CN116218795A - 表达h9亚型禽流感双拷贝ha基因的ⅶ型致弱新城疫重组病毒、制备方法和用途 - Google Patents
表达h9亚型禽流感双拷贝ha基因的ⅶ型致弱新城疫重组病毒、制备方法和用途 Download PDFInfo
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明属于生物领域，公开了一种表达H9亚型禽流感双拷贝HA基因的Ⅶ型致弱新城疫重组病毒，所述病毒为rDHN3mF‑2HA、rDHN3mF‑HA，rDHN3mF‑2HA的序列如序列表SEQ IDNO：1所示；rDHN3mF‑HA的序列如序列表SEQ ID NO：2所示。本发明的病毒在rDHN3‑mF病毒基因组的P基因和M基因之间的非编码区之间表达全长膜结合HA和另外一个带有截短跨膜结构域的可溶性HA蛋白，获得了能高效稳定表达H9N2AIV的HA蛋白的新城疫基因VII型减毒株重组病毒rDHN3mF‑2HA，经WB验证表明增加HA的可溶性表达与原HA基因相比，其表达效率更高。该重组病毒有望进一步开发成抗新城疫和H9N2亚型禽流感病毒的二联疫苗；同时，本发明还提供了该病毒的制备方法、用途，此外还涉及关键质粒的制备方法。
Description
技术领域
本发明涉及生物领域，具体涉及一种表达H9亚型禽流感双拷贝HA基因的Ⅶ型致弱新城疫重组病毒、制备方法、用途，此外还涉及关键质粒的制备方法。
背景技术
禽流行性感冒(Avian Influenza,AI)是由禽流感病毒(Avian Influenza virus,AIV)引起的一种急性呼吸道传染病，简称为禽流感。禽流感病毒属于甲型流感病毒，通常以其表面蛋白、血凝素(HA)和神经氨酸酶(NA)的组合为特征，导致许多不同的亚型，例如H1N1、H5N6或H9N2[1]。根据禽流感的致病性，可将其分为高致病性禽流感和低致病性禽流感。其中，H9N2亚型AIV通过分子表征和病理分型均被归为低致病性禽流感。在中国，H9N2已取代H5N6和H7N9成为鸡和鸭的主要AIV亚型[2]。家禽感染H9N2亚型AIV后一般呈亚临床症状，如咳嗽、打喷嚏、罗音、喘鸣等轻度呼吸系统症状[3]，还可对被感染鸡免疫系统产生明显影响，导致其免疫抑制[4]和继发感染，在肉鸡和青年鸡中易与其它病原体合并感染，引起死淘率升高，其高感染率和高传染性威胁着全球家禽业经济发展。此外，H9N2亚型病毒还能通过抗原漂移或抗原转移为新型的流感病毒提供基因，产生能够感染人类及其他动物的新型人兽共患病病毒，严重威胁公共卫生安全[5,6]。AIV属于正粘病毒科A型，包含一个分段负义RNA基因组，该基因组编码10个核心蛋白和多种辅助蛋白，其中HA蛋白是流感病毒粒子表面最主要成分，最重要功能是受体结合和膜融合，呈现禽流感病毒的中和抗体表位(neutralizing antibodies,NAbs)[7]，还可能在病毒粒子出芽和形态发生过程中发挥作用，并且还是宿主获得性免疫系统识别的主要对象，是流感病毒疫苗研制的主要靶抗原[8]。
新城疫(Newcastle disease,ND)是由新城疫病毒(Newcastle disease virus,NDV)引起的禽类高度接触性传染病，可导致呼吸系统、神经系统、消化道及内脏损伤，致死率可达100％。随着反向遗传技术的广泛应用，世界各地科学家己经证实了新城疫病毒作为控制人类和动物疾病的疫苗载体候选株的潜力，多项研究表明以新城疫为载体，可有效表达禽流感HA蛋白[9,10]。但许多H9N2亚型禽流感疫苗在动物与人体中诱导的血凝抑制(Hemagglutination inhibition,HI)与病毒中和抗体水平很低，限制了对疫苗免疫效力的大规模临床评价，因此，H9N2亚型禽流感疫苗的免疫原性有待提高。
文献1-10的来源如下：
[1].Peacock,T.,et al.,A Global Perspective on H9N2 Avian InfluenzaVirus.Viruses,2019.11(7).
[2].Bi,Y.,et al.,Dominant subtype switch in avian influenza virusesduring 2016-2019in China.Nat Commun,2020.11(1):p.5909.
[3].Kim,J.A.,et al.,H9N2 influenza viruses isolated from poultry inKorean live bird markets continuously evolve and cause the severe clinicalsigns in layers.Vet Microbiol,2006.118(3-4):p.169-76.
[4].Bano,S.,K.Naeem and S.A.Malik,Evaluation of pathogenic potentialof avian influenza virus serotype H9N2 in chickens.Avian Dis,2003.47(3Suppl):p.817-22.
[5].Lenny,B.J.,et al.,Replication Capacity of Avian Influenza A(H9N2)Virus in Pet Birds and Mammals,Bangladesh.Emerg Infect Dis,2015.21(12):p.2174-7.
[6].Quan,C.,et al.,New Threats from H7N9 Influenza Virus:Spread andEvolution of High-and Low-Pathogenicity Variants with High Genomic Diversityin Wave Five.J Virol,2018.92(11).
[7].Murin,C.D.,I.A.Wilson and A.B.Ward,Antibody responses to viralinfections:a structural perspective across three different envelopedviruses.Nat Microbiol,2019.4(5):p.734-747.
[8].Skehel,J.J.and D.C.Wiley,Receptor binding and membrane fusion invirus entry:the influenza hemagglutinin.Annu Rev Biochem,2000.69:p.531-69.
