WO2020052035A1 - 一种表达h5亚型ha的复制缺陷型重组h9n2禽流感病毒 - Google Patents

一种表达h5亚型ha的复制缺陷型重组h9n2禽流感病毒 Download PDF

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WO2020052035A1
WO2020052035A1 PCT/CN2018/114270 CN2018114270W WO2020052035A1 WO 2020052035 A1 WO2020052035 A1 WO 2020052035A1 CN 2018114270 W CN2018114270 W CN 2018114270W WO 2020052035 A1 WO2020052035 A1 WO 2020052035A1
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influenza virus
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李军伟
孙明宏
吴叔文
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青岛农业大学
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  • the invention belongs to the field of influenza virus vaccine technology research and development, and particularly relates to a replication-deficient recombinant H9N2 avian influenza virus expressing H5 subtype HA; that is, a surface protein blood capable of simultaneously expressing two subtypes of influenza A virus H9N2 and H5N1.
  • Recombinant influenza virus of lectin HA its preparation method and application.
  • Avian influenza is a type of infectious disease of avian respiratory diseases and systemic sepsis caused by Avian influenza virus (AIV).
  • the H5N1 subtype avian influenza virus is a highly pathogenic virus that is highly contagious and seriously harms birds, humans, and other animals.
  • the H9N2 subtype is a low-pathogenic avian influenza virus, which is widely present throughout the world. The harm is long-lasting and difficult to control, especially the mixed infection leads to higher mortality.
  • the H9N2 subtype influenza virus is also an internal gene donor of H5N1, H7N9 and other subtype influenza viruses, which can form new recombinant viruses, which seriously endangers the development of China's poultry industry and public health.
  • H9N2 and H5N1 subtype influenza viruses cannot be ignored.
  • vaccine immunization is still the most important and effective measure for the prevention and control of avian influenza.
  • Only inactivated vaccines have been approved for H5N1 and H9N2 avian influenza viruses, and there is no protection against H9N2 and H5N1 avian influenza at the same time.
  • the live attenuated vaccine of the virus, and the traditional live attenuated vaccine of the influenza virus are mainly low temperature adaptive strains, and there is a risk of virulence revert mutation.
  • the invention provides a replication-deficient recombinant H9N2 avian influenza virus expressing H5 subtype HA, a preparation method thereof and application in vaccine preparation.
  • the present invention first provides a replication-deficient recombinant H9N2 avian influenza virus expressing H5 subtype HA.
  • the preparation method is as follows:
  • the packaging signal region at the 5 ′ end is 203 base pairs at the 5 ′ end of the neuraminidase NA of the H9N2 virus;
  • the packaging signal region at the 3 ′ end is 195 base pairs at the 3 ′ end of the neuraminidase NA of the H9N2 virus;
  • nucleotide sequence of the DNA fragment for synthesizing influenza A virus NA ps- HA-NA ps is SEQ ID NO: 1;
  • the plasmid is preferably a plasmid pHW2000;
  • the recombinant plasmid constructed in 2) is transfected into 293T cells with the recombinant plasmids expressing H9N2 PB2, PB1, PA, HA, NP, M, and NS gene fragments, and can stably express H9N2 influenza virus neuraminic acid.
  • the replication-deficient recombinant H9N2 avian influenza virus expressing H5 subtype HA prepared by the present invention is used for preparing an attenuated vaccine.
  • the invention rescues a replication-deficient type A influenza virus divalent attenuated live vaccine strain.
  • a recombinant virus constructed can stably express influenza A virus H9N2 and H5N1 subtypes simultaneously.
  • Type of surface protein hemagglutinin HA.
  • the attenuated virus not only has good genetic stability but also cannot be replicated in experimental animals, so it is not pathogenic. At the same time, it can induce the body to produce a strong level of mucosal immune response and cellular immune response, maintaining a strong and durable immunogen. Sex. The most important thing is that this vaccine candidate strain can produce immune protection against both influenza A virus H9N2 and H5N1 subtype influenza viruses. Therefore, it has great social significance for the prevention and control of influenza A.
  • Figure 1 Technical roadmap for implementing the present invention
  • Figure 2 Gel electrophoresis of DNA sequences for the synthesis of NA ps -HA-NA ps .
