CN108018300A - 区分免疫和感染动物h7亚型禽流感疫苗株及其制备方法和应用 - Google Patents
区分免疫和感染动物h7亚型禽流感疫苗株及其制备方法和应用 Download PDFInfo
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- CN108018300A CN108018300A CN201711168682.7A CN201711168682A CN108018300A CN 108018300 A CN108018300 A CN 108018300A CN 201711168682 A CN201711168682 A CN 201711168682A CN 108018300 A CN108018300 A CN 108018300A
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Abstract
本发明公开了区分免疫和感染动物H7亚型禽流感疫苗株及其制备方法和应用,高致病H7禽流感不仅给畜牧业带来巨大经济损失,而且严重威胁公共卫生学的安全。虽然常规的H7亚型禽流感全病毒灭活疫苗效果确实,价格低廉,应用面广等优点,但在血清学上无法区分免疫和感染动物,给禽流感的监测和净化带来巨大障碍。本发明用首次B型流感的NA作为标签,成功建立一种构建区分免疫和感染H7亚型禽流感疫苗株的方法。该发明对H7亚型禽流感的防控和净化具有重要的应用价值和公共卫生学意义。
Description
技术领域
本发明属于基因工程疫苗技术领域,涉及一种制备区分免疫和感染H7亚型禽流感疫苗株的方法及应用。
背景技术
禽流感病毒属于正黏病毒科,流感病毒属。流感病毒根据抗原性不同可分为A,B,C型,其中A型流感宿主广泛(包括禽类,人,猪等)致病力较强危害巨大。B型流感仅感染人和海豹,致病力相对较低。C型流感病毒,仅在人与猪中发现。A型和B型流感的基因组可以分为PB2,PB1,PA,NP,HA,NA,M,NS共8个基因片段。宿主被感染后,可以对HA,NA,M1和NP产生大量抗体。其中HA可以直接诱导主要中和抗体。以往的研究发现A型和B型病毒诱导的针对HA,NA,M1和NP的四种主要抗体在血清学上无交叉反应性。根据A型流感病毒粒子的表面囊膜蛋白血凝素(HA)和神经氨酸酶NA不同,将A型流感分为众多亚型(HnNn)。其中HA有18种亚型,NA有11种亚型。不同亚型HA蛋白间序列同源性在40%-80%之间(Air G M.Proceedings ofthe National Academy of Sciences of the United States of America,1981,78(12):7639.Nobusawa E,et al.Virology,1991,182(2):475-485)。B型流感没有亚型之分,各个毒株基因相似性较高。根据HA的抗原性和基因型不同,B型流感病毒目前仅分为两个群,分别为Victoria群(依据B/Victoria/2/1987命名)和Yamagata群(依据B/Yamagata/16/1988命名)。禽类中几乎存在所有A型流感的各种亚型,在病毒的储存和进化中伴有重要作用。禽流感全球肆虐给养禽业带来巨大经济损失,随着禽流感病毒对人类的逐渐适应,禽流感感染人的事例也逐渐增多。相比季节性型人流感,禽流感感染人具有发病严重,死亡率高的特征,严重威胁公共卫生安全。在众多禽流感亚型中,高致病性H7亚型禽流感危害巨大,不仅造成巨大经济损失而且可以直接感染人。高致病性H7禽流感可以在数天内导致家禽100%死亡,而且可以直接感染人。人感染后症状严重,死亡率高。
目前疫苗是防控禽流感最有效的方法之一。以鸡胚高度适应株PR8内部基因为背景,外部基因(HA,NA)替换为针对流行株的构建的疫苗株具有安全,有效和廉价等优点,在中国使用最为广泛,在禽流感疫苗的防控中起到重要作用。但是这种全病毒灭活疫苗不能在血清学上区分免疫动物和感染动物,给禽流感病毒的监测和净化带来巨大障碍。由于HA蛋白负责与受体结合,协助病毒入侵细胞;NA蛋白是破坏HA于受体的结合,协助病毒从细胞释放。因此保持HA-NA之间的功能平衡(HA-NA的匹配性),直接影响流感病毒的复制能力,生长特性(Mitnaul L J,Matrosovich M N,Castrucci M R,et al.Balanced Hemagglutininand Neuraminidase Activities Are Critical for Efficient Replication ofInfluenza A Virus[J].Journal of Virology,2000,74(13):6015-20.)。