[9].Nagy,A.,et al.,Recombinant Newcastle disease virus expressingH9HA protects chickens against heterologous avian influenza H9N2 viruschallenge.Vaccine,2016.34(23):p.2537-45.
[10].Zhang,X.,et al.,Generation and Evaluation of RecombinantThermostable Newcastle Disease Virus Expressing the HA of H9N2 AvianInfluenza Virus.Viruses,2021.13(8).
发明内容
本发明的目的是提供一种表达H9亚型禽流感双拷贝HA基因的Ⅶ型致弱新城疫重组病毒、制备方法、用途，此外还涉及关键质粒的制备方法。
本发明的病毒在rDHN3-mF病毒基因组的P基因和M基因之间的非编码区之间表达全长膜结合HA和另外一个带有截短跨膜结构域的可溶性HA蛋白，获得了能高效稳定表达H9N2 AIV的HA蛋白的新城疫基因VII型减毒株重组病毒rDHN3mF-2HA，经WB验证表明增加HA的可溶性表达与原HA基因相比，其表达效率更高。该重组病毒有望进一步开发成抗新城疫和H9N2亚型禽流感病毒的二联疫苗。
本发明的具体方案如下：一种表达H9亚型禽流感双拷贝HA基因的Ⅶ型致弱新城疫重组病毒，所述病毒为rDHN3mF-2HA、rDHN3mF-HA，rDHN3mF-2HA的序列如序列表SEQ ID NO：1所示；rDHN3mF-HA的序列如序列表SEQ ID NO：2所示。
在上述的表达H9亚型禽流感双拷贝HA基因的Ⅶ型致弱新城疫重组病毒中，采用质粒pBR322-DHN3mF-HA或质粒pBR322-DHN3mF-2HA转染细胞，得到表达H9亚型禽流感双拷贝HA基因的Ⅶ型致弱新城疫重组病毒；
所述质粒pBR322-DHN3mF-2HA的制备方法如下：
将F1片段、F2片段、2HA片段、F4片段同源重组得到质粒pBR322-DHN3mF-2HA；
2HA片段的序列如SEQ ID NO：20所示；
F1片段、F2片段、F4片段参考CN114703207A重组质粒的制备方法和重组病毒中的SEQ ID NO：1、SEQ ID NO：20、SEQ ID NO：5。
在上述的表达H9亚型禽流感双拷贝HA基因的Ⅶ型致弱新城疫重组病毒中，所述细胞为BHK-21细胞。
同时，本发明还公开了一种如上任一所述的表达H9亚型禽流感双拷贝HA基因的Ⅶ型致弱新城疫重组病毒的制备方法，其特征在于，将BHK-21细胞用Lipofectamine LTX DNATransfection Reagents转染试剂盒转染；
转染试剂为：
A液：Opti-MEM Medium 150μl、Lipofectamine LTX Reagent 15μl；
B液：
Opti-MEM DNA 175μl、质粒pBR322-DHN3mF-HA或质粒pBR322-DHN3mF-2HA 4μg、质粒pXJ40-NP 2.5μg、质粒pXJ40-P 1.25μg、质粒pXJ40-L 1.25μg、质粒pXJ40-DE33μg、PLUSReagent 3.5μl；
质粒pXJ40-L、质粒pXJ40-DE3的制备方法参考CN111926025A一株经过密码子替换的基因Ⅶ型新城疫病毒的拯救方法中说明书的123-127段；
pXJ40-L的序列如下：
cgcgccattcgccattcaggctacgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgcca
gggtcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatg
acgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgccca
cttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggc
attatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatg
gtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccat
tgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattga
cgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctgg
agacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcggccgggaacggtgcat
tggaacggacccctaggcttttgcaaaaagctccggatcgatcctgagaacttcagggtgagtttggggacccttga
ttgttctttctttttcgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgg
gaagatgtcccttgtatcaccatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattg
tctcctcttattttcttttcattttctgtaactttttcgttaaactttagcttgcatttgtaacgaatttttaaatt
cacttttgtttatttgtcagattgtaagtactttctctaatcacttttttttcaaggcaatcagggtatattatatt
gtacttcagcacagttttagagaacaattgttataattaaatgataaggtagaatatttctgcatataaattctggc
tggcgtggaaatattcttattggtagaaacaactacatcctggtcatcatcctgcctttctctttatggttacaatg
atatacactgtttgagatgaggataaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttct
tctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattgtaatacgac
tcactatagggcgaattcggatccggatggttgggaggacgacattgcgccaatcatctcccataatgcttagagtc
aagctgaacattagcataaatcaggatcccgtgttgttgggcaaccgcaatccgacaatgctgacatgattgttctg
agtctcgctcactgtcactttattaagaaaaaacacaagaagcattgacatataagggaaaataaccaacaagagag
aacacgggtaggacatggcgggctccggtcctgagagggcagagcaccagatcatcctaccagagtcacatttatcc
tctccattggtcaagcacaaattgttatactactggaaattaaccgggctaccgcttcctgatgaatgcgactttga
tcatctcattatcagcaggcaatggaagagaatactggagtcagccactcctgacacagagagaatgataaaacttg
ggcgggcggtgcaccagactctcaaccacaattccaagatgactggagtgctccatcccaggtgtttagaagaactg
gctagtattgaggtccctgattcaactaacaaattccggaagattgaaaagaagatccagattcacaacacaagata
tggagacctgttcacaaagctgtgcgtgcaagttgagaagaaattgctagggtcatctctgtctaataatgtcccac
gatcagaggaattcaacagcatccgtacagatccggcattctggtttcactcaaaatggtccagagccaagttcgcg
tggctccatataaaacaagtccaaaggcatctgattgtagcagcaaggacaaggtctgcagtcaacaagttagtaac
attaaatcataagataggccatgtctttattactcctgagcttgtcattgtgacacacacagacgagaacaagttca
catgtctcacccaggaacttgtattgatgtatgcggatatgatggaaggcagggacatggtcaatataatatcttct
