CN109097341B - 一种同时表达ha和hef的复制缺陷型重组流感病毒 - Google Patents
一种同时表达ha和hef的复制缺陷型重组流感病毒 Download PDFInfo
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Abstract
本发明提供一种复制缺陷型A、D二价流感病毒弱毒活疫苗制备方法。本发明构建了一种重组病毒株，在没有改变基因稳定性前体下，构建的重组病毒可稳定的同时表达A型流感病毒H1N1亚型的表面蛋白血凝素HA和D型流感病毒唯一的表面蛋白血凝素酯酶融合蛋白HEF。所构建的弱毒株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H1N1亚型和D型流感病毒均可产生免疫保护作用。那么对于A型和D型流感的预防与控制具有巨大的社会意义。
Description
技术领域
本发明属于流感病毒疫苗技术研发领域，具体涉及一种可同时表达A型流感病毒H1N1表面蛋白血凝素HA和D型流感病毒唯一表面蛋白血凝素酯酶融合蛋白HEF的重组流感病毒的制备方法及用途。
背景技术
流感是由流感病毒(Influenza Virus)引起的严重危害人和动物呼吸道系统的传染病。跟据其核蛋白抗原性不同将流感病毒分为A、B、C三型(IAV、IBV、ICV)，感染人的流感病毒主要是A型和B型。D型流感病毒(IDV)是近年来发现的一种新型流感病毒，感染人和哺乳动物。
目前流感疫苗是预防和控制流感病毒流行的主要方法，常用的流感疫苗有灭活苗和弱毒苗：灭活苗存在问题主要是抗原性差，只能诱导抗体免疫；弱毒流感疫苗是低温适应流感病毒株，能同时诱导较强的抗体免疫应答和细胞免疫应答反应，但是目前批准的弱毒流感疫苗毒株只能表达一个亚型的HA，并且低温适应毒株研发的周期长，还有潜在的毒力回复突变的潜在危险；而且目前还没有针对D型流感病毒的疫苗。
发明内容
本发明所要解决的技术问题：研究开发一种复制缺陷型A、D二价流感病毒弱毒活疫苗制备方法。
本发明首先提供一种复制缺陷型重组流感病毒的制备方法，所述方法包括如下的步骤：
1)合成包含有D型流感病毒表面糖蛋白-血凝素酯酶融合蛋白HEF的开放阅读框、A型H1N1流感病毒NA片段3′端非编码区的202个核苷酸和5′端非编码区的185个核苷酸的DNA片段，将合成的DNA片段插入到载体质粒pHW2000上，构建成重组质粒pHW-NA-HEF；
其中DNA片段，其一种具体的序列为SEQ ID NO:1；
2)将步骤1)构建的重组质粒pHW-NA-HEF与用于表达A型流感病毒亚型H1N1的PB2、PB1、PA、HA、NP、M、NS基因的重组质粒共同转染293T细胞和稳定表达流感病毒H1N1亚型神经氨酸酶NA的MDCK细胞；
所述的NA基因，其核苷酸序列为SEQ ID NO：2；
3)收集转染后293T细胞和MDCK细胞的上清培养液；
4)在稳定表达A型流感病毒H1N1亚型神经氨酸酶NA基因的MDCK细胞系上拯救并扩增重组流感病毒；
本发明另一个方面提供一种重组病毒株，该病毒株是用上述方法构建的；
构建的重组病毒株能够用来制备疫苗；
所述的疫苗，为弱毒活疫苗。
本发明构建了一种重组病毒株，在没有改变基因稳定性前体下，构建的重组病毒可稳定的同时表达A型流感病毒H1N1亚型的表面蛋白血凝素HA和D型流感病毒唯一的表面蛋白血凝素酯酶融合蛋白HEF。所构建的弱毒株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H1N1亚型和D型流感病毒均可产生免疫保护作用。那么对于A型和D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；其中a)为D型流感病毒表面蛋白HEF的开放阅读框插入到H1N1神经氨酸酶NA开放阅读框位置，并保留NA两端的非编码区的模拟图b)流感病毒H1N1亚型的8个基因片段模拟图
图2：A/Califronia/2009/07H1N1流感病毒神经氨酸酶NA的凝胶电泳图。
图3：合成NA-HEF的DNA序列凝胶电泳图。
图4：CA09-HEF病毒生长曲线图。
图5：不同浓度重组CA09-HEF病毒液攻毒后实验动物体重变化。
图6：不同浓度重组CA09-HEF病毒液攻毒后实验动物存活率。
图7：免疫接种重组CA09-HEF病毒液后实验动物体重变化。
图8：免疫接种重组CA09-HEF病毒液后实验动物存活率。
具体实施方式
本发明构建了可同时表达A型流感病毒H1N1表面糖蛋白血凝素HA和D型流感病毒表面糖蛋白HEF的复制缺陷型二价流感病毒弱毒株，可作为疫苗侯选株。本发明所构建的重组病毒只能在表达流感病毒NA的稳定细胞系上复制，从而大大提高了本病毒作为弱毒疫苗的安全性。
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
1、将A型流感病毒亚型H1N1的PB2、PB1、PA、HA、NP、M、NS的RNA片段体外反转录制成cDNA，以cDNA为模板扩增流感病毒的基因片段，分别克隆到表达载体质粒pHW2000上。即得7个质粒分别为pHW-PB2、pHW-PB1、pHW-PA、pHW-HA、pHW-NP、pHW-M、pHW-NS。
2、构建质粒pHW-NA-HEF
新构建质粒pHW-NA-HEF(图1)：首先合成DNA序列NA-HEF(Genscript公司)，该序列保留了A型流感病毒H1N1NA片段3′端非编码区的202个核苷酸和5′端非编码区的185个核苷酸的包装信号。利用PCR技术进行目的片段NA(202nt)-ORF(HEF)-NA(185nt)的扩增(如图2)。
上游引物序列：TATTGGTCTCAGGGAGCAAAAGCAGGAGT、
下游引物序列：ATATGGTCTCGTATTAGTAGAAACAAGGAGTTTTTT；