因此选择匹配度较好的HA和NA是研制优异疫苗株的关键之一(Murakami S,et al.Growth Determinantsfor H5N1Influenza Vaccine Seed Viruses in MDCK Cells[J].Journal of Virology,2008,82(21):10502.)。为了保证疫苗株HA和NA有较好的匹配度,通常情况下HA和NA基因都来源于同一株病毒。引入异源的NA可能打破HA-NA之间的功能性平衡,从而降低病毒的生长和复制能力,甚至导致重组病毒无法救获。一般而言,这样的风险会随着引入NA基因相似性(与同源NA相比)降低而不断增加。不同亚型的NA之间的替换,会影响获救重组病毒的复制和生长等生物学特性。这也是自然界里为什么只存在少数优势亚型组合(如常见H9N2,H5N1,H7N9等)而不是HA-NA的随机组合(如罕见H9N1,H5N9等)(Wagner R et al,Functional balance between haemagglutinin and neuraminidase in influenzavirus infections[J].Reviews in Medical Virology,2002,12(3):159)。Rudneva et al用N1基因和不同亚型HA组合,发现获救的H3,H4,H10和H13的重组病毒在鸡胚上的生长特性比其野生型的病毒更差(Rudneva I A et al.Influenza A virus reassortants withsurface glycoprotein genes of the avian parent viruses:effects of HA and NAgene combinations on virus aggregation.[J].Archives of Virology,1993,133(3-4):437-450.由于B型NA蛋白和A型差异很大(相似性<30%),引入B型NA获得A/B型嵌合病毒的成功几率很小。此外获救的A/B型NA嵌合病毒可能存在生长性能的缺陷,需要在体外连续传代适应。而连续传代有引起抗原变异的风险,从而导致制备的疫苗株的抗原性与原有的野生流行株有较大差异。截止目前,含有H7亚型HA和含有B型NA的嵌合病毒没有任何成功救获的报道。
现有的H7亚型全病毒灭活疫苗虽然具有免疫效果确实,价格低廉等的优点,但是无法在血清学上区分免疫动物和感染动物(DIVA)严重影响了对病毒流行性的监测,阻碍对H7亚型禽流感在养殖场的彻底净化,给公共卫生和食品安全带来持续风险。因此,制备出能够区分免疫和感染的H7亚型新型禽流感疫苗株为目前所需。
发明内容
为了解决上述存在的问题,本申请首次用B型流感病毒的NA基因作为标记,研制出一种制备区分免疫和感染H7新型禽流感疫苗的方法。此外本发明通过对NS基因的部分缺失和HA的致弱修饰使得获救的疫苗株的安全性明显优于普通的疫苗株。因此本发明提供制备安全有效,生产成本低而且可以通过血清学方法区分免疫和感染动物的H7禽流感疫苗的方法,具有重要的应用价值和突出的公共卫生安全意义。
本发明的目的在于提供一种区分免疫和感染的H7亚型禽流感疫苗株及其应用。
本发明的另一目的在于提供一种制备区分免疫和感染的H7亚型禽流感疫苗株方法。
本发明所采取的技术方案是:
标签基因序列在制备区分免疫和感染A型流感病毒的H7亚型禽流感疫苗株中的应用,所述标签基因序列含有编码B型流感病毒NA蛋白胞外区氨基酸序列的DNA序列,或者含有编码与该胞外区氨基酸序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性氨基酸序列的DNA序列;
或者,所述标签基因序列含有B型流感病毒NA基因中编码胞外区氨基酸序列的DNA序列,或者含有与该DNA序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性的序列;
或者,所述标签基因序列为编码B型流感病毒NA蛋白的DNA序列,或者为编码与该NA蛋白氨基酸序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性氨基酸序列的DNA序列;
或者,所述标签基因序列为B型流感病毒NA基因的DNA序列,或者为与该DNA序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性的序列。
进一步的,所述H7亚型禽流感疫苗株中还含有H7亚型HA基因或突变的H7亚型HA基因;所述突变的H7亚型HA基因能够将野生型HA蛋白中的氨基酸序列VPKGKRTARGLF突变为VPSSRSRGLF或VPKGRGLF。
进一步的,所述B型流感病毒包括Victoria群和Yamagata群的B型流感病毒。
进一步的,所述B型流感病毒具体包括但不限于病毒株B/Massachusetts/2/2012,B/Brisbane/60/2008,B/Yamagata/16/1988,B/Malaysia/2506/04。
