CN101624580B - 一种表达人工修饰合成的甲型h1n1流感病毒ha-na-m1基因的重组杆状病毒 - Google Patents
一种表达人工修饰合成的甲型h1n1流感病毒ha-na-m1基因的重组杆状病毒 Download PDFInfo
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Abstract
本发明涉及病毒学领域。具体涉及一种人工修饰合成的含有甲型H1N1流感病毒主要免疫原性基因HA-NA-M1的重组杆状病毒,该毒株QP-Ac-HNM1属于杆状病毒(Baculovirus),保藏在中国典型培养物保藏中心(CCTCC),保藏号为CCTCC-V200912。该重组病毒可以同时表达甲型H1N1流感病毒HA、NA和M1蛋白,形成病毒样颗粒。该病毒样颗粒可以用来研制成疫苗,预防人和猪感染甲型H1N1流感病毒。
Description
技术领域
本发明涉及病毒学领域。更具体地,本发明涉及一种新的含有人工修饰合成的甲型H1N1流感病毒主要免疫原性基因HA-NA-M1和标记基因eGFP的重组杆状病毒株,这种重组毒株标记基因eGFP和甲型H1N1流感病毒HA-NA-M1蛋白,并包装成流感样病毒颗粒。该病毒颗粒研制的疫苗可以预防人和猪甲型H1N1流感病毒的感染。
背景技术
甲型H1N1流感(Influenza virus H1N1)是引起人和猪的呼吸道疾病的重要原发病原之一,常常与其他病原混合感染造成更严重的损失。该病毒属于正粘病毒科流感病毒属,根据病毒核蛋白抗原性不同,分为甲、乙和丙三个型(国际称其为A、B和C三个型)。甲型流感病毒(influenza virus,IV)为有囊膜的RNA病毒,其囊膜主要由血凝素(HA)、神经氨酸酶(NA)以及基质蛋白(M1)构成。
甲型流感病毒危害巨大,不但影响畜牧业(尤其养禽业)健康发展,更重要的是可以引起人类的流感大暴发,产生重要的公共卫生危害。人类历史上发生过3次流感大暴发,即1918年由H1N1亚型流感病毒引起的西班牙流感、1957年由H2N2亚型流感病毒引起的香港流感和1968年由H3N2亚型流感病毒引起的亚洲流感。其病原均由当时存在于禽类的甲型流感病毒直接突变或和人的流感病毒在猪体内重组而来,如1918年西班牙流感病毒H1N1的HA和NA基因分别具有猪流感病毒和禽流感病毒的分子特征。2009年4月出现甲型H1N1流感病毒是一种新型变异流感病毒。这种新的甲型流感病毒HA、NP和NS基因来源于与1918年人流感病毒密切相关的经典猪流感病毒;NA和M来自于1979年引入的欧洲猪流感病毒;PB1来自人的H3N2;PB2和PA来源于禽流感,该流感在1998年前后引入到猪体(Chang LY,et al.Novel Swine-originInfluenza Virus A(H1N1):The First Pandemic ofthe 21st Century.J Formos Med Assoc,2009;108(7):526-623)。
目前甲型H1N1流感几乎从美洲蔓延至世界各地,传播途径既可以动物传人,也可以人传人。截至2009年7月6日,目前全球已有94512人感染,429人死亡(http://www.who.int/csr/don/2009_07_06/en/index.html);高效疫苗和抗病毒药物预防控制甲型H1N1流感病毒有效措施。现阶段治疗甲型H1N1流感病毒最有效途径是抗病毒药物治疗,目前使用较多的是oseltamivir(又称Tamiflu)和zanamivir,但是目前在丹麦、日本、美国、中国香港等国家和地区发现抗Tamiflu耐药毒株(http://www.who.int/csr/disease/swineflu/notes/h1n1_antiviral_resistance_20090708/en/index.html),从而增加了该疾病带来的危险,该领域迫切需要有效的疫苗来预防控制该甲型H1N1流感病毒。迄今为止,还没有商品化的疫苗问世,而传统的季节性流感疫苗不能有效预防该新型流感病毒。据WHO报道常规灭活疫苗目前正在加紧研制,还没有问世(http://www.who.int/csr/disease/swineflu/notes/ h1n1_vaccine_20090713/en/index.html),新型疫苗还未见报道。因此,本领域迫切需要开发出具有高效、安全的针对甲型H1N1流感的基因工程疫苗。
发明内容
本发明的一个目的就是提供编码甲型H1N1人工修饰合成的免疫原性基因cDNA序列。
本发明的另一个目的是提供一种转移载体pFBDPHmHNM1P10eGFP,该载体含有编码具有甲型H1N1流感病毒免疫原性基因HA-NA-M1的cDNA序列和标记基因eGFP。
本发明的另一个目的就是提供一种表达人工修饰合成的甲型H1N1流感病毒HA-NA-M1的重组杆状病毒,它含有人工修饰合成的甲型H1N1流感病毒主要免疫原性基因HA-NA-M1以及标记基因eGFP的重组杆状病毒株,该毒株被命名为QP-Ac-HNM1,属于杆状病毒(Baculovirus),于2009年7月20送交湖北省武汉市武汉大学内的中国典型培养物保藏中心(CCTCC)保藏,其保藏号为CCTCC-V200912。
申请人根据密码子的偏爱性人工合成了一种来源于甲型H1N1流感病毒编码的主要免疫原性蛋白HA、NA和M1的核苷酸序列,如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示。
更详细的技术方案见《具体实施方式》所述。
本发明的有益效果是:
1.本发明根据密码子的偏爱性,人工修饰合成了甲型H1N1流感病毒HA、NA和M1基因,提高了这些基因在杆状病毒中表达水平。
2.本发明所构建的杆状病毒转移载体含有绿色荧光标记基因,提高了重组杆状病毒的筛选效率,便于监控外源蛋白的表达。
3.本发明是将合成HA、NA和M1基因分别置于杆状病毒PH启动子下串连表达,表达的蛋白在昆虫细胞中形成流感样病毒颗粒(VLP)。
4.本发明的重组杆状病毒所表达包装形成的流感样病毒颗粒(VLP)可以刺激小鼠诱导产生免疫反应。
附图说明
序列表SEQ ID NO:1是本发明人工合成的HA基因核苷酸序列;
序列表SEQ ID NO:2是本发明人工合成的NA基因核苷酸序列;
序列表SEQ ID NO:3是本发明人工合成的M1基因核苷酸序列;
图1:显示了表达绿荧光蛋白基因的杆状病毒载体pFBD-gpA-P10eGFP物理图谱;
图2:显示表达人工修饰合成的甲型H1N1流感病毒HA-NA-M1基因杆状病毒载体pFBDPHmHNM1P10eGFP的物理图谱;