  • Figure 3 A830-HA virus growth curve.
  • Figure 4 Survival graph of experimental animals after challenge with recombinant A830-HA virus solution of different concentrations.
  • Figure 5 Survival graph of experimental animals after immunization with recombinant A830-HA virus solution.
  • the present invention constructs a replication-deficient recombinant avian influenza virus that can simultaneously express type A bird flu virus H9N2 and H5N1 subtype surface glycoprotein hemagglutinin HA, and uses it to prepare a live attenuated vaccine.
  • Example 1 DNA fragment and plasmid construction for synthesis of influenza A virus NA ps -HA-NA ps
  • a plasmid containing the seven fragments of influenza A virus H9N2, pHW-PB2, pHW-PB1, pHW-PA, pHW-HA, pHW-NP, pHW-M, and pHW-NS was used.
  • the above seven plasmids constitute a reverse genetic operating system with influenza A virus H9N2 as a backbone.
  • the DNA sequence NA ps -HA-NA ps (Genscript Corporation) was synthesized, which retained 203 nucleotides at the 3 'non-coding region of the influenza A virus H9N2NA fragment and 195 nucleotides at the 5' non-coding region. Acid packaging signal.
  • Upstream primer sequence TATTGGTCTCAGGGAGCAAAAGCAGGAGT
  • Downstream primer sequence ATAGTGTCTCGTATTAGTAGAAACAAGGAGTTTTTTTT
  • the plasmid pHW2000 and the PCR product NA ps -HA-NA ps were digested with BsmB1 and Bsa1, respectively; the two digested fragments were ligated with T4 DNA ligase to form the plasmid pHW-NA-HA.
  • the DNA sequence of the newly synthesized target fragment NA (203nt) -ORF (HA) -NA (195nt) is SEQ ID NO: 1:
  • Construction of a cell line An MDCK cell line capable of stably expressing A / Chicken / Shandong / 830/2014 (H9N2) influenza virus surface glycoprotein neuraminidase NA.
  • G418 selects stable expression cell lines:
  • the optimal concentration of G418-selected MDCK cells was determined to be 500 ⁇ g / mL.
  • G418 Take 1g of G418 dissolved in 1mL of 1M HEPES solution, add ultrapure water to 10mL, filter, and save at 4 °C for later use.
  • Solution A Add 100 ⁇ L of opti-MEM culture solution to a sterile 1.5 mL centrifuge tube, then add 2 ⁇ g of plasmid pD2EGFP-NA, mix gently, and let stand at room temperature for 5 minutes.
  • Solution B Add 100 ⁇ L of opti-MEM culture solution to a sterile 1.5 mL centrifuge tube, and then add 6-8 ⁇ L of Lipofectamine 2000 (Invitrogen) transfection reagent. Then slowly add solution A to solution B and let stand at room temperature for 20 minutes.
  • the culture medium can be replaced with 10% fetal bovine serum (Fetal Bovine Serum, FBS) and 500 ⁇ g / mL G418 MEM medium Screening of cells, the culture medium was replaced once every 4-5 days until all other cells died, and only the cells with positive clones were successfully selected.
  • FBS Fetal Bovine Serum
  • Cell line culture 293T cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium containing 10% fetal bovine serum (FBS) and 1% double antibody (Penicillin-Streptomycin Solution) (PS). Incubate in a 37 ° C incubator.
  • the modified MDCK cells stably expressing neuraminidase NA
  • MEM medium containing 10% fetal bovine serum (Fetal Bovine Serum (FBS)) and 1% double antibody (Penicillin-Streptomycin Solution) (PS) at 37 ° C. Box culture.
  • FBS Fetal Bovine Serum
  • PS Penicillin-Streptomycin Solution
  • Cell line transfection 293T cells and modified MDCK cells were spread on a 6-well cell culture plate at a ratio of 1: 1, 7x10 5 cells per well, and transfected until the cell growth density reached 60% -70%. Before transfection, the original DMEM culture medium in the 6-well cell culture plate was aspirated and discarded, washed twice with Phosphate Buffer Saline (PBS), and replaced with 2 mL of fresh Opti-MEM culture medium. Transfection reagent Lipofectamine 2000 (Invitrogen) was used to transfer eight plasmids pHW-PB2, pHW-PB1, pHW-PA, pHW-HA, pHW-NP, pHW-NA-HA, pHW-M to 293T cells and MDCK cells.
