CN106434574A - 一种流感病毒表达载体的构建方法 - Google Patents

一种流感病毒表达载体的构建方法 Download PDF

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CN106434574A
CN106434574A CN201610849296.3A CN201610849296A CN106434574A CN 106434574 A CN106434574 A CN 106434574A CN 201610849296 A CN201610849296 A CN 201610849296A CN 106434574 A CN106434574 A CN 106434574A
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蒋文明
侯广宇
李金平
朱琳
陈继明
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CHINA ANIMAL HEALTH AND EPIDEMIOLOGY CENTER
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Abstract

本发明属于生物技术领域，确立了一种流感病毒表达载体的构建方法，利用NS1基因可以容纳外源基因的特性以及口蹄疫病毒2A蛋白和猪捷申病毒1型的2A蛋白的自裂解特性，将外源基因插入到NS1基因内部，构建成重组NS基因。利用反向遗传技术，获得重组病毒。此重组病毒可用于表达外源蛋白，可作为二价疫苗使用，在生产上有较大的开发和应用价值。

Description

一种流感病毒表达载体的构建方法
技术领域
本发明属于生物技术领域；具体说来，本发明确立了一种流感病毒表达载体的构建方法，用于表达外源蛋白，可作为单价苗或二价疫苗使用，在生产上有较大的开发和应用价值。
背景技术
禽流感是由禽流感病毒引起的一种禽类传染病。禽流感病毒根据血凝素（HA）和神经氨酸酶（NA）的不同，可分为16个HA亚型和9个NA亚型。高致病性禽流感是由某些H5或H7亚型禽流感病毒引起的严重威胁禽类的烈性传染病。
2003年以来，H5亚型高致病性禽流感一直在包括中国在内的多个国家和地区流行，给养禽业造成了严重的经济损失。目前，疫苗仍然是防控该病的重要手段。2005年，我国开始实施针对禽流感的大规模强制性免疫政策，并取得了显著成效。随着病毒的不断变异，疫苗也从Re-1更新到目前的Re-6、Re-7、Re-8。我们国家的这些禽流感疫苗都是使用基因重组技术生产的，将母本病毒PR8的六个内部基因（PB2、PB1、PA、NP、M、NS）和流行毒的两个外部基因（HA和NA）进行重组，获得重组病毒用于疫苗的生产。目前市场上使用的是Re-6+Re-7+Re-8三价苗或Re-6+Re-8二价苗。流感病毒有八个基因片段，NS基因是最小的片段，可以表达两种蛋白：NS1和NEP。其中，NS1基因可以容许外源基因的插入，可作为基因操作的靶基因。口蹄疫病毒（FMDV）的2A蛋白和猪捷申病毒1型（PTV-1）的2A蛋白具有自裂解功能，可以将表达的目的蛋白裂解成完整的个体。本专利利用NS1基因和2A蛋白的这些特性，提供了一种构建流感病毒表达载体的方法。
发明内容
本发明研究建立了一种构建流感病毒表达载体的方法。NS基因全长890bp，其中，nt27-719编码NS1蛋白（230aa），nt27-56和nt529-864表达NEP蛋白（121aa）。首先利用PCR或基因合成的方法，将口蹄疫病毒的2A蛋白（羧基端20aa）基因、目的蛋白基因（不含终止密码子）、猪捷申病毒1型的2A蛋白（羧基端20aa）基因、nt27-56四者串联，然后插入到NS基因的第nt246和nt529之间（替换nt246-528），这样可以表达出NS1蛋白的N端73aa、完整的目的蛋白和完整的NS2蛋白。然后，将重组的NS基因插入到双向表达载体中，与表达PR8病毒的其他五个内部基因和任一病毒的HA和NA基因共转染293T细胞，利用反向遗传技术获得重组病毒。
具体实施方式
下面通过实施例，说明本发明的技术方案，但本发明的保护范围不限于这个实施例。
本实施例利用基因合成技术，人工合成口蹄疫病毒的2A蛋白（羧基端20aa）、绿色荧光蛋白（GFP，不含终止密码子）、猪捷申病毒1型的2A蛋白（羧基端20aa）、nt27-56四者串联基因，然后与NS基因的nt1-245和nt529-890片段进行重叠延伸PCR，将该片段插入到NS基因的第nt246和nt529之间，获得NS基因与外源基因的重组体。然后，将该基因进行Esp3I酶切，克隆到双向表达载体中。然后与表达PR8病毒的其他五个内部基因和PR8病毒的HA和NA基因共转染293T细胞，72h后将转染细胞悬液接种10日龄SPF鸡胚，获得重组病毒。将重组病毒接种MDCK细胞，48h后通过荧光显微镜可以观察到有绿色荧光蛋白表达，同时重组病毒能凝集鸡的红细胞，说明重组病毒拯救成功，不仅表达HA蛋白，还能表达外源蛋白（GFP）。将该病毒免疫动物，不仅产生针对HA蛋白的抗体，还产生针对GFP蛋白的抗体。

Claims (4)

1.利用PCR或基因合成的方法，将口蹄疫病毒2A蛋白基因、目的蛋白基因（不含终止密码子）、猪捷申病毒1型的2A蛋白基因、nt27-56四者串联插入到PR8病毒NS基因的nt246-nt529之间，构建重组NS基因；利用反向遗传技术获得重组病毒。
2.如权利要求1所述的方法，构建重组病毒，用作表达其他亚型流感病毒HA全长或部分片段。
3.如权利要求1所述的方法，构建重组病毒，用作表达其他病毒主要保护性抗原蛋白。
4.如权利要求2和3所述的病毒，用于疫苗的生产。
CN201610849296.3A 2016-09-26 2016-09-26 一种流感病毒表达载体的构建方法 Pending CN106434574A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111363728A (zh) * 2020-01-20 2020-07-03 武汉大学 携带乙型肝炎病毒基因的重组甲型流感病毒、宿主细胞及其制备方法与应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101575608A (zh) * 2008-05-07 2009-11-11 中国科学院上海生命科学研究院 针对禽流感病毒的重组联合dna疫苗
WO2011014504A1 (en) * 2009-07-27 2011-02-03 Mount Sinai School Of Medicine Of New York University Recombinant influenza virus vectors and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101575608A (zh) * 2008-05-07 2009-11-11 中国科学院上海生命科学研究院 针对禽流感病毒的重组联合dna疫苗
WO2011014504A1 (en) * 2009-07-27 2011-02-03 Mount Sinai School Of Medicine Of New York University Recombinant influenza virus vectors and uses thereof

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TONY VELKOV ET AL.: "The antigenic architecture of the hemagglutinin of influenza H5N1 viruses.", 《MOLECULAR IMMUNOLOGY》 *
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崔宏锐等: "利用反向遗传技术构建表达绿色荧光蛋白的H9N2禽流感重组病毒", 《中国动物传染病学报》 *
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111363728A (zh) * 2020-01-20 2020-07-03 武汉大学 携带乙型肝炎病毒基因的重组甲型流感病毒、宿主细胞及其制备方法与应用

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Application publication date: 20170222