acagcagcacatctcaggaacctatccgagaaaattgatgatattctgcggttagtagatgctctggcaaaggactt
aggtaatcaagtctatgatgttgtagcattaatggagggatttgcatacggtgccgttcagctgcttgagccatcag
gtacatttgcaggagatttctttgcatttaacctacaggagctcaaaaacacgttaatcgaacttctccccaataat
atagcggaagcagtaactcacgctattgccactgtattctctggattagaacagaaccaagcagctgagatgttgtg
cttgctgcgtttgtggggtcatccattgcttgagtctcgtagtgcagcaagagcagtcaggagccagatgtgcgcac
caaagatggtagactttgatatgatcctccaggtattatctttctttaaaggaacaatcatcaatggatacagaaaa
aagaactcaggtgtgtggccacgcgtcaaagtagatacaatatatggaaatatcattgggcagctacatgctgattc
agcagagatctcacatgatgtcatgttgagggagtacaagagtttatccgctcttgaatttgagccatgtatagatt
atgaccctgttaccaatctaagcatgttcctaaaagacaaggcaatcgcacatcctagtgataactggctcgcctca
tttaggcggaacctactctctgaggaccagaagaaacagataaaggaggcaacttcaactaaccgcctcctgataga
gttcttagaatcaaatgattttgatccatataaagaaatggaatacctgacaaccctcgagtacctaagagatgaca
gtgtggcagtatcgtactcactcaaagagaaagaggtgaaagtgaatggacggatttttgctaaattaacaaagaaa
ctaaggaattgccaggtaatggcagaaggaattctagctgaccagattgcacctttcttccagggaaatggggtcat
tcaagatagcatatccttgacaaagagtatgttagcaatgagtcaactgtcctttaacagcaataagaaacgtatcg
ctgactgcaaagagagggtttcctcaaaccgcaatcatgatcccaagagcaagaatcgtagaagagttgccaccttt
atcacgactgacctacaaaagtattgtcttaactggagatatcagacagtcaaactattcgcccatgccatcaatca
gctgatgggcctacctcatttctttgagtggattcatcttaggctgatggacactacaatgtttgtaggggatcctt
tcaatcctccaagtgacccgaccgactgtgatctatcaagagtcccaaatgatgatatatatattgtcagtgctaga
gggggcattgagggactctgtcagaagctatggacgatgatctcaattgctgcaatccaacttgccgcagcaagatc
tcattgtcgagttgcctgcatggtacaaggtgacaatcaagtaatagctgtaacgagagaggtgagatcagatgatt
ccccggatatggtattgacgcagttgcatcaggctagtgataatttcttcaaggaattgattcatgtcaatcatctg
attggccataacctgaaggatcgtgaaaccattagatcagacacattcttcatatacagcaaacgaatattcaaaga
tggagcaatactcagtcaggtcctcaaaaattcatctaaattggtgctaatatcaggtgaccttagcgaaaacactg
taatgtcctgtgccaacattgcatccactgtagcacgactatgtgagaatgggcttcctaaggacttctgttactat
ttgaactacctaatgagttgcgtgcagacatattttgattcagagttttctattactcacagctcacagtcagattc
caaccagtcctggattgaggatatctctttcgtacactcatacgtgttaacccctgcccaactggggggactgagta
accttcaatactcaaggctctacacaaggaatattggcgacccagggaccactgcctttgcagaggtcaagcgacta
gaagcagtggggttgttgagtcccagcatcatgactaacatcttaaccaggccacctggcaatggagattgggccag
cctatgcaacgacccatactcttttaattttgagactgttgcaagcccaaatattgtcctcaagaaacatacacaga
aagtcctatttgagacatgttcaaaccctttattatccggggtacatacagaggacaatgaggcagaagagaaagca
ttggctgaattcttactcaatcaagaagtgattcacccacgtgtcgcacatgctatcatggaagcaagctctgtggg
taggagaaagcaaattcaagggcttgttgacacaacgaacactgtgattaagattgcactgactaggaggcccctcg
gtatcaaaagactgatgcggataatcaattactcaagcatgcatgcaatgttgttcagggatgatattttcttatcc
actagatccaaccacccattagtttcttctaatatgtgctcgctgacgctagcagattatgctcggaacagaagctg
gtcacccctgacagggggcaggaaaatactgggtgtatccaaccccgataccatagaacttgtggagggagagattc
tcagcgtcagtggagggtgcacaaaatgtgacagcggagatgagcagtttacttggttccatcttccaagcaatata
gagttgactgatgacaccagcaaaaatcccccgatgagagtgccatatctcgggtcgaagactcaagagagaagagc
cgcctcacttgcgaaaatagcccatatgtcaccacatgtgaaagcagcactaagggcatcatccgtgttaatctggg
cttatggggacaatgaagtgaactggactgctgctcttaatattgcaaggtctcgatgcaacataagctcagagtat
cttcggctattgtcacccctgcccacagctgggaatctccaacatagattggatgatggcataacccagatgacatt
tacccctgcatctctctacagagtgtcgccttacattcacatatccaatgattctcaaaggctgttcaccgaagaag
gggtcaaagagggaaacgtggtttaccagcaaattatgctcttgggtttatctctaattgagtcactcttcccaatg
acaacaaccagaacatacgatgagatcacattacacctccacagtaaatttagctgctgtatccgagaagcgcctgt
cgcagttcctttcgagctcctcggactggtaccggaattaaggatggtaacctcaaataagttcatgtatgatccta
gccctatatcagagagggattttgcgagacttgacttagctatattcaagagttatgagcttaacttggaatcatat
cccacgctggagctaatgaacattctttcgatatctagcgggaaattgattggccaatctgtggtttcttatgatga
agatacttctataaagaatgatgctataatagtgtatgacaacacacggaattggattagtgaggcacagaactcag
atgtggtccgcctgtttgagtatgcagcactcgaagtgctcctcgactgtgcttatcaactctactatctgagggta
aggggtctaaacaacatcgtcctatacatgaatgacttatataagaacatgccagggatcctactctccaatattgc