图3:是杆状病毒载体pFBDPHmHNM1P10eGFP的酶切鉴定的电泳图谱;
图4:是在荧光显微镜下(4A)和光镜下(4B)重组杆状病毒感染sf9细胞结果的图片。
具体实施方式
实施例1:一种表达人工修饰合成的甲型H1N1流感病毒HA-NA-M1基因重组杆状病毒QP-Ac-HNM1的构建。
1、甲型H1N1流感病毒主要免疫原性基因的合成。
参照甲型H1N1流感病毒A/California/04/2009(H1N1)毒株的HA、NA和M1基因序列,根据密码子的偏爱性人工合成HA、NA和M1基因的序列,其中NA和M基因合成到一起,同时在NA基因N端加上杆状病毒多角体基因的启动子(PH),在其C端引SV40病毒的poly(A)序列,在M1基因的N端加入启动子PH。
2、杆状病毒转移载体pFBDPHmHNM1P10eGFP的构建。
根据Invitrogen公司Bac-to-Bac Baculovirus Expression System(试剂盒)中杆状病毒载体pFastBacDual骨架,加将鸡的globin的polyA插入到StuI和SalI之间,将绿荧光蛋白基因eGFP插入到P10下游的NcoI和KpnI之间,获得改造的杆状病毒载体pFBD-gpA-P10eGFP,在该载体的基础上,用BamHI和EcoRI分别酶切mHA基因和载体pFBD-gpA-P10eGFP,回收mHA基因和载体pFBD-gpA-P10eGFP,用T4DNA连接酶连接过夜,转化DH5α感受态细胞,涂布含有氨苄青霉素抗性平皿,37℃培养16小时,提取重组质粒进行酶切鉴定后获得载体pFBDPHmHAgpA-P10eGFP;再用XhoI和HindIII分别酶切mNA-mM1基因和pFBDPHmHAgpA-P10eGFP,回收mNA-Mm1基因和载体pFBDPHmHAgpA-P10eGFP,然后连接,提取重组质粒,酶切鉴定,获得最终杆状病毒转移载体pFBDPHmHNM1P10eGFP。
3、重组病毒杆状病毒QP-Ac-HNM1的构建。
(1)重组穿梭载体(Bacmid)的构建
取测序鉴定正确的杆状病毒转移载体pFBDPHmHNM1P10eGFP和pFBD-gpA-P10eGFP 2μL与100μLDH10Bac大肠杆菌感受态细胞混合,冰浴30min后,于42℃ 45s水浴进行热激,然后冰浴2min,加入900μL LB液体培养基(氯化钠浓度为10%),37℃振摇培养4h,按10-1、10-2、10-3稀释后,各取100μL涂布于三抗高盐LB平板(卡那霉素、庆大霉素和四环素,使用浓度按Invitrogen公司Bac-to-Bac BaculovirusExpression System(试剂盒)操作说明书),37℃培养24-48h,通过蓝白斑筛选、纯化阳性菌落。
(2)重组穿梭载体(Bacmid)的提取
无菌挑取含重组Bacmid的阳性菌落于高盐LB液体培养基中,培养至细菌对数生长期时,收集菌体用0.3mL溶液I(50mmol/L葡萄糖,10mmol/L EDTA,25mmol/L Tris-Cl(pH8.0))重悬,加入0.3mL溶液II(0.2mol/LNaOH,1%SDS,现用现配)轻微混匀,室温静置5min,缓慢加入0.3mL溶液III(3mol/L的乙酸钾,pH5.0)混匀,冰浴5-10min,14000r/min离心10min,上清加到0.5mL异丙醇中,混匀冰浴5-10min,室温14000r/min离心15min,70%乙醇洗涤沉淀,干燥后溶于40μL TE中,可立即使用或-20℃保存,避免反复冻融。
(3)重组杆状病毒获得
利用脂质体介导转染法(按Invitrogen公司Bac-to-Bac Baculovirus Expression System(试剂盒)操作说明书操作),将筛选、纯化后的重组穿梭载体(Bacmid)转染到sf9昆虫细胞(购自武汉大学中国典型物保藏中心)中,于28℃培养,待出现细胞病变和发绿色荧光后,收集细胞培养上清即可获得重组杆状病毒。
实施例2:重组杆状病毒QP-Ac-HNM1鉴定、VLP疫苗制备和免疫原性实验。
1、重组杆状病毒QP-Ac-HNM1鉴定。
将sf9细胞接种6孔细胞培养板,待细胞长成80-90%单层时,用1个MOI重组杆状病毒QP-Ac-HNM1接种,同时设对照杆状病毒,逐日观察。感染72小时后90%以上的sf9细胞发亮绿荧光。
取400μl培养上清和细胞悬液,用胰蛋白酶在56℃下处理4个小时,用等体积的酚/氯仿抽提二次,取上清加入1/10体积的3M醋酸纳和2倍体积的无水乙醇沉淀,离心弃上清,用500μl70%乙醇洗沉淀一次,自然干燥后加入50μl灭菌的去离子水重悬作为PCR模板。PCR鉴定所用的上游引物为:5’---ttttcccgggatccaccatgaaggccatc---3’,下游引物为:5’---ttttctcgaggattccgggtcactgtgag---3’。
PCR扩增条件如下:
LA Taq酶 0.5μl
10×LA buffer 5.0μl
模板 5.0μl
特异性上游引物 1.0μl
特异性下游引物 1.0μl
dNTP 2.0μl
ddH2O 35.5μl
反应条件
94℃ 3min
72℃ 3.0min
72℃ 10min
4℃ ∞
PCR扩增产物经0.8%琼脂糖凝胶电泳检测,结果表明能够扩增出一条2597bp特异的目的带,大小与预期一致,表明外源基因已经成功的插入到重组杆状病毒中。
2、重组杆状病毒QP-Ac-HNM1 VLP疫苗制备。
参考Pushko P等人的方法(Influenza virus-like particles comprised of the HA,NA,and M1proteins of H9N2 influenza virus induce protective immune response in BALB/c mice.Vaccine2005,23:5751-5759)提取甲型H1N1流感病毒样颗粒(VLP)。在刚长成单层的sf9细胞上,以3-5pfu/cell的感染复数接种重组杆状病毒QP-Ac-HNM1,72小时后通过荧光显微镜检测90%以上细胞发绿色荧光后收集病变细胞并超声波裂解,总体积为300ml。在4℃条件下8000g离心30min,然后用蔗糖浓缩到原体积的1/10,上清中即含有表达的VLP颗粒。将上清27,000rpm/min,超速离心3小时,沉淀重悬于2ml的PBS中,并用1ml的注射器反复吹打,然后病毒悬液用20-60%蔗糖密度梯度进行27,00rpm/min超速离心16小时,最上层的蛋白带即为流感病毒样颗粒VLP。获得的VLP用pH7.2的PBS重悬稀释,27,000rpm/min,超速离心3小时,沉淀重悬于2ml的PBS中,测定所获得的VLP蛋白浓度,配成1mg/ml浓度,然后用等体积的氟氏完全佐剂进行乳化制备成VLP疫苗,存储于4℃备用。
