【作者】 颜艳；

【导师】 何维明； 王希良；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2007， 硕士

【摘要】 流感(Influenza)是由流行性感冒病毒(Influenza virus)感染引起的急性呼吸道传染病。流感病毒属于正粘病毒科,可感染人和多种动物。其中A型流感病毒对人类威胁最大,曾引起人类历史上4次流感大流行。A型流感病毒抗原性易发生变异,抗原的漂移和转换给流感的防治带来了较大困难。接种疫苗仍然是预防和控制流感的主要手段。目前使用的疫苗包括全病毒疫苗、裂解疫苗和减毒活疫苗。减毒活疫苗可通过鼻腔接种诱导局部的黏膜免疫应答和系统免疫应答,是流感疫苗发展的新方向。反向遗传学的快速发展,为开发流感病毒疫苗提供了新手段,1999年以后Neumann等、Hoffmann等分别建立了完全以质粒为基础的流感病毒反向遗传操作系统,在开发新型流感疫苗、快速制备疫苗候选株等方面显示了良好应用前景。本研究选择冷适应、温度敏感、减毒的A/Ann Arbor/6/60 (H2N2)(MDV-A)流感病毒株作为骨架病毒,运用8质粒系统拯救出冷适应减毒的A型人流感病毒,建立A型流感病毒冷适应拯救体系,并运用于06～07 A型流感病毒推荐疫苗组分株的拯救,为流感病毒新型疫苗的开发奠定了基础。1.MDV-A 6个内部骨架基因双向转录/表达重组质粒的构建与验证设计一对引物,以pcDNA3.1(+)质粒为模板扩增多聚A(polyA)信号序列,取代双向转录/表达载体pHW2000上牛生长激素(BGH)的多聚腺苷酸信号序列,构建双向转录/表达载体pAD3000。人工合成的MDV-A株6个内部基因片段,克隆至双向转录/表达载体pAD3000,构建6个polⅠ-polⅡ系统的转录/表达质粒,经测序验证6个内部基因片段均在设计位点引入突变,期望6个质粒通过同一个载体,转染后利用细胞中的polⅠ和polⅡRNA聚合酶,实现MDV-A 6个内部基因vRNA和mRNA的转录和表达。用MDV-A任意一个内部基因重组质粒与A/PR/8/34的基因组合,即7+1组合形式共转染COS-1细胞,37℃或33℃、5%CO2培养48h后将转染上清液接种10日龄SPF鸡胚,96h后检测鸡胚尿囊液血凝效价。实验结果表明,6种组合均能产生有血凝性的流感病毒,说明双向转录/表达载体pAD3000能正常工作,含有MDV-A 6个内部基因片段的质粒,在同一载体上实现了vRNA和mRNA的转录和表达,并能装配成病毒,为下一步拯救MDV-A冷适应骨架病毒毒株打下了基础。2冷适应减毒重配A型流感病毒rMDV-A的拯救及部分生物学特性的鉴定用MDV-A 6个内部基因质粒与A/PR/8/34的表面HA和NA基因质粒组合,共转染COS-1细胞,33℃、5%CO2培养48 h,转染上清液接种10日龄SPF鸡胚,96h后检测鸡胚尿囊液血凝效价,得到有血凝性的重组A型流感病毒rMDV-A,电镜形态观察rMDV-A,与野生型流感病毒相似。rMDV-A在鸡胚中传至4代后,收集尿囊液,RT-PCR扩增6个内部基因片段,测序,序列分析与MDV-A 6个内部基因片段序列一致,说明rMDV-A能在鸡胚中稳定增殖。rMDV-A在不加胰酶的情况下感染MDCK细胞,能引起细胞变圆、脱落等病变。收集PR8和rMDV-A病毒在33℃、37℃、38℃和39℃四个温度培养的鸡胚尿囊液,测定两种病毒在MDCK细胞上空斑形成单位(PFU),结果表明rMDV-A效价随着温度的升高有明显下降趋势。到39℃时,病毒效价急剧下降,可见rMDV-A对温度敏感,较低温度(如33℃)更适于rMDV-A病毒的繁殖。rMDV-A的拯救和鉴定,说明成功建立了A型流感病毒冷适应减毒株拯救体系,为开发A型流感病毒减毒疫苗奠定了基础。3.06～07 A型流感病毒冷适应候选疫苗株的拯救RT-PCR扩增06～07 A型流感病毒推荐疫苗组分株A/New Caledonia/20/99(H1N1)和A/Wisconsin/67/2005(H3N2)的HA和NA基因,连接pGEM-T载体并测序,得到序列正确的HA和NA基因,并克隆至双向转录/表达载体pAD3000上,分别与MDV-A 6个内部基因共转染COS-1细胞,33℃、5%CO2培养48 h,转染上清液接种10日龄SPF鸡胚,96h后检测鸡胚尿囊液血凝效价,得到有血凝性的重组A型流感病毒rMDV-A-H1,rMDV-A-H3,拯救出06～07 A型流感病毒冷适应候选疫苗株,为06～07流感病毒减毒三价疫苗的开发与生产奠定基础。本研究运用8质粒拯救系统建立了A型流感病毒冷适应拯救体系,为流感病毒新型疫苗的开发提供了新的手段。更多还原