agccacgatatcccaccccctcattcactcaaggttgaatgcagtaggtctaattaatcatgacgggtcacaccagc
ttgcagatatagactttgtcgaggtgtctgcgaaattgttagtctcctgcactcgacgcgtggtctcaggtttatat
gcagggagtaagtatgatctgctgtttccatctgtcttagatgataacctgaatgagaagatgcttcaactaatttc
ccggttatgctgcttgtacacagtgctctttgctacaacaagagaaatcccaaaaataaggggcctatcagcagaag
agaaatgctcaatactcactgagtatctattgtcggatgctgtaaaaccgttgcttaggtccgaacaattgagttct
atcatgtctcccaacataatcacgttcccagccaatctatactacatgtctaggaagagccttaatttgatcagaga
acgagaggacagagatactatcttgtcgttgttgttccctcaggagtcactgcttgagcttcgcccagtacgggaca
ttggtgctcgagtgaaagacccgtttacccgacaacccgcatctttcatacaagagctggatctgagtgccccagca
aggtacgacgcgtttacactgagtaagatttgcttcgagcacacactaccgaacccaagggaagattacctagtacg
atacttgttcagaggagtagggactgcttcatcttcttggtataaggcgtctcatcttctatccatatctgaggtta
ggtgtgcaagacatgggaactctttatacttagcggaaggaagcggagccatcatgagtcttcttgaattgcatata
ccacatgagaccatctattacaatacacttttctcgaatgagatgaaccctccacagcggcatttcggacctacacc
aacacagtttctaaactcggtcgtttataggaatctacaagcggaagtgccatgtaaagatggatatgtccaggagt
tctatccattatggagagagaatgcagaagaaagtgatctgacctcagataaggcagttggatatatcacatctgta
gtaccctacaggtctgtatcattactacattgtgacattgagattcctccagggtccagtcaaagcttattagatca
actggctactaatttatccctgattgccatgcattctgtgagagagggcggggtagtgatcatcaaggtactgtatg
caatggggtactacttccacttactcatgaatttattcactccatgttccacgaaaggatacatactttccaatggc
tacgcctgtagaggggatatggagtgttacctgatattcgttatgggctgcttaggcgggcccactttcgtgcacga
agtggtaaggatggcaaaagctctaatacaacgacacggtacacttctatctaaatcagatgaaatcacattgacta
agctatttacctcacagcagcgtcgtgtaacagatctcctatccagccctttaccgaagctaatgaggctcttaagt
gaaaacattgatgctgcactaattgaagccgggggacagcccgtccgtccattttgtgcagaaagtttggtgagcac
actaacaaatacgacccagacaactctgatcattgccagccacattgacacagtcatccggtccgtgatttacatgg
aggctgagggtgacctcgccgacacagtgttcttattaactccttacaatctatccacagacggtaaaaagagaaca
tcacttaagcagtgcaccaaacagatcttggaagtcacaatattgggtctcagagccaaagacatcaataaaatagg
tgatgtaatcagcttagtactcagaggtgcgatttccctagaggacctcatcccattaaggacatacctgaagcaca
gtacctgtcctaaatacctgaaagcggtcctaggtattactaagctcaaagaaatgttcacaggtacttcgttattg
tacttgactcgcgctcaacaaaaattctacatgaaaactataggtaatgctgccaagggatattacagtaataatga
ctcttaaaggcaatcgtacaccaatcagttatcttcttaactgatgactccctcattgacttgattataccagatta
gaaaaaagttaaattctgactctttggaactcgtattcggattcagttagttaactttaagcaaaaatgcgcaaagt
cgtctctaatcacagctatgtcattcaccaaatctctgtttggtgcgcctgcaggagctcggtaccagatcttatta
aagcagaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatt
tttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggtcgactctagactctt
ccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggta
atacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccg
taaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtc
agaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgtt
ccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctg
taggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgct
gcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggt
aacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactag
aaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggca
aacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaa
gatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatt
atcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaa
cttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagtt
gcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcg
agacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctg
caactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttg
cgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttc
ccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttg
tcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatcc
gtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctc
ttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttctt
cggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatct
tcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataag
ggcgacacggaaatgttgaatactcatactcttcttttttcaatattattgaagcatttatcagggttattgtctca
tgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgcca
cctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcccctttcgtctcg
cgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggat
gccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatc
agagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatca
ggaaattgtaaacgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaatagg
ccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaag
agtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtga
accatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgat
ttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcg
ctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgt；
pXJ40-DE3的序列如下：
cgcgccattcgccattcaggctacgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgcca
gggtcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatg
acgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgccca
cttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggc
attatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatg
gtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccat
tgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattga
cgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctgg
agacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcggccgggaacggtgcat
tggaacggacccctaggcttttgcaaaaagctccggatcgatcctgagaacttcagggtgagtttggggacccttga
ttgttctttctttttcgctattgtaaaattcatgttatatggagggggcaaagttttcagggtgttgtttagaatgg
gaagatgtcccttgtatcaccatggaccctcatgataattttgtttctttcactttctactctgttgacaaccattg
tctcctcttattttcttttcattttctgtaactttttcgttaaactttagcttgcatttgtaacgaatttttaaatt
cacttttgtttatttgtcagattgtaagtactttctctaatcacttttttttcaaggcaatcagggtatattatatt
gtacttcagcacagttttagagaacaattgttataattaaatgataaggtagaatatttctgcatataaattctggc
tggcgtggaaatattcttattggtagaaacaactacatcctggtcatcatcctgcctttctctttatggttacaatg
atatacactgtttgagatgaggataaaatactctgagtccaaaccgggcccctctgctaaccatgttcatgccttct
tctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattgtaatacgac
tcactatagggcgaattcggatccgccatgaacacgattaacatcgctaagaacgacttctctgacatcgaactggc
tgctatcccgttcaacactctggctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagt
cttacgagatgggtgaagcacgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgct
gccgccaagcctctcatcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagc
taagcgcggcaagcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccatta
agaccactctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccatt
gaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaa
caagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtctactcg
gtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatgctcattgag
tcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagactatcgaactcgcacc
tgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgttccaaccttgcgtagttc
ctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgtcctctggcgctggtgcgtact
cacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacattgcgcaaaa
caccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcattgtccggtcgagg
acatccctgcgattgagcgtgaagaactcccgatgaaaccggaagacatcgacatgaatcctgaggctctcaccgcg
tggaaacgtgctgccgctgctgtgtaccgcaaggacaaggctcgcaagtctcgccgtatcagccttgagttcatgct
tgagcaagccaataagtttgctaaccataaggccatctggttcccttacaacatggactggcgcggtcgtgtttacg
ctgtgtcaatgttcaacccgcaaggtaacgatatgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggt
aaggaaggttactactggctgaaaatccacggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcat
caagttcattgaggaaaaccacgagaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagc
aagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgc
tcccttccgctggcgtttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtgg
tcgcgcggttaacttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattc
tacaagcagacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgag
aaagtcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttc
agtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagccagcta
ttgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgatttgggaatct
gtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggt
caaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcctgatggtttccctgtgt
ggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgcttacagcctaccatt
aacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgctcctaactttgtacacagccaaga
cggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacggaatcgaatcttttgcactgattcacgact
ccttcggtaccattccggctgacgctgcgaacctgttcaaagcagtgcgcgaaactatggttgacacatatgagtct
tgtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagcact
tccggctaaaggtaacttgaacctccgtgacatcttagagtcggacttcgcgttcgcgtgactcgaggcggccgccc
cgggctgcaggagctcggtaccagatcttattaaagcagaacttgtttattgcagcttataatggttacaaataaag
caatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatg
tatcttatcatgtctggtcgactctagactcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctg
cggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacat
gtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgccccc
ctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgttt
ccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttc
gggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggct
gtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaaga
cacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagtt
cttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttacct
tcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcag
cagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacga
aaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaa
gttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatc
tcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggctt
accatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagc
cagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaa
gctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctc
gtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaa
aagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggca
gcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcatt
ctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaa
ctttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagt
tcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaac
aggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcttttttcaat
attattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaata
ggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataa
aaataggcgtatcacgaggcccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctc
ccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttgg
cgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaatac
cgcacagatgcgtaaggagaaaataccgcatcaggaaattgtaaacgttaatattttgttaaaattcgcgttaaatt
tttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgag
atagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaa
aaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaag
cactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaa
gggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgc
cgcgcttaatgcgccgctacagggcgcgt。
最后，本发明还公开了一种如上任一所述的表达H9亚型禽流感双拷贝HA基因的Ⅶ型致弱新城疫重组病毒作为疫苗的用途。
本发明相对于现有技术具有如下的优点及效果：
本发明在rDHN3-mF病毒基因组的P基因和M基因之间的非编码区之间表达全长膜结合HA和另外一个带有截短跨膜结构域的可溶性HA蛋白，获得了能高效稳定表达H9N2 AIV的HA蛋白的新城疫基因VII型减毒株重组病毒rDHN3mF-2HA，经WB验证表明增加HA的可溶性表达与原HA基因相比，其表达效率更高。该重组病毒有望进一步开发成抗新城疫和H9N2亚型禽流感病毒的二联疫苗。
附图说明
图1为pXJ40载体大片段凝胶电泳图；其中M：DNA Marker；1-2：pXJ40-HA质粒双酶切大片段(4291bp)
图2为HA片段与1917bp的sHA产物，基因片段凝胶电泳图；其中M：DNA Marker；1-2：sHA片段(1917bp)；3-4：HA片段(2046bp。
图3为pXJ40-2HA质粒示意图；
图4为F1凝胶电泳图；其中M：DNA Marker；1-4：F1-HA PCR片段(1471bp)。
图5为F2基因片段凝胶电泳图；其中M：DNA Marker；1-4：F2 PCR片段(4815bp)；
图6为F3-HA基因片段凝胶电泳图；其中M：DNA Marker；1-4：F3-HA片段(1731bp)；
图7为F3-2HA基因片段凝胶电泳图；其中M：DNA Marker；1-2：F3-2HA片段(3869bp)；
