犬副流感病毒分子克隆的构建及微型基因组建模

发布时间：2018-03-17 01:10

本文选题：犬副流感病毒　切入点：微型基因组　出处：《中国农业科学院》2015年硕士论文　论文类型：学位论文

【摘要】：犬副流感病毒(canine parainfluenza virus,CPIV)是引起犬的自限性气管支气管炎,犬窝咳的主要病因。临床上,CPIV单独感染不引起致死性疾病,只表现为温和的呼吸道症状,而这种情况并不常见;常见的是CPIV与其他病原的混合感染,使病情加剧,大幅度提高死亡率。自1967年首次分离得到该病毒以来,世界各地陆续有相关报道,目前该病毒感染范围已波及全世界,在我国亦出现了普遍流行趋势。在各类病毒疫苗的开发研究中,最为重大的科学进展是反向遗传技术。目前,CPIV的基因工程疫苗尚未见报道,但对人副流感病毒(human parainfluenza virus,HPIV)和牛副流感病毒3型(Bovine parainfluenza virus type3,BPIV3)已有较多的研究。随着负链RNA病毒反向遗传学技术的日趋成熟,副流感病毒(parainfluenza virus,PIV)作为活病毒表达载体方面有广阔的应用前景,HPIV和BPIV3的现有研究成果对CPIV的研究有借鉴意义。本实验室以构建CPIV的感染性分子克隆为目的。根据Gen Bank(KC237064.1)中PIV5 1168-1毒株序列设计引物,应用RT-PCR法对购自ATCC的CPIV HRB-V病毒株分段扩增,克隆到pHW2000载体中并进行测序,经序列拼接获得全基因组序列。与Gen Bank登录的PIV代表毒株进行对比分析。基因序列分析表明,HRB-V株基因组全长15246nt,含5'-末端、3'-末端和7个ORF,具有PIV基因组结构的典型特征;将结构蛋白F、HN以及全基因序列与参考毒株进行比较分析并做遗传进化树,结果表明:HRB-V与CPI-和CPI+属同一分支,亲缘性相近。同源性分析结果表明:HRB-V与各病毒株的F基因的核苷酸同源性在96.5%~98.8%之间,氨基酸同源性为94.2%~98%;HN基因的核苷酸同源性在98.1%~99.5%之间,氨基酸同源性为97%~99.3%。本研究系统了解了CPIV的基因组结构及遗传进化特点。参考已知的HRB-V病毒株序列设计引物,RT-PCR扩增NP、P和L基因,分别克隆入真核表达载体pCI-neo中,构建辅助质粒;同时使eGFP取代病毒整个编码区,只保留两个末端与病毒复制、转录和病毒粒子包装相关的调控序列,将其反向克隆入转录载体pVAX1中,构建该病毒的微型基因组质粒,应用此微型基因组对辅助质粒的表达产物进行功能鉴定。结果表明NP、P和L三种蛋白的正常表达并发挥RNA聚合酶作用,为病毒拯救奠定基础。本研究成功构建了CPIV的分子克隆,并进行系统分析;同时构建了病毒辅助质粒及微型基因组,鉴定了辅助质粒的RNA聚合酶功能。以上研究为实验室构建CPIV反向遗传操作技术平台奠定基础。
[Abstract]:Canine parainfluenza virus (CPIV) is the main cause of canine self-limited tracheobronchial bronchitis and burrow cough in dogs. Clinical infection of canine parainfluenza virus alone does not cause fatal diseases, but shows mild respiratory symptoms, which is not common. It is common to see a mixture of CPIV and other pathogens, which exacerbates the disease and dramatically increases the mortality rate. Since the virus was first isolated in 1967, there have been reports from around the world that the virus has spread to the whole world. The most important scientific progress in the development and research of various viral vaccines is reverse genetic technology. At present, the genetic engineering vaccine of CPIV has not been reported. However, more studies have been done on human parainfluenza virus (HPIV) and bovine parainfluenza virus type 3 (BPIV3). Parainfluenza virus (PIV) as a living virus expression vector has a broad application prospect. The existing research results of HPIV and BPIV3 can be used for reference in the study of CPIV. The purpose of our laboratory is to construct the infectious molecular clone of CPIV. According to Gen. The sequence of PIV5 1168-1 strain in KC237064.1) was designed. The CPIV HRB-V virus strain purchased from ATCC was amplified by RT-PCR, cloned into pHW2000 vector and sequenced. The whole genome sequence was obtained by sequence splicing, and compared with the PIV representative strain registered by Gen Bank. The gene sequence analysis showed that the whole genome of HRBV strain was 15246nt in length, which contained 5H-terminal 3 terminal and 7 ORFs, and had the typical characteristics of PIV genomic structure. The structure protein FHN and the whole gene sequence were compared with the reference strain and the genetic evolution tree was made. The results showed that the two species belong to the same branch as CPI- and CPI. The results of homology analysis showed that the nucleotide homology of F gene and amino acid homology were 96.5% and 98.8%, respectively, and the nucleotide homology of HN gene was 98.1% and 99.5%, respectively. The amino acid homology was 970.In this study, the genomic structure and genetic evolution characteristics of CPIV were studied. The nucleotide sequences of HRB-V virus were amplified by RT-PCR and cloned into eukaryotic expression vector pCI-neo, respectively, and the auxiliary plasmids were constructed. At the same time, eGFP was used to replace the whole coding region of the virus, and only two regulatory sequences related to virus replication, transcription and virus particle packaging were retained, and then cloned into the transcriptional vector pVAX1 to construct the microgenome plasmid of the virus. The microgenome was used to identify the function of the expression product of the auxiliary plasmid. The results showed that the three proteins expressed normally and played the role of RNA polymerase, which laid the foundation for virus rescue. In this study, the molecular cloning of CPIV was successfully constructed. At the same time, the virus auxiliary plasmid and microgenome were constructed, and the function of RNA polymerase of the auxiliary plasmid was identified, which laid a foundation for the laboratory construction of CPIV reverse genetic manipulation platform.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.655

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