【Abstract】 Influenza is a highly contagious respiratory disease caused by Influenza virus. Influenza virus, a member of the family Orthomyxoviridae, can infect humans and many kinds of animals. Influenza A virus that had caused fourth pandemic in 20th century is a mass threaten to humans. The fragmented genome of the influenza A virus is more vulnerable to genetic variations which leads antigenic shift and drift. This phenomenon makes it’s more difficult to prevent and control this disease. Compare to other measures, vaccination remains the most effective approach to prevent this disease. Currently, influenza vaccines used in humans contains“inactivated”vaccines、split vaccine and live attenuated vaccines. Live attenuated vaccines, an intranasal vaccine, can induce both local mucosa immune response and system immune response. Due to their safety and efficacy, the live attenuated vaccines are seen as viable alternatives to the“inactivated vaccines”. Reverse genetic systems entirely based on cloned cDNAs of influenza virus established by Neumann, Hoffmann, et al. since 1999 is being employed to specially design“recombinant”A and B viruses, and it has opened the way to develop live attenuated vaccines. In our research , cold-adapted、temperature sensitive and attenuated influenza A virus A/Ann Arbor/6/60 (H2N2) is used as a master donor virus(MDV-A) to generate an attenuated recombinants influenza A virus. This system is also used to rescue the strains for the manufacture of seasonal influenza vaccine for 2006/2007.1. Construction of six transcription/expression plasmids contained six inner gene of MDV-APolyA signal sequence was amplified from plasmid pcDNA3.1(+)by PCR to replace the bovine growth hormone polyadenylation signals of pHW2000, the reconstructed vector named as pAD3000.Six inner fragments contained five mutations of MDV-A were artificial synthesized. This six cDNAs were separately cloned into pAD3000 to construct six transcription/expression plasmids for the purpose of synthesizing of vRNAs and mRNAs by polⅠand polⅡfrom one template after being transfected into eukaryotic cells such as COS-1. All plasmids were sequenced to ensure it’s the same with the consensus MDV-A sequence except the five mutations.The COS-1 cell was cotransfected with eight plasmids which contained one plasmid of one fragment from MDV-A and seven plasmids had other seven fragments from PR8. The transfected cells were incubated at 35℃or 33℃,48 hours after transfection, the supernatant and cos-1 cells transfected were inoculated into the allantoic cavity of 10-day old specific-pathogen-free (SPF) chicken eggs . The HA titer were determined. A series 7+1 reassortant virus was rescued. It showed that all the six plasmids contained MDV-A genes were shown to be functionally expressed. With this progress, a cold-adapted、temperature sensitive and attenuated strain would be rescued next.2. Generation of cold-adapted attenuated reassortant influenza virus rMDV-A by reverse genetic and identification some of its biological characterizationsSix plasmids contained MDV-A genes were transfected into COS-1 cells in combination with plasmids incorporating the HA and NA genes of A/PR/8/34. The transfected cells were incubated at 33℃,48 hours after transfection, the supernatant and cos-1 cells transfected were inoculated into the allantoic cavity of 10-day old specific-pathogen-free (SPF) chicken eggs .The reassortant rMDV-A virus was determined by HA assay. The rescued viruses were indistinguishable from the wild-type in morphology. Recovered rMDV-A viruses was passed in eggs for four generations, and then its six inner segments were amplified by RT-PCR, each fragments was cloned into pGEM-T vector and sequenced. MDCK cells were infected with rMDV-A virus without trypsin. Temperature sensitivity of rMDV-A was determined by plaque assay on MDCK cells. The results showed that the inner gene of the fourth generation reassortant virus was the same with the consensus MDV-A sequence. Recovered rMDV-A viruses could replicate in eggs stably. The MDCK cells infected with rMDV-A became rounding, shedding and formed plaque. Plaque assay showed that rMDV-A have significant reduction in plaque at 39℃compared with 33℃. Recovered rMDV-A viruses was sensitive to temperature, lower temperature such as 33℃was more suitable for its replication. This reverse genetics system provided a new experimental tool for development of new-type vaccine candidates, for example, live-attenuated vaccine candidates.3. Rescue of the seasonal influenza vaccine composition strains for the season 2006/2007 by cold-adapted attenuated reverse genetic systemThe HA and NA gene of A/New Caledonia/20/99(H1N1)、A/Wisconsin/67/2005(H3N2) were amplified by RT-PCR, each fragments was cloned into pGEM-T vector and sequenced. The correct genes were then be cloned to pAD3000. This two plasmids contained HA and NA genes were transfected into COS-1 cells in combination with six plasmid incorporating the inner genes of MDV-A respectively. The transfected cells were incubated at 33℃,48 hours after transfection, the supernatant and cos-1 cells transfected were inoculated into the allantoic cavity of 10-day old specific-pathogen-free (SPF) chicken eggs .The recovered virus named rMDV-A-H1 and rMDV-A-H3.Generation of this two ressortant virus is the base of the development of trivalent vaccine for 2006/2007.Overall, this reverse genetics system provided a new experimental tool for development of new-type vaccine candidates, for example, live-attenuated vaccine candidates.更多还原