  • PBS Phosphate Buffer Saline
  • PHW-NS PHW-NS.
  • TPCK-trypsin Tosylsufonyl Phenylalanyl Chloromerthyl Ketone-trypsin
  • MDCK cells stably expressing NA were cultured in a MEM medium containing 10% Fetal Bovine Serum (FBS) and 1% double antibody (Penicillin-Streptomycin Solution (PS)) in a 37 ° C incubator.
  • FBS Fetal Bovine Serum
  • PS Penicillin-Streptomycin Solution
  • PBS Phosphate Buffer Saline
  • the cells were replaced with 2 mL containing 0.2% bovine serum white Protein (Bovine Serum Actin, BSA) and 1 ⁇ g / mL TPCK in MEM culture medium, and then the supernatant was collected at -24, 48, 72 hours after infection and stored at -80 ° C.
  • BSA bovine serum white Protein
  • MDCK cells stably expressing NA are evenly spread in two 6-well plates, 8 ⁇ 10 5 cells per well.
  • Preparation of 1.6% low-melting agarose Weigh 0.18g of agarose and dissolve it in 15mL of ultrapure water. After it is completely dissolved in a microwave oven for 40s, put it in a 42 ° C water bath and keep it at a constant temperature.
  • Safety test The safety of recombinant virus A830-HA in SPF chickens was studied by using the survival rate of SPF chickens after challenge.
  • Healthy 4-5 week old SPF chickens were randomly divided into 4 groups of 8 birds each. With three different titers comprises 10 5, 10 6, 10 7 PFU of recombinant A830-HA influenza virus after intranasal vaccination challenge, the survival rate in each group were observed daily statistics SPF chickens. As shown in Figure 4, nasal challenge with A830-HA recombinant influenza virus with different titers does not affect the survival rate of SPF chickens, indicating that the recombinant virus A830-HA does not affect the body when no exogenous NA exists Infectious.
  • Healthy 4-5 week old SPF chickens were randomly divided into 4 groups of 8 birds each.
  • the first group was a negative control group, immunized with PBS; the second group was a positive control, immunized with 10PFU / A830 virus solution in the nasal cavity, and the third and fourth groups were 10PFU / 100 and 100PFU / A830-HA respectively. Recombinant virus solution was inoculated.
  • the blood of the experimental SPF chickens in the above four groups was collected, and the serum was separated; the hemagglutination inhibition test and the virus neutralization test were performed.
  • the 11th well is a red blood cell control well without adding virus solution; the 12th well is an antigen control with virus solution.
  • the agglutination phenomenon is that red blood cells are tiled on the bottom wall of the V-shaped tube; unagglutinated red blood cells appear as obvious red dots in the V-shaped blood coagulation pores.
  • the reaction plate was tilted at a 45-degree angle, the result was judged to be 100% inhibition when the red blood cells flowed in a teardrop shape without agglomerated particles.