3、重组杆状病毒QP-Ac-HNM1 VLP疫苗免疫原性初步实验。
10只雌性6-8周龄的Blab/C小鼠分为两组,每组5只。第一组5只小鼠作为阴性对照,两后腿肌肉各注射PBS加氟氏完全佐剂乳化的对照疫苗100μl。第二组5只小鼠两后腿肌肉各注射VLP疫苗100μl,共含有2μgVLP蛋白。首免28天后用相同剂量加强免疫一次。分别于0天、14天、28天、42天采血,检测其血凝抑制抗体。血凝抑制抗体是用8个血凝单位的H1N2猪流感病毒进行检测(注:H1N2猪流感病毒的HA基因与甲型H1N1流感病毒的HA基因同源性达到95%)。结果如下表所示:初步表明VLP疫苗可以诱导小鼠产生免疫反应。
表1重组杆状病毒QP-Ac-HNM1 VLP疫苗免疫原性
尽管本发明的内容是结合本实施例进行说明,但是不能认为是对本发明范围的限制,本发明的范围由所附权利要求书限定。另外,本领域的技术人员在所附权利要求书限定的范围内对本发明进行各种改动或修饰,这些改动或修饰形式同样落在本发明范围内。
序列表
<110>华中农业大学
<120>一种表达人工修饰合成的甲型H1N1流感病毒HA-NA-M1基因的重组杆状病毒
<130>
<141>2009-07-14
<160>6
<170>PatentIn version 3.1
<210>1
<211>1701
<212>DNA
<213>甲型流感病毒(Influenza A virus)
<220>
<221>gene
<222>(1)..(1701)
<223>
<220>
<221>CDS
<222>(1)..(1701)
<223>
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Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn
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Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile
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325 330 335
aac atc cct tcc atc cag tcc aga ggc ctg ttc ggc gcc atc gcc ggt 1056
Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly
340 345 350
ttc atc gag ggc ggc tgg aca ggc atg gtg gac ggt tgg tac ggt tac 1104
Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr
355 360 365
cac cat cag aac gag cag ggc tcc ggt tac gct gcc gac ctg aag agc 1152
His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser
370 375 380
acc cag aac gcc atc gac gag atc acc aac aag gtg aac tcc gtg atc 1200
Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile
385 390 395 400
gag aag atg aac acc cag ttc acc gct gtg ggt aag gag ttc aac cac 1248
Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His
405 410 415
ctg gaa aag cgc atc gag aac ctc aac aag aag gtt gac gac ggt ttc 1296
Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe
420 425 430
ctg gac atc tgg acc tac aac gcc gag ctg ctg gtg ctg ctg gaa aac 1344
Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn
435 440 445
gag cgc acc ctg gac tac cac gat tcc aac gtg aag aac ctg tac gag 1392
Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu
450 455 460
aag gta cgc agc cag ctg aag aac aac gcc aag gaa atc ggc aac ggc 1440
Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly
465 470 475 480
tgc ttc gag ttt tac cac aag tgc gac aac acc tgc atg gag agc gtc 1488
Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val
485 490 495
aag aac ggc acc tac gac tac cca aag tac tcc gag gaa gcc aag ctc 1536
Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu
500 505 510
aac cgc gag gaa atc gac ggc gtg aag ctg gag tcc aca cgc atc tac 1584
Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr
515 520 525
cag att ctg gcc atc tat tcc act gtg gcc agc tcc ctg gtg ctg gtg 1632
Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Val