图8为SmaI酶切pBR322-DHN3mF凝胶电泳图，其中M：DNA Marker；1-2：pBR322-FDHN3SmaI酶切后DNA片段(13399bp)
图9为重组质粒rDHN3-mF-HA单菌落的PCR检测鉴定图，其中M：DNA Marker；1-9：rDHN3-mF-HA单菌落(阳性菌落：2306bp)；
图10为重组质粒rDHN3-mF-2HA单菌落的PCR检测鉴定图，其中M：DNA Marker；1-9：rDHN3-mF-2HA单菌落(阳性菌落：4046bp)；
图11为重组质粒的酶切鉴定图，其中M：DNA Marker；1-2：重组质粒rDHN3-mF-HA；3：重组质粒rDHN3-mF-2HA；
图12为pBR322-FDHN3-HA质粒示意图；
图13为pBR322-FDHN3-2HA质粒示意图；
图14为rDHN3mF-HA重组病毒尿囊液感染BHK-21细胞放大图；
图15为rDHN3mF-2HA重组病毒尿囊液感染BHK-21细胞放大图；
图16为SPF鸡胚尿囊液感染BHK-21细胞放大图；
图17为重组病毒F基因的PCR检测鉴定图，其中，M:DNA marker；A：重组病毒rDHN3mF-HA用引物对F-F/R的扩增条带大小为815bp；B：重组病毒rDHN3mF-HA-2P用引物对F-F/R的扩增条带大小为815bp；
图18为重组病毒HA基因的PCR检测鉴定图，其中，M:DNA marker；A：重组病毒rDHN3mF-HA用引物对HA-F/HA-R的扩增条带大小为1731bp；右B：重组病毒rDHN3mF-2HA用引物对HA-F/HA-R的扩增条带大小为3869bp；
图19为重组病毒rDHN3mF-HA与重组病毒rDHN3mF-2HA鸡胚连续传代HA基因检测电泳图，其中1:阴性对照；2-4:分别为第5代、第10代、第15代重组病毒rDHN3mF-HA的HA基因(1731bp)；5-7:分别为第5代、第10代、第15代重组病毒rDHN3mF-2HA的2HA基因(3869bp)；
图20为重组病毒的NDV蛋白检测结果，其中，M：蛋白分子量；1：阴性对照；2：转染pXJ40flag-HA质粒的细胞蛋白样品；3：感染rDHN3mF鸡胚尿囊液细胞蛋白样品；4：感染rDHN3mF-HA尿囊液细胞蛋白样品；5：感染rDHN3mF-2HA尿囊液细胞蛋白样品；
图21为重组病毒的HA蛋白检测结果，其中，M：蛋白分子量；1：阴性对照；2：转染pXJ40flag-HA质粒的细胞蛋白样品；3：感染rDHN3mF鸡胚尿囊液细胞蛋白样品；4：感染rDHN3mF-HA尿囊液细胞蛋白样品；5：感染rDHN3mF-2HA尿囊液细胞蛋白样品；
图22为重组病毒的β-actin蛋白检测结果，其中，M：蛋白分子量；1：阴性对照；2：转染pXJ40flag-HA质粒的细胞蛋白样品；3：感染rDHN3mF鸡胚尿囊液细胞蛋白样品；4：感染rDHN3mF-HA尿囊液细胞蛋白样品；5：感染rDHN3mF-2HA尿囊液细胞蛋白样品；
图23为等比例放大图21、22图的WB条带图；
具体实施方式
下面结合实施例，对本发明作进一步的描述，但不构成对本发明的任何限制，任何在本发明权利要求范围所做的有限次的修改，仍在本发明的权利要求范围内。
第一部分试剂和仪器
主要仪器设备
电热恒温培养箱HZ-100(一恒科学仪器有限公司，中国上海)；三孔电热恒温水槽DK-8D(一恒科学仪器有限公司,中国上海；海尔BCD-579WE冰箱(海尔，中国上海)；CO2恒温培养箱Forma 371(Thermo公司，美国)；超净工作台SW-CJ-2FD(苏州安泰空气技术有限公司，中国江苏)；生物安全柜1300SERIES A2(Thermo公司，美国)；倒置光学显微镜(Nikon公司，日本)；高速离心机Centrifuge 5804R(Eppendorf公司，德国)；移液器Research plus(Eppendorf公司，德国)；PCR仪C1000 Touch(Bio-Rad公司，美国)；电泳仪PowerPac Basic(Bio-Rad公司，美国)；垂直电泳槽MiniProtean Tetra(Bio-Rad公司，美国)；凝胶成像系统2500(R)(Tanon公司，中国上海)；超纯水仪Milli-Q(Millipore公司，美国)；超低温冰箱Forma 994(Thermo公司，美国)；核酸蛋白分析仪Nano Drop 2000(Thermo公司，美国)；生化培养箱LRH-250(一恒科学仪器有限公司，中国上海)；分析天平BSA224S(Sartorius公司，德国)；涡旋振荡器(Thermo公司，美国)；Azure Biosystems C600多功能分子成像系统(AzureBiosystems公司，美国)。
主要试剂和材料
TIAN prep Mini Plasmid Kit(DP103-03)购自天根生化科技(北京)有限公司；Gel Extraction Kit(D2500-02)购自OMEGA；HiScriptIII 1st Strand cDNA SythesisKit(+gDNA wiper)(R312)购自南京诺维赞生物制品有限公司；Agarose(E0301)购自TSINGK；0.25％ Trypsin-EDTA(25200-056)，DMEM basic(C11995500BT)购自Gibco；Lipofectamine LTX and Plus Reagent(15338-100)购自Invitrogen；FBS(10099-141C)购自Gibco；Premix-Taq(RR902A)购自TAKARA；Pen Strep penicillin Streptomycin(15140-122)购自Gibco；2×Phanta Flash Master Mix(Dye Plus)(P520)购自南京诺维赞生物制品有限公司；常用限制性内切酶购自TAKARA；ClonExpress Multis One Step Cloning Kit(C113)购自南京诺维赞生物制品有限公司；Trizol 15596-026购自Invitrogen；氯仿，异丙醇，无水乙醇等生化试剂购自宁波萃英化学技术有限公司；10xSDS-PAGE电泳液购自碧云天；SDS-PAGE凝胶配制试剂盒、BeyoECL Plus(超敏ECL化学发光试剂盒)购自碧云天；0.22μm NC膜(3米/卷)购自PALL公司；Goat Anti-Rabbit IgG H&L
preadsorbed(ab6940)购自CST；兔抗鸡IgG-HRP(SE235)购自solarbio；TBS缓冲液购自博士德生物公司；AIV-HA多抗与NDV高免血清(本实验室自制)；/>
Anti-β-Actin Mouse MonoclonalAntibody(HC201-01)购自全式金公司。pXJ40Flag-HA由本实验室构建；pBR322-DHN3mF，pXJ40-NP，pXJ40-P，pX J40-L，pXJ40-DE3均为华农(肇庆)生物技术研究院提供。
本发明涉及的引物参考表1。
表1引物序列表
第二部分实验步骤与结果
1.pXJ40Flag-2HA质粒的构建
(1)用BamHI和KpnI限制性核酸内切酶对质粒pXJ40Flag-HA进行线性化酶切，胶回收得到4291bp大片段，4291bp大片段的基因片段凝胶电泳图可参考图1；
pXJ40Flag-HA质粒的构建参考CN114703207A重组质粒的制备方法和重组病毒，说明书77-96段；
(2)以pBR322-DHN3mF-HA质粒为模板，分别用引物2HA-F1/2HA-R1、2HA-F2/2HA-R2扩增HA片段与删减跨膜区与胞内区的sHA片段，胶回收得到大小分别为2046bp的HA片段与1917bp的sHA片段，基因片段凝胶电泳图可参考图2。
sHA片段的序列可参考序列SEQ ID NO：19。
pBR322-DHN3mF-HA质粒的构建参考CN114703207A重组质粒的制备方法和重组病毒，说明书97-134段；
(3)利用同源重组方法将HA片段与sHA片段克隆至线性化载体pXJ40Flag上，将质粒命名为pXJ40Flag-2HA，pXJ40Flag-2HA的结构图如图3。
载体pXJ40Flag参考CN110592108B一种针对II类VII型流行NDV株DHN3的感染性重组克隆方法。
2.构建表达H9N2亚型AIV-HA基因的新城疫病毒重组质粒pBR322-DHN3mF-AIV-HA与-pBR322-DHN3mF-AIV-2HA
(1)以pBR322-FDHN3载体为模板用分别用引物，FDHN3-F1/FDHN3-SmaI-R1；FDHN3-SmaI-F2/FDHN3-R2扩增长度为1471bp的F1，长度为4815bp的F2 PCR片段。
分别以pXJ40flag-HA或pXJ40flag-2HA载体为模板用引物HA-F/HA-R扩增长度为1731bp与3869bp的F3 RCR片段。(引物序列详见表1)
PCR反应液的配制参考表2；
表2PCR反应液配方表
PCR反应条件：