  • Table 2 Hemagglutination inhibition test to measure antibody titers against wild A830 virus serum

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Abstract

一种制备表达H5N1亚型HA的复制缺陷重组H9N2禽流感病毒的方法，包括构建同时稳定表达H9N2和H5N1亚型的表面糖蛋白血凝素HA的复制缺陷型重组禽流感病毒，在可稳定表达神经氨酸酶H9N2亚型NA的MDCK细胞中复制、包装出重组病毒粒子。该病毒粒子可用于制备弱毒疫苗。

Description

一种表达H5亚型HA的复制缺陷型重组H9N2禽流感病毒 技术领域
本发明属于流感病毒疫苗技术研发领域，具体涉及一种表达H5亚型HA的复制缺陷型重组H9N2禽流感病毒；即一种可同时表达A型流感病毒H9N2和H5N1两种亚型的表面蛋白血凝素HA的重组流感病毒，及其制备方法和应用。
背景技术
禽流感是由A型流感病毒(Avian influenza virus,AIV)引起的禽类呼吸系统疾病和全身败血症的一类传染病。H5N1亚型禽流感病毒是一种高致病性病毒，极具传染性，严重危害禽类、人类以及其他动物。H9N2亚型是一种低致病性禽流感病毒，其广泛存在于世界范围内，危害持久并难以控制，特别是混合感染导致较高的死亡率。同时H9N2亚型流感病毒还是H5N1、H7N9等亚型流感病毒的内部基因供体，可形成新的重组病毒，严重危害我国养禽业的发展和公共卫生健康。因此，对于H9N2和H5N1亚型流感病毒的研究与防治不容忽视。目前，疫苗免疫仍是防控禽流感最重要和最有效的措施，针对H5N1亚型和H9N2亚型禽流感病毒已经批准使用的只有灭活苗，也没有可同时保护H9N2和H5N1亚型禽流感病毒的弱毒活疫苗，并且传统的流感病毒弱毒活疫苗主要是低温适应株，存在毒力回复突变的危险。
发明内容
本发明提供一种表达H5亚型HA的复制缺陷型重组H9N2禽流感病毒，及其制备方法和在疫苗制备中的应用。
本发明首先提供一种表达H5亚型HA的复制缺陷型重组H9N2禽流感病毒，其制备方法如下：
1)在H9N2病毒的神经氨酸酶NA的5′端的包装信号区和3′端的包装 信号区内插入H5N1亚型的血凝素HA基因的开放阅读框，形成用于合成A型流感病毒NA ps-HA-NA PS的DNA片段；
所述的5′端的包装信号区为H9N2病毒的神经氨酸酶NA的5′端203个碱基对；
所述的和3′端的包装信号区，为H9N2病毒的神经氨酸酶NA的3′端的195个碱基对；
更进一步的，所述的用于合成A型流感病毒NA ps-HA-NA ps的DNA片段，其核苷酸序列为SEQ ID NO:1；
2)将1)中构建的用于合成A型流感病毒NA ps-HA-NA ps的DNA片段克隆到质粒上形成重组质粒；
所述的质粒，优选为质粒pHW2000；
3)构建表达H9N2流感病毒神经氨酸酶NA基因的MDCK细胞系；
4)将2)中构建的重组质粒，与表达H9N2的PB2、PB1、PA、HA、NP、M、NS各个基因片段的重组质粒共同转染到293T细胞和可稳定表达H9N2流感病毒神经氨酸酶NA基因的MDCK细胞系中，并收集转染后293T细胞和表达NA的MDCK细胞的上清培养液
5)在稳定表达H9N2亚型禽流感病毒神经氨酸酶NA的MDCK细胞系上扩增4)中拯救出的重组流感病毒。
本发明所制备的表达H5亚型HA的复制缺陷型重组H9N2禽流感病毒用于制备弱毒疫苗。
本发明拯救出了一种复制缺陷型A型流感病毒二价弱毒活疫苗株，基于在没有改变基因稳定性的前体下，构建的重组病毒可稳定的同时表达A型流感病毒H9N2和H5N1亚型的表面蛋白血凝素HA。该弱毒不仅具有良好的基因稳定 性而且在实验动物体内不能复制，所以并不具备致病性，同时还可诱导机体产生很强的粘膜免疫应答和细胞免疫应答水平，保持强烈且持久的免疫原性。最关键的是，该疫苗侯选株可对A型流感病毒H9N2和H5N1亚型流感病毒均可产生免疫保护作用。因此对于A型流感的预防与控制具有巨大的社会意义。
附图说明
图1：本发明实施的技术路线图；