进一步的,所述标签基因序列两端还含有包装信号序列,所述包装信号为H1亚型NA的包装信号,或者为与H1亚型NA的包装信号具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的包装信号序列。
进一步的,所述标签基因序列两端还含有包装信号序列,其中5′端包装信号序列包括非编区序列、胞内区序列、跨膜区序列。
进一步的,所述胞内区序列编码5~7个氨基酸,氨基酸序列位于胞内。
进一步的,跨膜区序列编码24~32个氨基酸,氨基酸序列位于跨膜区。
进一步的,所述标签基因序列的5′端包装信号序列为SEQ ID NO:3,或者为与SEQIDNO:3具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的序列。
进一步的,所述标签基因序列两端还含有包装信号序列,其中3′端包装信号序列为SEQID NO:4,或者为与SEQ ID NO:4具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的序列。
一种制备区分免疫和感染A型流感病毒的H7亚型禽流感疫苗株的方法,包括以下步骤:将标签基因序列与H7亚型禽流感病毒的HA基因或突变的H7亚型HA基因;通过反向遗传操作系统获救得到重组疫苗株,即区分免疫和感染A型流感病毒的H7亚型禽流感疫苗株;
所述突变的H7亚型HA基因能够将野生型HA蛋白中的氨基酸序列VPKGKRTARGLF突变为VPSSRSRGLF或VPKGRGLF;
所述标签基因序列含有编码B型流感病毒NA蛋白胞外区氨基酸序列的DNA序列,或者含有编码与该胞外区氨基酸序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性氨基酸序列的DNA序列;
或者,所述标签基因序列含有B型流感病毒NA基因中编码胞外区氨基酸序列的DNA序列,或者含有与该DNA序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性的序列;
或者,所述标签基因序列为编码B型流感病毒NA蛋白的DNA序列,或者为编码与该NA蛋白氨基酸序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性氨基酸序列的DNA序列;
或者,所述标签基因序列为B型流感病毒NA基因的DNA序列,或者为与该DNA序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性的序列。
进一步的,所述标签基因序列两端还含有包装信号序列。
进一步的,所述标签基因序列的5′端包装信号序列为SEQ ID NO:3,或者为与SEQIDNO:3具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的序列。
进一步的,所述标签基因序列的3′端包装信号序列为SEQ ID NO:4,或者为与SEQIDNO:4具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的序列。
进一步的,所述反向遗传操作系统获救过程中还用到6个PR8内部基因,即ΔNS或野生型NS与PB2、PB1、PA、NP、M;其中ΔNS为突变的NS基因,ΔNS的核苷酸序列如SEQ ID NO:5所示。
区分免疫和感染A型流感病毒的H7亚型禽流感疫苗株,其命名为H7亚型禽流感疫苗候选株Re-Mu2H7-DIVA-ΔNS,已保藏于中国典型培养物保藏中心,保藏编号为CCTCCNO:V201742。
上述所述疫苗株在制备禽流感疫苗中的应用。
申请人将本发明疫苗株Re-Mu2H7-DIVA-ΔNS保藏于中国典型培养物保藏中心,保藏单位地址为中国武汉武汉大学,保藏中心于2017年10月19日收到申请人提供的疫苗株。保藏中心给予该培养物的保藏号为CCTCC NO:V201742,提议的分类命名为H7亚型禽流感疫苗候选株Re-Mu2H7-DIVA-ΔNS,已于2017年10月28日鉴定保藏的疫苗株是存活的。
本发明的有益效果是:
(1)本申请首次用B型流感病毒的NA基因作为标签,研制出一种制备区分免疫和感染的H7新型禽流感疫苗的方法。
(2)本发明成功构建了区分免疫动物和感染动物的H7亚型禽流感疫苗株,在该疫苗株中,NA基因和HA基因具用很好的兼容性,表现出很好的复制和生长等生物学特性,不需要在体外的传代适应,避免了传代适应引起的抗原变异的缺点。即使续传3代,仍然保持对鸡胚低致病力和高滴度生长的特性。本发明具有重要的应用价值和突出的公共卫生安全意义。
(3)高致病H7禽流感不仅给畜牧业带来巨大经济损失,而且严重威胁公共卫生学的安全。虽然常规的H7亚型禽流感全病毒灭活疫苗效果确实,但在血清学上无法区分免疫和感染产生的抗体,给禽流感的监测和净化带来巨大障碍。本发明首次用B型流感的NA作为标签,成功构建区分免疫和感染H7亚型禽流感疫苗株,对H7亚型禽流感的防控和净化具有重要的意义和应用价值。
