猪圆环病毒2型CC12毒株感染性克隆构建及小鼠感染模型初步研究

发布时间：2018-03-01 06:27

本文关键词： PCV2 分离鉴定感染性克隆病毒拯救小鼠模型　出处：《吉林大学》2015年硕士论文　论文类型：学位论文

【摘要】：猪圆环病毒病（Porcine circovirus diseases）是由猪圆环病毒2型引起的一种严重危害养猪业发展的新发传染病，以断奶仔猪生长发育不良、消瘦、呼吸困难、皮下淋巴结高度肿大、出血和坏死，母猪繁殖障碍为主要特征。本研究应用PCR扩增病毒基因组序列和免疫学等技术方法确诊长春市某规模化养猪场暴发的一起断奶仔猪病因未明疫病为猪圆环病毒2型感染，并应用病毒分离技术获得一株猪圆环病毒2型毒株，命名为PCV2CC12毒株。序列测定与比对分析发现CC12毒株的基因组存在独特的突变。与目前已知PCV2毒株相比，CC12毒株基因组第607位核苷酸由腺嘌呤（A）突变为鸟嘌呤（G），该突变导致所编码的Rep蛋白第186位氨基酸由天冬酰胺（N）突变为丝氨酸（S）。此外，，CC12毒株编码的衣壳结构蛋白Cap亦存在两个少见突变，包括59位氨基酸由精氨酸（R）突变为赖氨酸（K），190位氨基酸由丙氨酸（A）突变为苏氨酸（T）。核苷酸和氨基酸序列比较分析发现CC12毒株与目前国内猪群流行的毒株相似，属于PCV2b基因型。该结果为防控该病提供了流行病学理论依据。根据CC12毒株的完整基因组序列，设计合成引物，以PCR扩增病毒的全基因组序列，并将其插入pBluescript II SK载体构建获得CC12株单拷贝基因组重组质粒，并以此构建了正向双拷贝重组质粒。将构建的感染性克隆以脂质体介导方法转染PK15细胞后，经过连续盲传8代后，应用PCR方法、间接免疫荧光技术（IFA）、免疫过氧化物酶单层细胞试验（IPMA）和免疫印迹技术（Westernblot）检测到病毒核酸分子和病毒Cap结构蛋白表达，表明CC12毒株拯救成功。我们将拯救的CC12毒株命名为rCC12。rCC12与亲本病毒具有相似的TCID50病毒滴度和生物学特性。该结果为研究PCV2复制机理、致病机制及病毒与宿主细胞间的相互作用提供了有效的工具和平台。将CC12病毒通过腹腔注射途径接种昆明小鼠，通过检测不同时间病毒在体内的分布及其引起的病理损伤与诱导的抗体水平，建立病毒感染小鼠模型。免疫组织化学检测PCV2病毒抗原的结果发现，小鼠感染初期病毒主要分布于肝脏、肾脏、脾脏、淋巴结、心脏、肺脏和胸腺；而感染35天后，PCV2主要集中在淋巴结，这与PCR检测结果基本一致；组织病理学观察发现，感染小鼠肺脏、肝脏、胸腺、淋巴结、脾脏均出现程度不一的炎性病变或坏死；ELISA检测抗体结果显示，小鼠接种PCV2后第7天可以检测到抗体，第28天抗体水平达到最高，随后呈现下降趋势。
[Abstract]:Porcine circovirus disease (Porcine circovirus disease) is a new infectious disease caused by porcine circovirus type 2, which seriously endangers the development of pig industry. It is characterized by poor growth, wasting, dyspnea, high swelling of subcutaneous lymph nodes, hemorrhage and necrosis in weaned piglets. In this study, PCR amplification virus genomic sequence and immunology were used to confirm that an outbreak of weaning piglets caused by an outbreak in a large scale pig farm in Changchun was due to the infection of porcine circovirus type 2. A porcine circovirus type 2 strain, named PCV2CC12 strain, was obtained by virus isolation technique. Sequence determination and alignment analysis showed that there was a unique mutation in the genome of the CC12 strain. Compared with the known PCV2 strain, the 607 nucleotide mutation in the genome of the CC12 strain was transformed from adenine to guanine guanine, which resulted in the coding of. The 186th amino acid of Rep protein was mutated from asparagine to serine, and there were two rare mutations in capsid structure protein Cap encoded by CC12 strain. The amino acids of 59 amino acids were mutated from arginine R) to lysine kapok (190 amino acids from alanine A) to threonine tassel. The comparison of nucleotide and amino acid sequences showed that the CC12 strains were similar to those prevalent in swine populations in China. The results provide an epidemiological basis for the prevention and control of the disease. According to the complete genome sequence of CC12 strain, the primers were designed and synthesized, the whole genome sequence of the virus was amplified by PCR, and inserted into pBluescript II SK vector to construct the single copy genome recombinant plasmid of CC12 strain. The positive double copy recombinant plasmid was constructed. The constructed infectious clone was transfected into PK15 cells by liposome mediated method. After 8 consecutive generations of blind transmission, PCR method was used. The expression of viral nucleic acid molecules and viral Cap structural proteins was detected by indirect immunofluorescence assay (IFA), immunoperoxidase monolayer assay (IPMA) and Western blot (Western blot). The results showed that the CC12 strain was successfully saved. We named the rescued CC12 strain rCC12.rCC12 having similar titers and biological characteristics of TCID50 virus to parent virus. This result was used to study the replication mechanism of PCV2. The pathogenicity mechanism and the interaction between virus and host cells provide an effective tool and platform. The CC12 virus was inoculated into Kunming mice by intraperitoneal injection. The distribution of the virus in the body, the pathological damage caused by the virus and the induced antibody level were detected by measuring the distribution of the virus in the body at different time points. The PCV2 virus antigen was detected by immunohistochemistry. The results showed that the virus mainly distributed in liver, kidney, spleen, lymph node, heart, lung and thymus. After 35 days of infection, PCV2 was mainly concentrated in lymph nodes, which was consistent with the results of PCR. Histopathological observation showed that the infected mice were infected with lung, liver, thymus and lymph nodes. The results of Elisa showed that antibodies could be detected on the 7th day after inoculation of PCV2, reached the highest level on the 28th day, and then showed a downward trend.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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