CN106434575A - 流感病毒作为表达载体的构建方法 - Google Patents

流感病毒作为表达载体的构建方法 Download PDF

Info

Publication number
CN106434575A
CN106434575A CN201610849405.1A CN201610849405A CN106434575A CN 106434575 A CN106434575 A CN 106434575A CN 201610849405 A CN201610849405 A CN 201610849405A CN 106434575 A CN106434575 A CN 106434575A
Authority
CN
China
Prior art keywords
gene
virus
recombinant
protein
influenza virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201610849405.1A
Other languages
English (en)
Inventor
蒋文明
侯广宇
李金平
朱琳
陈继明
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CHINA ANIMAL HEALTH AND EPIDEMIOLOGY CENTER
Original Assignee
CHINA ANIMAL HEALTH AND EPIDEMIOLOGY CENTER
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CHINA ANIMAL HEALTH AND EPIDEMIOLOGY CENTER filed Critical CHINA ANIMAL HEALTH AND EPIDEMIOLOGY CENTER
Priority to CN201610849405.1A priority Critical patent/CN106434575A/zh
Publication of CN106434575A publication Critical patent/CN106434575A/zh
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16141—Use of virus, viral particle or viral elements as a vector
    • C12N2760/16143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32311—Enterovirus
    • C12N2770/32334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

本发明属于生物技术领域，确立了一种构建流感病毒表达载体的方法，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

流感病毒作为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，本发明确立了一种构建流感病毒表达载体的方法，用于表达外源蛋白，可作为单价或二价疫苗使用，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nt402和nt529之间（替换nt402-528），这样可以表达出NS1蛋白的N端125aa、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nt1-401和nt529-890片段进行重叠延伸PCR，将该片段插入到NS基因的第nt402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nt402-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。
CN201610849405.1A 2016-09-26 2016-09-26 流感病毒作为表达载体的构建方法 Pending CN106434575A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610849405.1A CN106434575A (zh) 2016-09-26 2016-09-26 流感病毒作为表达载体的构建方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610849405.1A CN106434575A (zh) 2016-09-26 2016-09-26 流感病毒作为表达载体的构建方法

Publications (1)

Publication Number Publication Date
CN106434575A true CN106434575A (zh) 2017-02-22

Family

ID=58169415

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610849405.1A Pending CN106434575A (zh) 2016-09-26 2016-09-26 流感病毒作为表达载体的构建方法

Country Status (1)

Country Link
CN (1) CN106434575A (zh)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011014504A1 (en) * 2009-07-27 2011-02-03 Mount Sinai School Of Medicine Of New York University Recombinant influenza virus vectors and uses thereof
CN103492574A (zh) * 2011-02-22 2014-01-01 加州理工学院 使用腺相关病毒（aav）载体递送蛋白

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011014504A1 (en) * 2009-07-27 2011-02-03 Mount Sinai School Of Medicine Of New York University Recombinant influenza virus vectors and uses thereof
CN103492574A (zh) * 2011-02-22 2014-01-01 加州理工学院 使用腺相关病毒（aav）载体递送蛋白

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
SANDRA GOHRBANDT ET AL.: ""Amino acids adjacent to the haemagglutinin cleavage site are relevant for virulence of avian influenza viruses of subtype H5"", 《JOURNAL OF GENERAL VIROLOGY》 *
TONY VELKOV ET AL.: ""The antigenic architecture of the hemagglutinin of influenza H5N1 viruses"", 《MOLECULAR IMMUNOLOGY》 *
刘锴: "《传染性法氏囊病毒的反向遗传操作研究》", 30 September 2013 *
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》 *
秦成峰 等: "《人类流感与禽流感》", 31 July 2007 *
蒋文明等: ""H5N6亚型禽流感病毒反向遗传疫苗株的构建及免疫保护试验"", 《中国动物检疫》 *
龙进学等: ""禽流感病毒NS第263～277位核苷酸缺失降低其抗干扰素能力"", 《微生物学通报》 *

Similar Documents

Publication Publication Date Title
Szewczyk et al. Introduction to molecular biology of influenza a viruses
Gao et al. The influenza A virus PB2, PA, NP, and M segments play a pivotal role during genome packaging
Steel et al. Live attenuated influenza viruses containing NS1 truncations as vaccine candidates against H5N1 highly pathogenic avian influenza
Tian et al. Protective efficacy of the H5 inactivated vaccine against different highly pathogenic H5N1 avian influenza viruses isolated in China and Vietnam
Samal et al. Coordinate deletion of N-glycans from the heptad repeats of the fusion F protein of Newcastle disease virus yields a hyperfusogenic virus with increased replication, virulence, and immunogenicity
JP2015119730A5 (zh)
Rohaim et al. Chickens expressing IFIT5 ameliorate clinical outcome and pathology of highly pathogenic avian influenza and velogenic newcastle disease viruses
WO2016134678A1 (zh) 新的甲型流感病毒哺乳动物细胞适应株及其制备和应用
CN104962581A (zh) 一种表达非洲猪瘟病毒p72蛋白的重组病毒疫苗株
KR102277763B1 (ko) 개선된 바이러스 생산을 위한 조류 세포
CN104232594A (zh) 重组类禽型h1n1流感病毒灭活疫苗株(js40/pr8)及其制备方法和应用
EP2716752A1 (en) Canine influenza recombinant virus, preparation method therefor and application thereof
US20140286993A1 (en) Immune methods against influenza viruses and combinatorial vaccines thereof
CN108348596B (zh) 用牛免疫缺陷病毒Gag蛋白的重组病毒样颗粒
Pei et al. Codon optimization of G protein enhances rabies virus-induced humoral immunity
CN102373183A (zh) 禽流感和新城疫混合病毒样颗粒、制备方法和应用
JP2007282636A5 (zh)
CN106434574A (zh) 一种流感病毒表达载体的构建方法
CN108329394A (zh) 一种鸭短喙矮小综合征疫苗及其制备方法
KR102091281B1 (ko) 재조합 인플루엔자 a 바이러스 h5n6주 및 이를 포함하는 고병원성 인플루엔자 a 바이러스 백신 조성물
CN103333916A (zh) 适应鸡新城疫病毒增殖的bhk细胞系的建立方法及其应用
Gao et al. It is not just AIV: from avian to swine-origin influenza virus
CN101857872A (zh) 甲型流感病毒抗原决定区更换方法
CN106434575A (zh) 流感病毒作为表达载体的构建方法
Sun et al. Evolution of the PB1 gene of human influenza A (H3N2) viruses circulating between 1968 and 2019

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20170222

RJ01 Rejection of invention patent application after publication