附图说明
图1为人工合成A/B嵌合型NA基因的结构示意图;
图2为pFLu载体图谱及流感病毒基因片段的克隆示意图;
图3为免疫荧光检测抗Re-Mu2H7-DIVA-ΔNS的血清与A型流感NA的反应性。
具体实施方式
下面结合具体实施例和附图,对发明进行详细说明,但本发明的实施方案不局限于此。未注明的常规实验方法请参考《分子克隆实验指南第三版》(萨姆布鲁克主编,科学出版社,2002)。
实施例1禽流感疫苗株Re-MuH7-DIVA-ΔNS病毒的制备方法
(1)构建低致病性HA突变基因
pFlu载体是一种双向转录载体,既可以在人polI启动子先转录完整病毒的RNA,又可以在CMV启动下转录病毒mRNA,从而合成病毒蛋白(Hoffmann et al.,PNAS,USA 97,6108-6113,2000)。
人工合成的野生型H7亚型禽流感中的HA基因(KY855526),通过定点突变技术将该野生型HA氨基酸序列中高致病性特征序列(VPKGKRTARGLF)中的(KRTA)删除,获得相应的低致病性的Mu1HA基因序列;或者将高致病性特征序列(VPKGKRTARGLF)突变为(VPSSRSRGLF),获得相应的低致病性的Mu2HA基因序列;将突变后的Mu1HA、Mu2HA基因通过位点克隆到pFlu载体获得重组质粒pFlu-Mu1HA和pFlu-Mu2HA,构建原理图如图2所示。
(2)构建低致病性A/B嵌合型NA基因
构建图1所示人工合成A/B嵌合型NA基因,其中含有B型流感病毒NA中编码胞外区蛋白氨基酸序列(SEQ ID NO:1)的DNA序列(SEQ ID NO:2)作为标签基因序列,SEQ ID NO:2所示的含B型NA胞外区的序列来自B型流感病毒Yamagata群中的B/Massachusetts/2/2012(Ping J et al,PNAS,2016,113(51):E8296-E8305),标签基因序列两端还含有包装信号序列,其中5′端包装信号序列(SEQ ID NO:3)包括非编区序列、胞内区序列和跨膜区序列,3′端包装信号序列为SEQ ID NO:4。通过BsmBI位点将嵌合NA插入pFlu载体获得重组质粒pFlu-PR8-BNA。
(3)Re-MuH7-DIVA-ΔNS疫苗株的获得
为了保证疫苗株的安全性,我们对病毒野生型NS1基因进行修改,修改后的突变基因ΔNS的核苷酸序列如SEQ ID NO:5所示,含突变基因ΔNS的病毒丧失拮抗干扰素的功能,因此只能在干扰素缺失的细胞或干扰素系统发育不全的鸡胚生长繁殖,因此具有良好的安全性。
采用经典“6+2”流感反向遗传操作系统拯救重组疫苗株Re-MuH7-DIVA-ΔNS。将6个PR8内部基因pFlu-PR8-PB2,pFlu-PR8-PB1,pFlu-PR8-PA,pFlu-PR8-NP,pFlu-PR8-M,pFlu-PR8-ΔNS和2个外部基因pFlu-Mu1-HA/pFlu-Mu2-HA,pFlu-PR8-BNA各0.5ug分别共转染到293T细胞(Lipofectamine 3000)。转然后24h更换含有终浓度为0.5ug/ml TPCK-Trypsin的培养液,并在转然后48h收集细胞上清,将细胞上清按照0.2ml/枚通过尿囊腔接种8日龄SPF鸡胚。接种后的鸡胚在37℃温箱内培养48h。收集鸡胚尿囊液(F0代),分别获得,疫苗株Re-Mu1H7-DIVA-ΔNS和Re-Mu2H7-DIVA-ΔNS,并测定其是否有血凝价。如果没有血凝价,将收获病毒盲传一代,再测其是否有血凝价。
实施例2含不同低致病修饰的突变基因Mu1HA、Mu2HA的疫苗株在鸡胚上的生长特性
将实施例1所得疫苗株Re-Mu1H7-DIVA-ΔNS和Re-Mu2H7-DIVA-ΔNS分别在8日龄SPF鸡胚上连续传代(F0-F3)。接种后48小时收获每代病毒并测其血凝价(HA效价)。
检测结果如表1所示,从中可以看出,Re-Mu2H7-DIVA-ΔNS的生长特性明显优于Re-Mu1H7-DIVA-ΔNS。由于Re-Mu1H7-DIVA-ΔNS和Re-Mu2H7-DIVA-ΔNS重配病毒的基因背景几乎一样,仅在对高致病性野生型HA的修饰不同。因此Mu2-HA的修饰方式更有利于H7亚型禽流感在鸡胚上的生长,达5log2~6log2。传代过程未见任何鸡胚致死,说明重组病毒对鸡胚表现为低致病性或者无致病性,具有良好的安全性。取F0和F3病毒通过RT-PCR扩增人工合成A/B嵌合型NA基因,经过测序证明嵌合NA基因可以稳定遗传给子代病毒。
综上所述,获救的Re-Mu2H7-DIVA-ΔNS株变为低致病力或者无致病力,只能在干扰素系统缺失的细胞系或干扰素系统发育不全的低日龄鸡胚上生长繁殖,具有良好的安全性。在8日龄SPF鸡胚培养48小时,其HA效价可达到为6log2。由于Re-Mu2H7-DIVA-ΔNS株的NS1部分缺失,虽然在鸡胚上的生长滴度要低于正常非缺失的病毒,但是其安全性比非缺失的野生型病毒更好。