530 535 540
gtc tcc ctg ggc gcc atc agc ttc tgg atg tgc tct aac ggc tcc ctc 1680
Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu
545 550 555 560
cag tgt cgc atc tgc atc taa 1701
Gln Cys Arg Ile Cys Ile
565
<210>2
<211>566
<212>PRT
<213>甲型流感病毒(Influenza A virus)
<400>2
Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn
1 5 10 15
Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr
20 25 30
Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn
35 40 45
Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val
50 55 60
Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly
65 70 75 80
Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile
85 90 95
Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe
100 105 110
Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe
115 120 125
Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp
130 135 140
Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser
145 150 155 160
Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro
165 170 175
Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val
180 185 190
Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu
195 200 205
Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser
210 215 220
Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln
225 230 235 240
Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys
245 250 255
Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe
260 265 270
Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro
275 280 285
Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn
290 295 300
Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys
305 310 315 320
Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg
325 330 335
Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly
340 345 350
Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr
355 360 365
His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser
370 375 380
Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile
385 390 395 400
Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His
405 410 415
Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe
420 425 430
Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn
435 440 445
Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu
450 455 460
Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly
465 470 475 480
Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val
485 490 495
Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu
500 505 510
Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr
515 520 525
Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Val
530 535 540
Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu
545 550 555 560
Gln Cys Arg Ile Cys Ile
565
<210>3
<211>1410
<212>DNA
<213>甲型流感病毒(Influenza A virus)
<220>
<221>gene
<222>(1)..(1410)
<223>
<220>
<221>CDS
<222>(1)..(1410)