图2：合成NA ps-HA-NA ps的DNA序列凝胶电泳图。
图3：A830-HA病毒生长曲线图。
图4：不同浓度重组A830-HA病毒液攻毒后实验动物存活率图。
图5：免疫接种重组A830-HA病毒液后实验动物存活率图。
具体实施方式
本发明构建了可同时表达A型禽流感病毒H9N2和H5N1亚型表面糖蛋白血凝素HA的复制缺陷型重组禽流感病毒，并用其来制备弱毒活疫苗。
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍：
实施例1用于合成A型流感病毒NA ps-HA-NA ps的DNA片段及质粒构建
使用含A型流感病毒H9N2七个片段的质粒pHW-PB2、pHW-PB1、pHW-PA、pHW-HA、pHW-NP、pHW-M、pHW-NS。上述七个质粒构成以A型流感病毒H9N2为骨架的反向遗传操作系统。
1、构建用于合成A型流感病毒NA ps-HA-NA ps的DNA片段
合成A型流感病毒NA-HA(ORF)的DNA序列：用A型流感病毒A/wild duck/Hunan/021/2005(H5N1)亚型的血凝素HA的开放阅读框(Open Reading Frame,ORF)去替换A型流感病毒A/Chicken/Shandong/830/2014(H9N2)中神经 氨酸酶NA的开放阅读框，同时保留NA 5’端的包装信号区(203个碱基对)和3’端的包装信号区(195个碱基对)，合成新的DNA序列命名为NA ps-HA(ORF)-NA ps(如图1)。
2、构建质粒pHW-NA-HA
首先合成DNA序列NA ps-HA-NA ps(Genscript公司)，该序列保留了A型流感病毒H9N2NA片段3′端非编码区的203个核苷酸和5′端非编码区的195个核苷酸的包装信号。利用PCR技术进行目的片段NA(203nt)-ORF(HA)-NA(195nt)的扩增(图2)。
上游引物序列：TATTGGTCTCAGGGAGCAAAAGCAGGAGT
下游引物序列：ATATGGTCTCGTATTAGTAGAAACAAGGAGTTTTTT
分别用BsmB1和Bsa1酶切质粒pHW2000和PCR产物NA ps-HA-NA ps；利用T4 DNA ligase连接以上两个酶切片段，即构成质粒pHW-NA-HA。
新合成的目的片段NA(203nt)-ORF(HA)-NA(195nt)的DNA序列为SEQ ID NO:1：
实施例2拯救重组病毒
构建细胞系：构建可稳定表达A/Chicken/Shandong/830/2014(H9N2)流感病毒表面糖蛋白神经氨酸酶NA的MDCK细胞系。
具体步骤如下：
G418筛选稳定表达细胞系：
筛选前，确定G418筛选MDCK细胞的最佳浓度为500μg/mL。
G418的配制：取1g G418溶于1mL 1M的HEPES溶液中，加超纯水至10mL，过滤，4℃保存备用。
(1)将A型流感病毒亚型H9N2表面糖蛋白神经氨酸酶NA的RNA片段体外 反转录制成cDNA，以cDNA为模板扩增流感病毒的NA基因片段，并克隆到载体质粒pD2EGFP-N1上。即得质粒pD2EGFP-NA。
(2)将MDCK细胞铺于6孔板，培养在含有10％胎牛血清的(Fetal Bovine Serum，FBS)和1％双抗(Penicillin-Streptomycin Solution，PS)的MEM培养液于37℃培养箱培养。(培养基和血清均购自Biological Industries公司)。待细胞生长密度达到60-70％左右进行转染。
(3)细胞转染前将6孔细胞培养板中的培养液吸弃掉，用PBS洗两遍，更换为新鲜的opti-MEM培养液，培养板放回培养箱。
(4)配制转染试剂：
Solution A：向一个无菌的1.5mL离心管中加入100μL的opti-MEM培养液，再加入2μg质粒pD2EGFP-NA，轻轻混匀，室温静置5分钟。
Solution B：向一个无菌的1.5mL离心管中加入100μL的opti-MEM培养液，再加入6-8μL Lipofectamine2000(Invitrogen公司)转染试剂。然后将solution A缓慢的加入到solution B中，室温静置20分钟。