表1.不同低致病修饰的疫苗株Re-Mu1H7-DIVA-ΔNS、Re-Mu2H7-DIVA-ΔNS在鸡胚上的生长特性
申请人将本发明疫苗株Re-Mu2H7-DIVA-ΔNS保藏于中国典型培养物保藏中心,保藏单位地址为中国武汉武汉大学,保藏中心于2017年10月19日收到申请人提供的疫苗株。保藏中心给予该培养物的保藏号为CCTCC NO:V201742,提议的分类命名为H7亚型禽流感疫苗候选株Re-Mu2H7-DIVA-ΔNS,已于2017年10月28日鉴定保藏的疫苗株是存活的。
实施例3区分免疫和感染A型流感病毒的H7亚型禽流感疫苗株Re-MuH7-DIVA-ΔNS的制备方法
实施例3中的制备方法同实施例1,除了在构建图1所示人工合成A/B嵌合型NA基因时,B型流感病毒NA中编码胞外区蛋白氨基酸序列的DNA序列与实施例1中的不同外,其他均同实施例1。
在本实施例中,B型流感病毒NA中编码胞外区蛋白氨基酸序列(SEQ ID NO:6)的DNA序列如SEQ ID NO:7所示,作为标签基因序列,SEQ ID NO:7所示的序列来自B型流感病毒Victoria群中的B/Brisbane/60/2008(Ping J et al,PNAS,2016,113(51):E8296-E8305)。
下面对本发明制备的Re-MuH7-DIVA-ΔNS疫苗株作进一步的效果检测。
方法:将实施例1制得的Re-Mu2H7-DIVA-ΔNS疫苗株(NA胞外区基因来自Yamagata群的B/Massachusetts/2/2012)、实施例3制得的Re-MuH7-DIVA-ΔNS疫苗株(NA胞外区基因来自Victoria群中的B/Brisbane/60/2008)、对照组1的PR8-ΔNS病毒(NS缺陷的PR8病毒)、对照组2的PR8-WT病毒(PR8野生型病毒)分别按照0.2ml/枚通过尿囊腔接种8日龄SPF鸡胚。接种后的鸡胚在37℃温箱内培养48h。收集鸡胚尿囊液(F0代),测定其血凝效价。将F0代病毒稀释后接种10枚SPF鸡胚,培养48h后收获病毒定义为F1代。用同样的方法,将F1代病毒连续传代致F3代。
结果:检测结果如表2所示,从中可以看出,为了证明不同分支的B型NA基因是否能与H7亚型的HA(H7-BNA)良好匹配,我们选取了不同群的代表株B/Brisbane/60/2008(Victoria群)和Massachusetts/2/2012(Yamagata群)的NA基因进行研究,结果发现来自B型不同分支NA的基因(Victoria和Yamagata群)与H7均表现出良好的匹配性,实施例1和3所得Re-PR8-MuH7-ΔNS疫苗株不需要在鸡胚上传代适应就可接近自身的上限(5log2~6log2)。从表2中还可看出,虽然含突变型ΔNS的疫苗株比野生型的生长滴度低2log2~3log2,但是含突变型ΔNS的疫苗株的安全性更好。
表2.不同嵌合重组的H7亚型禽流感病毒在鸡胚的生长特性
来自不同群的B型流感病毒代表株B/Brisbane/60/2008(Victoria群)和Massachusetts/2/2012(Yamagata群)二者NA全基因核苷酸序列的同源性为94.9%,氨基酸序列同源性为94.9%;二者编码NA蛋白胞外区的DNA序列的同源性为95.1%,NA蛋白胞外区的氨基酸序列的同源性为94.6%。由于B型流感只分为Victoria和Yamagata群,本发明证明了来自这两个群的NA的代表株(实施例1和实施例3)均能和H7的HA有较好的兼容性,说明B型流感病毒的NA基因均可用于制备区分免疫和感染A型流感病毒的H7亚型禽流感疫苗株。
实施例4Re-MuH7-DIVA-ΔNS灭活疫苗的制备
收取上述实施例制备的Re-MuH7-DIVA-ΔNS疫苗株F0、F1、F2或F3代尿囊液50ml,用终浓度0.25%的福尔马林溶液37℃灭活24h。将灭活后的尿囊液加入2%的Tween-80,待充分溶解后与含有3%Span 80的白油乳化,乳化比例为1:3,剪切乳化速度12000rpm,3min。经剂型检验、粒度检验、粘度检验、稳定性检验,确定灭活油苗为乳白色的油包水型乳剂,粘度低,颗粒大小均匀,稳定性好,适宜注射。
实施例5Re-MuH7-DIVA-ΔNS灭活疫苗免疫动物的效果检测
方法:将10只3周龄SPF鸡免疫上述制备的Re-Mu2H7-DIVA-ΔNS疫苗,0.3ml/只,颈部皮下注射,免疫后21天采血,分离血清,测定HI抗体。
结果:实验表明Re-Mu2H7-DIVA-ΔNS刺激机体产生高水平的HI抗体,其HI效价在免疫后第3周的平均值(log2)为9.3±0.95。HA和HI试验参考GBT 18936-2003(高致病性禽流感诊断技术)。
实施例6 血清学实验