<223>
<400>3
atg aac cca aac cag aag atc atc acc att ggc tcc gtg tgc atg acc 48
Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Cys Met Thr
1 5 10 15
att ggc atg gcc aac ctg atc ctc cag atc ggc aac atc atc tcc atc 96
Ile Gly Met Ala Asn Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile
20 25 30
tgg atc agc cac tcc atc cag ctc ggc aac cag aac cag atc gag acc 144
Trp Ile Ser His Ser Ile Gln Leu Gly Asn Gln Asn Gln Ile Glu Thr
35 40 45
tgc aac cag agc gtc atc act tac gag aac aac acc tgg gtg aac cag 192
Cys Asn Gln Ser Val Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gln
50 55 60
acc tac gtg aac atc agc aac acc aac ttc gct gcc ggc cag tcc gtg 240
Thr Tyr Val Asn Ile Ser Asn Thr Asn Phe Ala Ala Gly Gln Ser Val
65 70 75 80
gtc tcc gtg aaa ctg gcc ggc aac tcc tct ctc tgc cct gtg agc ggc 288
Val Ser Val Lys Leu Ala Gly Asn Ser Ser Leu Cys Pro Val Ser Gly
85 90 95
tgg gct atc tac agt aaa gac aac agt gtg cgc atc ggt tcc aag ggc 336
Trp Ala Ile Tyr Ser Lys Asp Asn Ser Val Arg Ile Gly Ser Lys Gly
100 105 110
gat gtg ttc gtc atc cgc gag cca ttc atc tcc tgc tcc cct ctg gag 384
Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser Pro Leu Glu
115 120 125
tgc cgc acc ttc ttc ctg act cag ggc gcc ctg ctc aac gac aag cat 432
Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His
130 135 140
tcc aac ggc acc atc aaa gac cgc agc cca tac cgc acc ctg atg agc 480
Ser Asn Gly Thr Ile Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser
145 150 155 160
tgt cct atc ggt gag gtg cct tct cca tac aac tct cgc ttc gag tcc 528
Cys Pro Ile Gly Glu Val Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser
165 170 175
gtc gct tgg tcc gcc agc gct tgc cac gac ggc atc aac tgg ctg aca 576
Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Ile Asn Trp Leu Thr
180 185 190
atc ggc atc tct ggc cca gac aac ggc gcc gtg gct gtg ctg aag tac 624
Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr
195 200 205
aac ggc atc att acc gac acc atc aag agc tgg cgc aac aac atc ctg 672
Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu
210 215 220
cgc aca cag gag tct gaa tgt gcc tgt gtg aac ggt tct tgc ttc act 720
Arg Thr Gln Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr
225 230 235 240
gtg atg acc gac ggc cca agc aac ggc cag gcc tcc tac aag att ttc 768
Val Met Thr Asp Gly Pro Ser Asn Gly Gln Ala Ser Tyr Lys Ile Phe
245 250 255
cgc atc gag aag ggc aag atc gtc aag tcc gtc gag atg aac gcc cct 816
Arg Ile Glu Lys Gly Lys Ile Val Lys Ser Val Glu Met Asn Ala Pro
260 265 270
aac tac cac tat gag gaa tgc tcc tgt tac cct gac tct agc gag atc 864
Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ser Ser Glu Ile
275 280 285
acc tgt gtg tgc cgc gac aac tgg cac ggc tcc aac cgc cct tgg gtg 912
Thr Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val
290 295 300
tct ttc aac cag aac ctg gag tac cag atc ggc tac atc tgt agc ggc 960