(5)取出6孔板，将(4)中的混合液加到孔内，晃匀。
(6)转染6h后，将6孔板中的培养液吸弃掉，更换为新鲜的含有10％胎牛血清的(Fetal Bovine Serum，FBS)和1％双抗(Penicillin-Streptomycin Solution，PS)的MEM培养液，继续培养24h。
(7)转染24h后，将6孔细胞培养板置于荧光显微镜下观察，若能观察到细胞内有明显绿色荧光的表达(因载体质粒pD2EGFP-N1上有一段表达绿色荧光的基因序列EGFP)，则说明质粒pD2EGFP-NA成功转染入MDCK细胞内。
(8)细胞转染24h后，若显微镜下观察有绿色荧光的表达，即可将培养液换成 含有10％胎牛血清的(Fetal Bovine Serum，FBS)和500μg/mL G418的MEM培养液进行细胞的筛选，4-5天更换一次培养液，直至其他细胞全部死亡，只剩下阳性克隆的细胞即筛选成功。
(9)将筛选成功的细胞进行培养，验证是否可稳定传代，若能稳定传代，则证明稳定表达A/Chicken/Shandong/830/2014(H9N2)流感病毒表面糖蛋白神经氨酸酶NA的MDCK细胞系构建成功。
细胞系的培养：293T细胞培养在含有10％胎牛血清的(Fetal Bovine Serum，FBS)和1％双抗(Penicillin-Streptomycin Solution，PS)的DMEM(Dulbecco′s Modified Eagle′s Medium)培养液于37℃培养箱培养。改造的MDCK细胞(稳定表达神经氨酸酶NA)培养在含有10％胎牛血清的(Fetal Bovine Serum，FBS)和1％双抗(Penicillin-Streptomycin Solution，PS)的MEM培养液于37℃培养箱培养。(培养基和血清均购自Biological Industries公司)。
细胞系转染：将293T细胞与改造的MDCK细胞按1:1比例铺在6孔细胞培养板中，每孔7x10 5个细胞，待细胞生长密度达到60％-70％进行转染。转染前将6孔细胞培养板中原有的DMEM培养液吸弃掉，用磷酸盐缓冲液(Phosphate Buffer Saline，PBS)洗两遍，更换为2mL新鲜的Opti-MEM培养液。利用转染试剂Lipofectamine2000(Invitrogen公司)向293T细胞与MDCK细胞中转入八个质粒pHW-PB2、pHW-PB1、pHW-PA、pHW-HA、pHW-NP、pHW-NA-HA、pHW-M、pHW-NS。转染6小时后，弃掉6孔细胞培养板中培养液，更换为2mL新鲜的Opti-MEM培养液；转染24小时后，向培养液中加入1μg/mL甲苯磺酰基-L-氨基联苯氯甲基酮胰酶(Tosylsufonyl Phenylalanyl Chloromerthyl Ketone-trypsin,TPCK-trypsin)，继续培养48小时后，收集细胞上清液。
实施例3测试重组病毒的生长曲线
稳定表达NA的MDCK细胞培养在含有10％胎牛血清(Fetal Bovine Serum，FBS)和1％双抗(Penicillin-Streptomycin Solution，PS)的MEM培养液于37℃培养箱培养。(培养基和血清均购自Biological Industries公司。)将改造的MDCK细胞铺在6孔细胞培养板中，每孔3x10 5个细胞，待细胞生长密度达到90％进行病毒的扩增。在病毒扩增前，将改造的MDCK细胞用磷酸盐缓冲液(Phosphate Buffer Saline，PBS)洗两遍，用MoI＝0.001的重组病毒感染细胞，吸附1小时后更换为2mL含有0.2％牛血清白蛋白(Bovine Serum Actin,BSA)和1μg/mL的TPCK的MEM培养液，然后于感染后12、24、48、72小时收集上清液存于-80℃保存。
实施例4利用空斑实验测病毒的滴度
实验分为如下两组：
第一组用野生型A/Chicken/Shandong/830/2014(H9N2)流感病毒，第二组用拯救出的重组病毒A830-HA，在可稳定表达A/Chicken/Shandong/830/2014(H9N2)流感病毒NA的MDCK细胞中进行空斑实验。