将现有A型流感的N1,N2,N6,和N9基因通过KpnI和NheI位点克隆到pCAGGS真核表达质粒,命名为pCAGGS-N1,pCAGGS-N2,pCAGGS-N6,pCAGGS-N9。把1μg各个pCAGGS-N1,pCAGGS-N2,pCAGGS-N6,pCAGGS-N9质粒转染到预先在铺24孔细胞培养板上293T细胞。转染后30h,用免疫荧光方法检测以下7组鸡血清和N1,N2,N6,N9的反应性。
7组鸡血清的情况如下:
抗Re-Mu2H7-DIVA-ΔNS鸡血清:只免疫本发明Re-Mu2H7-DIVA-ΔNS灭活疫苗的鸡血清;
抗H7N9标准鸡血清:H7N9标准血清,购于哈尔滨兽医研究所。
抗H5+H7血清:免疫H5N1Re-8株+H7N9Re-1株全病毒灭活疫苗的临床血清。
抗N1鸡血清:周龄SPF鸡分别免疫(肌肉注射)100μg pCAGGS-N1,免疫4周后采集全血,制备血清。
抗N2鸡血清:周龄SPF鸡分别免疫(肌肉注射)100μg pCAGGS-N2,免疫4周后采集全血,制备血清。
抗N6鸡血清:周龄SPF鸡分别免疫(肌肉注射)100μg pCAGGS-N6,免疫4周后采集全血,制备血清。
抗N9鸡血清:周龄SPF鸡分别免疫(肌肉注射)100μg pCAGGS-N9,免疫4周后采集全血,制备血清。
免疫荧光方法如下:
1)在每细胞加入0.5ml的4%多聚甲醛固定20分钟,然后用PBS洗三遍。
2)0.2%Triton X 100通透10分钟,然后用PBS洗三遍。
3)5%BSA封闭1小时,然后用PBS洗三遍。
4)一抗用含有1%BSA的PBS稀释相应倍数(抗Re-Mu2H7-DIVA-ΔNS,抗H7N9标准,抗H5+H7,100倍;抗N1/N2/N6/N9,20倍),并在每孔加入0.5ml,37℃湿盒内孵育1小时,然后用PBS洗三遍。
5)鸡二抗(Alexa Fluor 594驴抗鸡IgY)用含有1%BSA的PBS稀释200倍,加入0.5ml于每孔,室温孵育0.5小时,然后用PBS洗三遍。
6)用荧光显微镜观察。
结果:在293T细胞分别表达流感N1,N2,N6和N9神经氨酸酶,用免疫荧光的方法,检测Re-Mu2H7-DIVA-ΔNS免疫后3周的血清是否与N1、N2、N6和N9反应,结果发现抗Re-Mu2H7-DIVA-ΔNS的血清不与N1,N2,N6和N9蛋白发生交叉反应(如表3和图3所示),现有全A型病毒疫苗(H5N1Re-8株+H7N9Re-1株)免疫的临床血清和H7N9标准血清能与N9蛋白发生强烈反应。此实验证实免疫Re-Mu2H7-DIVA-ΔNS疫苗不仅可以诱导高水平HI抗体而且可以区分免疫和感染动物,而且克服了现有H7亚型全病毒疫苗不能区分免疫和感染动物的缺点。
表3.比较不同抗原免疫的鸡血清和各NA亚型的反应性
注:N/A:不适用,ND:未检验
实施例7区分免疫和感染A型流感病毒的H7亚型禽流感疫苗株Re-MuH7-DIVA-ΔNS的制备方法
本实施例7中的制备方法同实施例1,除了在构建图1所示人工合成A/B嵌合型NA基因时,所用的B型流感病毒NA序列为编码NA全蛋白序列的DNA序列之外,其他均同实施例1,其中,该NA的DNA序列来自B型流感病毒Yamagata群中的B/Massachusetts/2/2012的NA全基因序列(Ping J et al,PNAS,2016,113(51):E8296-E8305)。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 宋, 家升
<120> 区分免疫和感染动物H7亚型禽流感疫苗株及其制备方法和应用
<130>
<160> 7
<170> PatentIn version 3.5
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acaggtgttg acatggctct gtaa 1224
Claims (17)
1.标签基因序列在制备区分免疫和感染A型流感病毒的H7亚型禽流感疫苗株中的应用,所述标签基因序列含有编码B型流感病毒NA蛋白胞外区氨基酸序列的DNA序列,或者含有编码与该胞外区氨基酸序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性氨基酸序列的DNA序列;
或者,所述标签基因序列含有B型流感病毒NA基因中编码胞外区氨基酸序列的DNA序列,或者含有与该DNA序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性的序列;
或者,所述标签基因序列为编码B型流感病毒NA蛋白的DNA序列,或者为编码与该NA蛋白氨基酸序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性氨基酸序列的DNA序列;
或者,所述标签基因序列为B型流感病毒NA基因的DNA序列,或者为与该DNA序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性的序列。
