理损伤等情况。攻毒后2天攻毒组的豚鼠开始出现打喷嚏、流鼻涕、眼分泌物增多及腹式呼吸的临床症状,实验过程中出现较高的致死率。攻毒后3天可以检出病毒血症,持续到攻毒后第7天,攻毒后第3天开始直到第14天均可以从肺组织中检测到病毒。剖检观察攻毒组豚鼠,豚鼠肺出现实变、淤血、肿大,病理组织学检测发现攻毒组豚鼠肺组织出现肺泡间隔增宽、肺泡融合、炎性细胞浸润等变化。HI试验表明豚鼠感染后第5天开始出现血凝抑制抗体,之后持续升高至21天仍保持较高的水平。以上结果表明CPIV3JS2013株对豚鼠具有较强的致病性,且豚鼠是一种研究CPIV3较为理想的实验动物。以本试验分离的CPIV3 JS2013株纯化的第4代病毒液为样本,提取总RNA,根据GenBank上已发表的各PIV3成员的全基因组序列,设计1对特异性引物扩增CPIV3 JS2013株N基因的部分片段。将其克隆到pMD18-T载体上,筛选并鉴定出阳性重组质粒PMD18-T-N,再将其克隆到原核表达载体质粒pET-32a(+)上,经菌液PCR、双酶切、测序鉴定出阳性重组质粒pET-32a-N,将其转化到大肠杆菌BL21感受态细胞,经IPTG诱导后,表达产物经SDS-PAGE电泳检测,表明CPIV3 JS2013株N基因获得高效表达。Western-blot检测表明本试验表达的重组N蛋白具有良好的反应原性。将该蛋白免疫小鼠制备多克隆抗体,IFA检测显示良好的效果。以上结果为今后建立CPIV3的诊断方法和疫苗的研制奠定了基础。
[Abstract]:Parainfluenza virus type 3 (Parainfluenza virus 3 type, PIV3) is an important pathogen of human and animal respiratory diseases. There are cattle PIV3 (BPIV3) and PIV3 (HPIV3), BPIV3 can cause serious respiratory disease of cattle, and have high morbidity and mortality, causing huge losses to the cattle industry but PIV3 infection in sheep at home and abroad are few reports on.2013 in the second half of Jiangsu, Anhui and more goat farm fattening goat appeared in respiratory tract disease, the clinical manifestations of severe than in the past, the traditional drug treatment the cure rate is low, suggesting that the particularity of the pathogen to us. This study showed severe respiratory symptoms collected from goat clinical samples after treatment were inoculated with MDBK cells, continuous passage of virus, virus plaque purification by further purification of the virus by RT-PCR and hemagglutination test, identification card The virus isolation success. By studying the characteristics of gene sequencing and confirmed that the pathogen is different from other PIV3, is a new member of PIV3, the virus named goat parainfluenza virus type 3 (Caprine parainfluenza 3 virus, CPIV3) JS2013 strains of pathogenic CPIV3JS2013 strains. In order to further explore the new isolated, this test the PIV3 susceptible animal study of this virus in guinea pig pathological injury situation and animal tissues in vivo replication. The toxic group attack 2 days after infection of guinea pigs began sneezing, runny nose, eye secretions and clinical symptoms of abdominal breathing have higher mortality during the experiment. After challenge 3 days can be detected in viremia, until seventh days after virus challenge, challenge third days after fourteenth days until we can detected the virus from lung tissue. Necropsy observation group of guinea pigs challenged guinea pig lung, real change, congestion, swelling, Histopathological detection of toxic groups of guinea pig lung tissue showed widened alveolar septum, alveolar fusion, inflammatory cell infiltration and other changes of.HI test showed that guinea pigs appeared fifth days after hemagglutination inhibition antibody continued to increase after 21 days still maintain higher level. These results showed that CPIV3JS2013 strains of guinea pigs with strong pathogenicity. The guinea pig is an experimental animal study of CPIV3 ideal. In this experiment. CPIV3 JS2013 strain of the fourth generation of purified virus liquid samples, extracted the total RNA, according to the whole genome sequence published in GenBank by PIV3 members, designed to amplify a fragment of the N gene of CPIV3 JS2013 strain 1 pairs of specific primers. It was cloned into the pMD18-T vector, screening and identification of positive recombinant plasmid PMD18-T-N, then cloned into prokaryotic expression vector pET-32a (+), the bacterium PCR, double enzyme digestion, sequencing Yang The recombinant plasmid pET-32a-N and transformed into Escherichia coli BL21 competent cells after induced by IPTG, the expression products were analyzed by SDS-PAGE showed that CPIV3 JS2013 strain N gene was expressed in.Western-blot test showed that the test expression of recombinant N protein has good immunogenicity. The response of mice immunized with polyclonal IFA antibody detection showed good results. These results laid the foundation for the development of diagnostic methods and the establishment of the CPIV3 vaccine.

【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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