新合成的目的片段NA(202nt)-ORF(HEF)-NA(185nt)的DNA序列为SEQ ID NO:1；
分别用BsmB1和Bsa1酶切质粒pHW2000和PCR回收产物NA-HEF；利用T4DNA ligase连接以上两个酶切片段的回收产物，即构成质粒pHW-NA-HEF。
实施例2拯救重组病毒
构建细胞系：构建可稳定表达A/Califronia/2009/07(H1N1)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H1N1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，即可将培养液更换为含有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/Califronia/2009/07(H1N1)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 ModifiedEagle′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)。
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7x105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-HEF、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(TosylsufonylPhenylalanyl 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。)将改造的MDCK细胞铺在6孔细胞培养板中，每孔3x105个细胞，待细胞生长密度达到50％-60％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 SerumActin,BSA)和1μg/mL的TPCK的MEM培养液，然后于感染后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/California/2009/04(H1N1)流感病毒，第二组用拯救出的重组病毒CA09-HEF，在可稳定表达A/California/2009/04(H1N1)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8x105个细胞。配制无血清2ⅹDMEM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，用加钙、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4所示，实验组CA09-HEF重组病毒的生长曲线跟对照组CA09病毒在细胞的生长曲线趋势一样，说明重组病毒CA09-HEF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：以攻毒后小鼠体重变化和存活率为指标研究重组病毒CA09-HEF在BALB/C小鼠体内的安全性。
将健康的6-8周龄的BALB/c雌鼠随机分成4组，每组8只。用3种不同滴度包括105、106、107PFU的CA09-HEF重组流感病毒经鼻腔攻毒后，每天观察统计每组中小鼠体重的变化和存活率。如图5和图6所示利用不同滴度的CA09-HEF重组流感病毒经鼻腔攻毒后，并不会影响BALB/c雌鼠的体重和存活率，说明在没有外源性NA存在时，重组病毒CA09-HEF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H1N1亚型和D型流感病毒中和实验的研究。
将健康的6-8周龄的BALB/C雌鼠随机分成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CA09病毒液鼻腔接种免疫，第三组和第四组分别用10PFU/只和100PFU/只CA09-HEF重组病毒接种免疫。免疫后第29天时，以上四组(表1)均用10ⅹLD50野生CA09病毒液攻毒，统计体重变化和存活率(如图7和图8所示使用10PFU/只和100PFU/只CA09-HEF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CA09-HEF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
采集以上四组中实验小鼠的血液，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CA09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CA09病毒血清抗体效价
由表2中血凝效价数据可知，2、3、4组中血凝效价都很高，说明血清抗体的浓度很高。
病毒微量中和实验：
(1)将HRT-18T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CA09-HEF病毒液。
(4)培养箱中取出培养HRT-18T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
由表3中的中和滴度数据可知，3、4组中和滴度很高，说明血清抗体的浓度很高。
Claims (6)
1.一种复制缺陷型重组流感病毒的制备方法，其特征在于，所述方法包括如下的步骤：