Ser Phe Asn Gln Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly
305 310 315 320
atc ttc ggc gac aac cca cgc cct aac gac aag aca ggc agc tgc ggt 1008
Ile Phe Gly Asp Asn Pro Arg Pro Asn Asp Lys Thr Gly Ser Cys Gly
325 330 335
cca gtg tcc tct aac ggt gcc aac ggc gtg aag ggc ttc tcc ttc aag 1056
Pro Val Ser Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser Phe Lys
340 345 350
tac ggc aac ggt gtg tgg atc ggt cgc act aag agc atc agt tct cgc 1104
Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Ile Ser Ser Arg
355 360 365
aac ggt ttc gag atg att tgg gac cct aac ggc tgg act ggc acc gac 1152
Asn Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Gly Thr Asp
370 375 380
aac aac ttc tcc atc aag cag gac atc gtg ggc atc aac gag tgg tcc 1200
Asn Asn Phe Ser Ile Lys Gln Asp Ile Val Gly Ile Asn Glu Trp Ser
385 390 395 400
ggc tac agc ggc agc ttc gtg cag cac cca gag ctg aca ggc ctg gac 1248
Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp
405 410 415
tgt atc cgc cct tgc ttc tgg gtg gaa ctg atc cgc ggt cgc cct aag 1296
Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys
420 425 430
gag aac aca atc tgg act agc ggc agc agc atc tcc ttc tgt ggt gtg 1344
Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val
435 440 445
aac agt gac act gtg ggt tgg tct tgg cca gac ggt gct gag ctg cca 1392
Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro
450 455 460
ttc acc atc gac aag taa 1410
Phe Thr Ile Asp Lys
465
<210>4
<211>469
<212>PRT
<213>甲型流感病毒(Influenza A virus)
<400>4
Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Cys Met Thr
1 5 10 15
Ile Gly Met Ala Asn Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile
20 25 30
Trp Ile Ser His Ser Ile Gln Leu Gly Asn Gln Asn Gln Ile Glu Thr
35 40 45
Cys Asn Gln Ser Val Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gln
50 55 60
Thr Tyr Val Asn Ile Ser Asn Thr Asn Phe Ala Ala Gly Gln Ser Val
65 70 75 80
Val Ser Val Lys Leu Ala Gly Asn Ser Ser Leu Cys Pro Val Ser Gly
85 90 95
Trp Ala Ile Tyr Ser Lys Asp Asn Ser Val Arg Ile Gly Ser Lys Gly
100 105 110
Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser Pro Leu Glu
115 120 125
Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His
130 135 140
Ser Asn Gly Thr Ile Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser
145 150 155 160
Cys Pro Ile Gly Glu Val Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser
165 170 175
Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Ile Asn Trp Leu Thr
180 185 190
Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr
195 200 205
Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu
210 215 220
Arg Thr Gln Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr
225 230 235 240
Val Met Thr Asp Gly Pro Ser Asn Gly Gln Ala Ser Tyr Lys Ile Phe
245 250 255
Arg Ile Glu Lys Gly Lys Ile Val Lys Ser Val Glu Met Asn Ala Pro