空斑实验：
(1)将稳定表达NA的MDCK细胞均匀平铺于两个6孔板中，每孔8x10 5个细胞。配制无血清2x DMEM培养液，用NAOH溶液调试PH值为7-7.5，用滤器过滤备用。配制1.6％低熔点琼脂糖：称取0.18g琼脂糖溶于15mL超纯水中，微波炉40s至完全溶解后，放入42℃水浴锅中恒温保存。
(2)稀释病毒：取八个1.5mL离心管，标记为1-8号，分别向离心管中加入900μL的无血清DMEM培养液；然后向1号管内加入100μL的病毒液，充分混匀，再从1号管中取100μL液体加入2号管中，混匀，依次倍比稀 释到8号管。
(3)取出6孔板，吸弃培养液，用含钙、镁离子的PBS洗一遍。第1-6孔分别加入稀释浓度为10 -3-10 -8稀释度的病毒液，放入37℃温箱孵育1h。期间每隔15分钟，取出轻轻晃匀，使病毒液充分吸收。
(4)配制维持液：向13.5mL的无血清2ⅹDMED培养液中，加入1200μL 5％BSA、300μL非必需氨基酸NEAA和37.5μL浓度为1mg/mL TPCK-trypsin储存液于37℃恒温放置。
(5)1h后取出6孔板，吸弃孔中剩余的病毒液，用加钙、镁离子的PBS洗一遍。混合1.6％低熔点琼脂糖和维持液，混匀后，向6孔板的每个孔中加入4mL。待凝固后，放入37℃恒温培养。
(6)3天后取出6孔板，将孔内的凝胶弃掉。每个孔加入500μL的考马斯亮蓝染料(Coomassie Brilliant blue)，室温静置5分钟，用水缓慢冲洗掉染料，观察统计每个孔的空斑数，并制作病毒的生长曲线图，如图3实验组A830-HA重组病毒的生长曲线跟对照组A830病毒在细胞的生长曲线趋势一样，说明重组病毒A830-HA在稳定表达NA的MDCK细胞系中具有良好的复制增殖能力。
实施例5动物实验
1.安全性试验：以攻毒后SPF鸡存活率为指标研究重组病毒A830-HA在SPF鸡体内的安全性。
将健康的4-5周龄的SPF鸡随机分成4组，每组8只。用3种不同滴度包括10 5、10 6、10 7PFU的A830-HA重组流感病毒经鼻腔接种攻毒后，每天观察统计每组中SPF鸡的存活率。如图4所示利用不同滴度的A830-HA重组流感病毒经鼻腔攻毒后，并不会影响SPF鸡的存活率，说明在没有外 源性NA存在时，重组病毒A830-HA对机体不具有感染性。
2.免疫保护实验：该复制缺陷型二价弱毒活疫苗对A型流感病毒H9N2和H5N1中和实验的研究。
将健康的4-5周龄的SPF鸡随机分成4组，每组8只。第一组为阴性对照组，用PBS免疫接种；第二组阳性对照，用10PFU/只的A830病毒液鼻腔接种免疫，第三组和第四组分别用10PFU/只和100PFU/只A830-HA重组病毒液接种免疫。免疫后第29天时，以上四组(表1)均用10ⅹLD 50野生A830病毒液攻毒，统计存活率(如图5所示使用10PFU/只和100PFU/只A830-HA重组病毒接种免疫的实验组，与阳性对照组一致，其体重跟存活率并无变化，说明A830-HA重组病毒对机体具有良好的免疫效果)。
表1：小鼠免疫攻毒列表
Figure PCTCN2018114270-appb-000001
采集以上四组中实验SPF鸡的血液，分离血清；进行血凝抑制实验和病毒中和试验。
血凝抑制实验测抗野生A830病毒血清抗体效价：
(1)将待检血清放置冰上融化备用。取V型96孔微量反应板，做好标记。
(2)用微量移液器向第1-10孔各加入25μL生理盐水，第11孔加50μL生理盐水。
(3)用微量移液器吸取25μL被检血清，放入第1孔，吸头浸于液体中缓慢吹吸几次，使被检血清与稀释液混合均匀，再吸取25μL液体小心地移至第2孔，如此连续稀释至第10孔，最后第10孔吸取25μL液体弃掉，被检血清稀 释倍数依次为1：2—1：1024。第11孔为红细胞对照，第12孔为抗原对照。
(4)第1-10孔每孔再各加入25μL含有4个单位的病毒液。第11孔为红细胞对照孔，不加病毒液；第12孔为抗原对照，加病毒液。
(5)置振荡器上振荡1-2min后，放37℃静止20min。
