新城疫病毒耐热株HR09的鉴定与反向遗传操作系统的构建

发布时间：2018-03-08 19:48

  本文选题：新城疫病毒　切入点：耐热株　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：新城疫病毒(Newcastle disease virus,NDV)是严重危害养禽业的烈性传染病——新城疫(Newcastle disease,ND)的病原。由于V4和1-2等耐热ND弱毒疫苗的贮存和运输不需要依赖冷链,在热带、亚热带国家和地区已经得到广泛应用。我国上世纪90年代引进了 NDV耐热毒株V4,但由于知识产权等的限制,至今未大规模应用于生产。本研究从保存的40多株NDV中筛选到1个耐热强毒株(HR09株);测定了该毒株的全基因组序列,并分析了其分子生物学特性;建立了该毒株的反向遗传操作系统,并对其F基因进行突变,得到NDV耐热弱毒株。本研究为进一步揭示NDV耐热毒株的耐热机制和创制耐热ND弱毒疫苗奠定了基础。1新城疫病毒耐热株HR09鉴定及全基因组序列测定将本室保存的40多个NDV分离株与V4、LaSota等参考毒株共同进行耐热性试验。以56℃耐热处理30 min时仍具有血凝性作为筛选标准,获得1个耐热毒株——HR09株。HR09株经蚀斑纯化后进行生物学特性鉴定,显示该毒株的MDT为57.6 h,ICPI为1.8,IVPI为1.56,符合NDV强毒株特征。设计10对引物,对HR09株全基因组序列进行扩增、测定和生物信息学分析,结果显示,该毒株基因组全长为15192 nt,推导的F蛋白裂解位点氨基酸序列为112RRQKRF117,HN蛋白长度为571个氨基酸残基,符合NDV强毒株特征。HR09株基因组GC含量为46.27%,与耐热的V4株、1-2株全基因组的核苷酸序列同源性为86.7%和85.4%,6个结构蛋白编码基因的核苷酸序列同源性介于83.1%～95.6%之间、推导的氨基酸序列同源性介于81.6%～98.0%之间。基于F基因全长所构建的进化树显示,HR09株属于基因ⅷ型毒株。2新城疫病毒耐热株HR09反向遗传操作系统的构建设计了多对引物,构建了 HR09株基因组的全长cDNA克隆以及含有该毒株NP、P和L基因的3个真核表达质粒,共转染BSR-T7/5细胞,拯救出病毒NDV/rHR09。对获救病毒的耐热性进行测定,显示其经56℃处理60 min时仍具有血凝活性,说明NDV/rHR09株与亲本株的耐热性相似。NDV/rHR09株的MDT值为60 h,ICPI值为1.625,仍为NDV强毒株。3新城疫病毒耐热弱毒株rAHR09的构建设计两对引物,经Overlap PCR的方法将NDV/rHR09毒株F基因裂解位点的112、115和117位碱性氨基酸突变成弱毒株特征的非碱性氨基酸,构建了转录载体prNDV/HR09,将转录载体与NP、P和L基因的真核表达质粒共转染BSR-T7/5细胞,拯救出病毒NDV/rAHR09。经56。C、45min处理,NDV/rAHR09株仍具有血凝性,MDT值120h,ICPI值为0.057。结果表明,NDV/rAHR09株具有耐热性,且毒力已明显致弱,可以作为耐热ND弱毒疫苗的候选毒株。
[Abstract]:Newcastle disease virus (NDV) is the pathogen of Newcastle disease (NDV), a severe infectious disease in poultry industry. Because the storage and transportation of heat-resistant NDV vaccines such as V4 and 1-2 do not depend on cold chains, they are in the tropics. Subtropical countries and regions have been widely used. In -10s, China introduced NDV heat-resistant strain V4, but due to the limitations of intellectual property rights, Up to now, no large scale application has been made. In this study, a heat-resistant and virulent strain HR09 was screened out of more than 40 strains of NDV preserved, the whole genome sequence of the strain was determined, and its molecular biological characteristics were analyzed. The reverse genetic operating system of the strain was established, and the F gene was mutated. NDV heat-resistant attenuated strain was obtained. This study laid a foundation for further revealing the heat-resistant mechanism of NDV heat-resistant strain and creating a heat-resistant ND attenuated vaccine. 1. The Newcastle disease virus heat-resistant strain was identified by HR09 and the whole genome sequence was sequenced. More than 40 NDV isolates were tested for heat resistance with reference strains V4 and LaSota. Hemagglutination at 56 鈩，

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