260 265 270
Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ser Ser Glu Ile
275 280 285
Thr Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val
290 295 300
Ser Phe Asn Gln Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly
305 310 315 320
Ile Phe Gly Asp Asn Pro Arg Pro Asn Asp Lys Thr Gly Ser Cys Gly
325 330 335
Pro Val Ser Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser Phe Lys
340 345 350
Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Ile Ser Ser Arg
355 360 365
Asn Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Gly Thr Asp
370 375 380
Asn Asn Phe Ser Ile Lys Gln Asp Ile Val Gly Ile Asn Glu Trp Ser
385 390 395 400
Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp
405 410 415
Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys
420 425 430
Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val
435 440 445
Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro
450 455 460
Phe Thr Ile Asp Lys
465
<210>5
<211>759
<212>DNA
<213>甲型流感病毒(Influenza A virus)
<220>
<221>gene
<222>(1)..(759)
<223>
<220>
<221>CDS
<222>(1)..(759)
<223>
<400>5
atg agc ctc ctg acc gag gtg gag acc tac gtg ctg tct atc atc cct 48
Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro
1 5 10 15
tcc ggc cct ctc aag gcc gag atc gcc cag cgc ctg gag agc gtg ttc 96
Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Ser Val Phe
20 25 30
gcc ggc aag aac aca gac ctg gag gct ctc atg gag tgg ctg aag aca 144
Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr
35 40 45
cgc cca atc ctg tcc cct ctg act aag ggc att ctg ggc ttc gtg ttc 192
Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe
50 55 60
acc ctc acc gtg cct agc gag cgc ggt ctc cag cgt cgc cgc ttc gtc 240
Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val
65 70 75 80
cag aac gcc ctg aac ggt aac ggc gac cct aac aac atg gac cgc gca 288
Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala
85 90 95
gtg aag ctg tac aag aag ctc aag cgc gag atc acc ttc cac ggc gcc 336
Val Lys Leu Tyr Lys Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala
100 105 110
aag gag gtg tcc ctg agc tac tca act ggt gcc ctg gcc agc tgc atg 384
Lys Glu Val Ser Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met
115 120 125
ggc ctc atc tac aac cgc atg ggc aca gtg acc aca gaa gct gcg ttc 432
Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Ala Ala Phe
130 135 140
ggt ctg gtg tgc gcc act tgt gaa cag att gct gac tcc cag cac cgc 480
Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg
145 150 155 160
tct cac cgc cag atg gct act acc acc aac cct ctg atc agg cac gag 528
Ser His Arg Gln Met Ala Thr Thr Thr Asn Pro Leu Ile Arg His Glu
165 170 175
aac cgc atg gtg ctg gcc agc act acc gcc aag gct atg gaa cag atg 576
Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met
180 185 190
gct ggc tcc agc gaa cag gcc gct gag gct atg gag gtg gct aac cag 624
Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Asn Gln
195 200 205
act agg cag atg gtg cac gcc atg cgc act atc ggc act cat cct agc 672
Thr Arg Gln Met Val His Ala Met Arg Thr Ile Gly Thr His Pro Ser
210 215 220
tcc agt gct ggt ctg aag gac gac ctc ctg gag aac ctc cag gct tac 720
Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr
225 230 235 240
cag aag cgc atg ggc gtg cag atg cag cgc ttc aag tga 759
Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys
245 250
<210>6
<211>252
<212>PRT
<213>甲型流感病毒(Influenza A virus)
<400>6
Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro
1 5 10 15
Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Ser Val Phe
20 25 30
Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr
35 40 45
Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe
50 55 60
Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val
65 70 75 80
Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala
85 90 95
Val Lys Leu Tyr Lys Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala
100 105 110
Lys Glu Val Ser Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met
115 120 125
Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Ala Ala Phe
130 135 140
Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg
145 150 155 160
Ser His Arg Gln Met Ala Thr Thr Thr Asn Pro LeuIle Arg His Glu
165 170 175
Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met
180 185 190
Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Asn Gln
195 200 205
Thr Arg Gln Met Val His Ala Met Arg Thr Ile Gly Thr His Pro Ser
210 215 220
Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr
225 230 235 240
Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys
245 250
Claims (2)
1.一种重组的杆状病毒株,其特征在于,它含有人工修饰合成的甲型H1N1流感病毒主要免疫原性基因HA-NA-M1以及标记基因eGFP,该毒株被命名为QP-Ac-HNM1,属于杆状病毒(Baculovirus),保藏在中国典型培养物保藏中心,其保藏编号为CCTCC-V200912。
2.权利要求1所述的重组杆状病毒株在制备甲型H1N1流感病毒基因工程疫苗中的应用。
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CN103122352B (zh) * | 2012-09-27 | 2015-02-11 | 华中农业大学 | 一种猪圆环病毒2型重组杆状病毒及制备方法和应用 |
CN104561049A (zh) * | 2015-01-22 | 2015-04-29 | 华中农业大学 | 一种表达猪细小病毒vp2蛋白的重组杆状病毒及制备方法与应用 |
US20180128545A1 (en) * | 2016-11-08 | 2018-05-10 | Berry Metal Company | Modular furnace cooling wall |
CN111526886A (zh) * | 2017-10-27 | 2020-08-11 | 国家血清研究所 | 多基因流感疫苗 |
CN109295014B (zh) * | 2018-10-30 | 2021-08-17 | 华中农业大学 | 一种非典型猪瘟病毒e2蛋白重组杆状病毒及其制备方法和应用 |
CN110845580A (zh) * | 2019-11-05 | 2020-02-28 | 中国农业科学院兰州兽医研究所 | 一种猪细小病毒样颗粒的组装及其免疫原性鉴定方法 |
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Peter Pushko,etc..Influenza virus-like particles comprised of the HA,NA,and M1 proteins of H9N2 influenza virus induce protective immune responses in BALB/c mice.《Vaccine》.2005,第23卷 * |
PeterPushko etc..Influenza virus-like particles comprised of the HA |
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