2.根据权利要求1所述的应用,其特征在于,所述H7亚型禽流感疫苗株中还含有H7亚型HA基因或突变的H7亚型HA基因;所述突变的H7亚型HA基因能够将野生型HA蛋白中的氨基酸序列VPKGKRTARGLF突变为VPSSRSRGLF或VPKGRGLF。
3.根据权利要求1任一所述的应用,其特征在于,所述B型流感病毒包括Victoria群和Yamagata群的B型流感病毒。
4.根据权利要求3所述的应用,其特征在于,所述B型流感病毒具体包括但不限于病毒株B/Massachusetts/2/2012,B/Brisbane/60/2008,B/Yamagata/16/1988,B/Malaysia/2506/04。
5.根据权利要求1所述的应用,其特征在于,所述标签基因序列两端还含有包装信号序列,所述包装信号为H1亚型NA的包装信号,或者为与H1亚型NA的包装信号具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的包装信号序列。
6.根据权利要求1所述的应用,其特征在于,所述标签基因序列两端还含有包装信号序列,其中5′端包装信号序列包括非编区序列、胞内区序列、跨膜区序列。
7.根据权利要求6所述的应用,其特征在于,胞内区序列编码5~7个氨基酸,氨基酸序列位于胞内。
8.根据权利要求6所述的应用,其特征在于,跨膜区序列编码24~32个氨基酸,氨基酸序列位于跨膜区。
9.根据权利要求6所述的应用,其特征在于,所述标签基因序列的5′端包装信号序列为SEQ ID NO:3,或者为与SEQ ID NO:3具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的序列。
10.根据权利要求1所述的应用,其特征在于,所述标签基因序列两端还含有包装信号序列,其中3′端包装信号序列为SEQ ID NO:4,或者为与SEQ ID NO:4具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的序列。
11.一种制备区分免疫和感染A型流感病毒的H7亚型禽流感疫苗株的方法,其特征在于,包括以下步骤:将标签基因序列与H7亚型禽流感病毒的HA基因或突变的H7亚型HA基因;通过反向遗传操作系统获救得到重组疫苗株,即区分免疫和感染A型流感病毒的H7亚型禽流感疫苗株;
所述突变的H7亚型HA基因能够将野生型HA蛋白中的氨基酸序列VPKGKRTARGLF突变为VPSSRSRGLF或VPKGRGLF;
所述标签基因序列含有编码B型流感病毒NA蛋白胞外区氨基酸序列的DNA序列,或者含有编码与该胞外区氨基酸序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性氨基酸序列的DNA序列;
或者,所述标签基因序列含有B型流感病毒NA基因中编码胞外区氨基酸序列的DNA序列,或者含有与该DNA序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性的序列;
或者,所述标签基因序列为编码B型流感病毒NA蛋白的DNA序列,或者为编码与该NA蛋白氨基酸序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性氨基酸序列的DNA序列;
或者,所述标签基因序列为B型流感病毒NA基因的DNA序列,或者为与该DNA序列具有至少90%同源性、或至少92%同源性、或至少95%同源性、或至少98%同源性的序列。
12.根据权利要求11所述的方法,其特征在于,所述标签基因序列两端还含有包装信号序列。
13.根据权利要求12所述的方法,其特征在于,所述标签基因序列的5′端包装信号序列为SEQ ID NO:3,或者为与SEQ ID NO:3具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的序列。
14.根据权利要求12所述的方法,其特征在于,所述标签基因序列的3′端包装信号序列为SEQ ID NO:4,或者为与SEQ ID NO:4具有至少80%同源性、或至少85%同源性、或至少90%同源性、或至少95%同源性的序列。
15.根据权利要求11所述的方法,其特征在于,所述反向遗传操作系统获救过程中还用到6个PR8内部基因,即ΔNS或野生型NS与PB2、PB1、PA、NP、M;其中ΔNS为突变的NS基因,ΔNS的核苷酸序列如SEQ ID NO:5所示。
16.区分免疫和感染A型流感病毒的H7亚型禽流感疫苗株,其命名为H7亚型禽流感疫苗候选株Re-Mu2H7-DIVA-ΔNS,已保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:V201742。
17.权利要求16所述的疫苗株在制备禽流感疫苗中的应用。