(6)每孔再加入25μL 0.8％红细胞悬浮液，放微量振荡器上振荡1-2min混匀，置37℃，15min后判定结果。
(7)结果判定：凝集现象表现为红细胞平铺在V型管底壁；未凝集的红细胞在V型血凝孔中呈现为明显的红色圆点。将反应板倾斜45度角后判定结果，当红细胞呈现泪滴状流淌且没有凝集颗粒时为100％抑制。
(8)结果结算：按Reed-Muench两氏或Karber法进行计算。
表2：血凝抑制实验测抗野生A830病毒血清抗体效价
Figure PCTCN2018114270-appb-000002
由表2中血凝效价数据可知，2、3、4组中血凝效价都很高，说明针对A/Chicken/Shandong/830/2014(H9N2)血清抗体的浓度很高，间接表明重组病毒A830-HA对于H9N2亚型流感病毒感染具有良好的免疫保护作用。
病毒微量中和实验：
(1)将MDCK细胞铺于96孔细胞培养板中。
(2)待检血清于56℃30分钟灭火。
(3)倍比稀释待检血清，向每管不同稀释倍数的血清中加入100PFU的重组A830-HA病毒液。
(4)培养箱中取出培养MDCK细胞的96孔细胞培养板，弃掉培养液，用PBS 洗两遍。
(5)将(3)中所述配制好的不同稀释倍数的血清与病毒混合液对应的加入到96孔细胞培养板中，做好标记，置于37℃培养箱中培养72小时。
(6)检测中和滴度。
表3：病毒微量中和实验
Figure PCTCN2018114270-appb-000003
由表3中的中和滴度数据可知，3、4组中和滴度很高，说明针对A/wild duck/Hunan/021/2005(H5N1)的血清抗体的浓度很高，表明重组病毒A830-HA对于H5N1亚型流感病毒感染具有良好的免疫保护作用。

Claims (9)

  1. 一种制备表达H5亚型HA的复制缺陷型重组H9N2禽流感病毒的方法，其特征在于，所述的方法如下：
    1)在H9N2病毒的神经氨酸酶NA的5′端的包装信号区和3′端的包装信号区内插入H5N1亚型的血凝素HA基因的开放阅读框，形成用于合成A型流感病毒NA-HA的DNA片段；
    2)将1)中构建的用于合成A型流感病毒NA-HA的DNA片段克隆到质粒上形成重组质粒；
    3)构建表达H9N2流感病毒神经氨酸酶NA基因的MDCK细胞系；
    4)将2)中构建的重组质粒，与表达H9N2的PB2、PB1、PA、HA、NP、M、NS各个基因片段的重组质粒共同转染到293T细胞和可稳定表达H9N2流感病毒神经氨酸酶NA基因的MDCK细胞系中，并收集转染后293T细胞和表达NA的MDCK细胞的上清培养液；
    5)在稳定表达H9N2亚型禽流感病毒神经氨酸酶NA的MDCK细胞系上扩增4)中拯救出的重组流感病毒。
  2. 如权利要求1所述的制备方法，其特征在于，所述的1)中5′端的包装信号区为H9N2病毒的神经氨酸酶NA的5′端的203个碱基对。
  3. 如权利要求1所述的制备方法，其特征在于，所述的1)中3′端的包装信号区为H9N2病毒的神经氨酸酶NA的3′端的195个碱基对。
  4. 如权利要求1所述的制备方法，其特征在于，所述的用于合成A型流感病毒NA-HA的DNA片段，其核苷酸序列为SEQ ID NO:1。
  5. 如权利要求1所述的制备方法，其特征在于，所述的2)中的质粒为质粒pHW2000。
  6. 一种表达H5亚型HA的复制缺陷型重组H9N2禽流感病毒，其特征在于， 所述的重组H9N2禽流感病毒是使用权利要求1所述的方法制备的。
  7. 权利要求6所述的重组H9N2禽流感病毒在制备疫苗中的应用。
  8. 如权利要求7所述的应用，其特征在于，所述的疫苗为弱毒疫苗。
  9. 一种弱毒疫苗，其特征在于，所述的弱毒疫苗中使用的抗原包含有权利要求6所述的重组H9N2禽流感病毒。
PCT/CN2018/114270 2018-09-14 2018-11-07 一种表达h5亚型ha的复制缺陷型重组h9n2禽流感病毒 WO2020052035A1 (zh)

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