1）合成包含有D型流感病毒表面糖蛋白-血凝素酯酶融合蛋白HEF的开放阅读框、A型H1N1流感病毒NA片段3′端非编码区的202个核苷酸和5′端非编码区的185个核苷酸的DNA片段，将合成的DNA片段连接到载体质粒pHW2000上，构建成重组质粒pHW-NA-HEF；其中D型流感病毒表面糖蛋白-血凝素酯酶融合蛋白HEF的开放阅读框插入到A型流感病毒H1N1 NA片段3′端非编码区的202个核苷酸和5′端非编码区的185个核苷酸的包装信号之间；形成的元件顺序为 202nt NA-HEF ORF-185nt NA；
2）将步骤1）构建的重组质粒pHW-NA-HEF与用于表达A型流感病毒亚型H1N1的PB2、PB1、PA、HA、NP、M、NS七个基因序列的质粒共同转染到293T细胞和稳定表达流感病毒H1N1亚型神经氨酸酶NA的MDCK细胞；
3）收集转染后293T细胞和MDCK细胞的上清培养液；
4）在稳定表达A型流感病毒H1N1亚型神经氨酸酶NA的MDCK细胞系上扩增拯救出的重组流感病毒CA09-HEF。
2.一种重组病毒株，其特征在于，所述的重组病毒株是用权利要求1所述的方法构建的。
3.权利要求2所述的重组病毒株在制备疫苗中的应用。
4.如权利要求3所述的应用，其特征在于，所述的疫苗，为弱毒活疫苗。
5.一种弱毒活疫苗，其特征在于，所述的疫苗，其中抗原包含有权利要求2所述的重组病毒株。
6.权利要求3中 所述的重组病毒株在制备中和抗体中的应用。
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| CN116144612B (zh) * | 2022-12-08 | 2023-11-10 | 广州医科大学附属第一医院（广州呼吸中心） | 重组乙型流感病毒及其制备方法与应用 |
| CN115998854A (zh) * | 2023-01-30 | 2023-04-25 | 南京市第二医院 | 一组流感病毒基因的非编码区在制备mRNA疫苗中的应用 |
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| CN103316357A (zh) * | 2013-02-05 | 2013-09-25 | 中国人民解放军军事医学科学院微生物流行病研究所 | 携带rsv基因的重组流感病毒嵌合疫苗及其制备方法 |
| CN103757032A (zh) * | 2014-01-28 | 2014-04-30 | 中国人民解放军第三0二医院 | 一种以流感病毒为载体的hcv嵌合疫苗及其制备方法 |
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| US8715940B2 (en) * | 1999-04-06 | 2014-05-06 | Wisconsin Alumni Research Foundation | Method of making recombinant influenza virus |
| PT1820853E (pt) * | 1999-04-06 | 2012-01-03 | Wisconsin Alumni Res Found | Vírus recombinante da gripe para vacinas e terapia genética |
| US8592197B2 (en) * | 2003-07-11 | 2013-11-26 | Novavax, Inc. | Functional influenza virus-like particles (VLPs) |
| US8227425B2 (en) * | 2007-01-23 | 2012-07-24 | Therapicon Srl | Antiviral compounds |
| EP2048237A1 (en) * | 2007-10-05 | 2009-04-15 | Avir Green Hills Biotechnology Research Development Trade Ag | Replication deficient Influenza virus for the expression of heterologous sequences |
| AU2009254501B2 (en) * | 2008-06-05 | 2014-07-31 | Ablynx N.V. | Amino acid sequences directed against envelope proteins of a virus and polypeptides comprising the same for the treatment of viral diseases |
| WO2011087839A1 (en) * | 2009-12-22 | 2011-07-21 | Baxter International Inc. | Vaccine to influenza a virus |
| KR20120132506A (ko) * | 2010-02-18 | 2012-12-05 | 마운트 시나이 스쿨 오브 메디슨 | 인플루엔자 바이러스 질환의 예방 및 치료에 사용되는 백신 |
| JP2013529913A (ja) * | 2010-06-02 | 2013-07-25 | アヴィール グリーン ヒルズ バイオテクノロジー リサーチ ディベロプメント トレード アクチエンゲゼルシャフト | Ｒｎａウィルスの作製のための方法及びヘルパーウィルス |
| EP2747778B1 (en) * | 2011-08-26 | 2017-12-06 | Wisconsin Alumni Research Foundation | Influenza viruses with mutant pb2 gene segment as live attenuated vaccines |
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