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EP (1) | EP3715458B1 (zh) |
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CN109402070A (zh) * | 2018-11-05 | 2019-03-01 | 扬州大学 | 一种重组h7n9亚型禽流感病毒株、灭活标记疫苗及其制备方法 |
WO2019100687A1 (zh) * | 2017-11-21 | 2019-05-31 | 宋家升 | 区分免疫和感染动物h7亚型禽流感疫苗株及其制备方法和应用 |
CN113827713A (zh) * | 2020-06-23 | 2021-12-24 | 普莱柯生物工程股份有限公司 | 一种抗h7亚型禽流感病毒的疫苗组合物、及其制备方法和应用 |
CN114438043A (zh) * | 2022-01-27 | 2022-05-06 | 浙江迪福润丝生物科技有限公司 | 一种区分感染和免疫的致弱流感病毒疫苗株及疫苗 |
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RU2757397C1 (ru) * | 2021-05-14 | 2021-10-15 | Федеральное государственное бюджетное научное учреждение "Институт экспериментальной медицины" (ФГБНУ "ИЭМ") | Реассортантный штамм вируса гриппа В/60/Вашингтон/2019/3676 (линия Виктория) для производства живой гриппозной интраназальной вакцины для взрослых и для детей |
RU2757396C1 (ru) * | 2021-05-14 | 2021-10-15 | Федеральное государственное бюджетное научное учреждение "Институт экспериментальной медицины" (ФГБНУ "ИЭМ") | Вакцинный штамм вируса гриппа В/60/Гонконг/2017/5584 (линия Виктория) для производства живой гриппозной интраназальной вакцины для взрослых и для детей |
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WO2019100687A1 (zh) * | 2017-11-21 | 2019-05-31 | 宋家升 | 区分免疫和感染动物h7亚型禽流感疫苗株及其制备方法和应用 |
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CN109402070B (zh) * | 2018-11-05 | 2020-07-31 | 扬州大学 | 一种重组h7n9亚型禽流感病毒株、灭活标记疫苗及其制备方法 |
CN113827713A (zh) * | 2020-06-23 | 2021-12-24 | 普莱柯生物工程股份有限公司 | 一种抗h7亚型禽流感病毒的疫苗组合物、及其制备方法和应用 |
CN113827713B (zh) * | 2020-06-23 | 2024-07-05 | 普莱柯生物工程股份有限公司 | 一种抗h7亚型禽流感病毒的疫苗组合物、及其制备方法和应用 |
CN114438043A (zh) * | 2022-01-27 | 2022-05-06 | 浙江迪福润丝生物科技有限公司 | 一种区分感染和免疫的致弱流感病毒疫苗株及疫苗 |
CN114438043B (zh) * | 2022-01-27 | 2023-12-15 | 浙江迪福润丝生物科技有限公司 | 一种区分感染和免疫的致弱流感病毒疫苗株及疫苗 |
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EP3715458A4 (en) | 2021-12-01 |
WO2019100687A1 (zh) | 2019-05-31 |
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US11510976B2 (en) | 2022-11-29 |
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