参考图4-图7：
图4为F1凝胶电泳图；其中M：DNA Marker；1-4：F1-HA PCR片段(1471bp)。
图5为F2基因片段凝胶电泳图；其中M：DNA Marker；1-4：F2 PCR片段(4815bp)；
图6为F3-HA基因片段凝胶电泳图；其中M：DNA Marker；1-4：F3-HA片段(1731bp)；
图7为F3-2HA基因片段凝胶电泳图；其中M：DNA Marker；1-2：F3-2HA片段(3869bp)；
注：F3-HA片段即为HA片段；F3-2HA片段即为2HA片段；
HA片段的序列参考CN114703207A重组质粒的制备方法和重组病毒中的SEQ IDNO：3；
用SmaI酶切pBR322-DHN3mF载体(本实验室提供)，通过胶回收纯化13399bp DNA片段F4，具体参考图8；
(2)将上述4个片段共同进行如下同源重组(重组试剂盒购自南京诺维赞(ClonExpressMultis One Step Cloning Kit，C113)；
同源重组试剂配方参考表3；
表3配方表
| 试剂 | 用量 |
| F1片段 | 30ng |
| F2片段 | 97ng |
| HA/2HA片段 | 30ng/78ng |
| F4片段 | 200ng |
| 5xCE MultiS Buffer | 4μl |
| Exnase MultiS | 2μl |
| ddH2O | up to 20μl |
在冰上制备好上述体系后在37℃反应30分钟。
(3)将重组产物转化进Trans2-Blue感受态细菌。
(a)在冰上解冻Trans2-Blue感受态细胞，取10μl重组物加入到50μl感受态细胞中，轻弹管壁混匀(请勿振荡混匀)，冰上静置30min。
(b)42℃水浴热激45sec后，立即置于冰上冷却2-3min。
(c)加入1ml SOC培养基(不添加抗生素)，37℃摇菌1h(转速200-250rpm)。
(d)将Amp+的LB平板固体培养基在37℃培养箱中预热。
(e)取100μl的细菌液用无菌涂板棒轻轻涂在含有AMP+抗性的平板上。
(f)37℃培养箱中倒置培养12-16h。
(4)用引物M-F2/P-R2进行菌落PCR鉴定，总共筛选8个菌落，有8个呈阳性，正确产物分别为2300bp与4046bp。
参考图9-10：
图9为重组质粒rDHN3-mF-HA单菌落的PCR检测鉴定图，其中M：DNA Marker；1-9：rDHN3-mF-HA单菌落(阳性菌落：2306bp)；
图10为重组质粒rDHN3-mF-2HA单菌落的PCR检测鉴定图，其中M：DNA Marker；1-9：rDHN3-mF-2HA单菌落(阳性菌落：4046bp)；
(5)HindIII酶切进行质粒鉴定。
分别取上述2个阳性菌落进行扩增培养，提取质粒DNA。
分别用HindIII(购自TAKARA)酶切，反应如下表4；
表4配方表
| 试剂 | 用量 |
| Buffer | 2μl |
| 质粒DNA | 400ng |
| HindIII | 2μl |
| ddH2O | up to 20μl |
正确的pBR322-DHN3mF-HA质粒用HindIII酶切后应分别产生1个12382bp，5654bp，3294bp的DNA片段。正确的pBR322-DHN3mF-2HA质粒用HindIII酶切后应分别产生1个12359bp，5654bp，3294bp，1763bp的DNA片段。
酶切结果如下图11，1号与2号pBR322-DHN3mF-HA质粒产生了正确的3个片段。3号pBR322-DHN3mF-2HA质粒产生了正确的4个片段。将1号质粒与3号质粒进行测序验证，结果证明该质粒含正确的插入序列，分别命名为pBR322-DHN3mF-HA与
pBR322-DHN3mF-2HA。
图12是pBR322-DHN3mF-HA的结构图谱；
图13是pBR322-DHN3mF-2HA的结构图谱。
2.转染BHK细胞,拯救病毒
(1)将BHK-21细胞培养于30mm瓶皿，24h之内用Lipofectamine LTX DNATransfection Reagents转染试剂盒，并严格按试剂盒指导方法进行转染。
(2)转染试剂配制如下：
A液：Opti-MEM Medium 150μl,Lipofectamine LTX Reagent 15μl,静置5min；
B液：Opti-MEM DNA 175μl，pBR322-DHN3mF-HA(pBR322-DHN3mF-2HA)4μg，pXJ40-NP 2.5μg，pXJ40-P 1.25μg，pXJ40-L 1.25μg，pXJ40-DE3 3μg，PLUS Reagent3.5μl，吹打混匀，静置5min。
pXJ40-NP、pXJ40-P的制备方法参考CN 110592108 A一种针对II类VII型流行NDV株DHN3的感染性重组克隆方法的说明书的127-130段记载。
取A液B液各150μl混合均匀，静置20min。取250μl混合液用于转染细胞。
(3)4天后将上述转染细胞(包括培养液)置-80度冻存。
(4)将该冻存细胞反复冻融3次，4度10000/rpm离心5分钟。取200μl上清液感染DF-1细胞。24h后可见大量细胞死亡。如下图14-17所示：图14为rDHN3mF-HA感染细胞A；图15为rDHN3mF-2HA感染细胞B；图15为未加病毒液的阴性对照细胞C。分别从A,B,C细胞提取总RNA用于下述病毒鉴定试验。
3.鉴定重组病毒
(1)RT-PCR
用上述A、B和C细胞总RNA进行RT-PCR。
RT:使用HiScriptIII 1st Strand cDNA Sythesis Kit(+gDNA wiper)，严格按照说明书进行。
A.Microtube管中配制下列去基因组DNA混合液，总量8μl。
表5配方表
| 试剂名称 | 使用量 |
| 模板RNA | 8μl |
| 5×gDNA wiper Mix | 2μl |
用移液器轻轻吹打混匀，42℃2min。
B.在上述Microtube管中配制下列反转录反应液。
表6配方表
| 试剂名称 | 使用量 |
| 上述混合溶液 | 10μl |
| 10×RT mix | 2μl |
| HiScript III Enzyme Mix | 2μl |
| Oligo(dT)20VN | 1μl |
| RNase-free ddH2O | 5μl |
置37℃，反应45min，85℃，反应5s。
PCR：分别用引物F-F/F-R和引物HA-F/HA-R进行鉴定。
PCR体系参考表7；
表7配方表
| Primer2 | 10pmol |
| cDNA模板 | 2μL |
| Premix Taq | 10μL |
| ddH2O | up to 20μL |
PCR反应条件：
分别取10μl PCR产物跑胶，结果如下图所示：图17为F-F/F-R引物，正确产物应为815bp；图18为M-F2/P-R2引物，正确产物分别应为2300bp与4046bp。
(2)扩增病毒
用上述rDHN3mF-HA与rDHN3mF-2HA的P0代病毒200μl接种SPF鸡胚并连续传代10次，收获各代鸡胚尿囊液于-80℃保存。
(3)RT-PCR和测序验证rDHN3mF-HA与rDHN3mF-2HA重组病毒的稳定性
分别从重组毒rDHN3mF-HA与rDHN3mF-2HA的F1、F5、F10代鸡胚尿囊液中提取RNA并用其进行RT-PCR。方法和前述一致。
PCR：引物为M-F2/P-R2；正确产物分别应为2300bp与4046bp。
结果如图19所示，各病毒的3个样本均产生了正确大小的PCR产物。将其分别送去测序，结果证实均为正确的DNA序列，提示该重组病毒是相对稳定的。
4.WB验证重组病毒HA蛋白的表达水平
用正常SPF鸡胚尿囊液接种细胞的蛋白样品(1)，转染pXJ40flag-HA质粒的细胞蛋白样品(2)，感染rDHN3mF鸡胚尿囊液细胞蛋白样品(3)，感染rDHN3mF-HA尿囊液细胞蛋白样品(4)和感染rDHN3mF-2HA尿囊液细胞蛋白样品(5)跑10％蛋白胶并进行WB检测。结果显示如下：
用NDV高免血清能检测到约55kD大小的蛋白，该蛋白在蛋白胶上显示与rDHN3-mF感染产物具相同的大小和形态。
参考图20，图20为重组病毒的NDV蛋白检测结果，其中，M：蛋白分子量；1：阴性对照；2：转染pXJ40flag-HA质粒的细胞蛋白样品；3：感染rDHN3mF鸡胚尿囊液细胞蛋白样品；4：感染rDHN3mF-HA尿囊液细胞蛋白样品；5：感染rDHN3mF-2HA尿囊液细胞蛋白样品；用兔抗HA多克隆抗体能检测到约75kDa大小的蛋白，该蛋白与HA蛋白的大小一致。
参考图21，图21为重组病毒的HA蛋白检测结果，其中，M：蛋白分子量；1：阴性对照；2：转染pXJ40flag-HA质粒的细胞蛋白样品；3：感染rDHN3mF鸡胚尿囊液细胞蛋白样品；4：感染rDHN3mF-HA尿囊液细胞蛋白样品；5：感染rDHN3mF-2HA尿囊液细胞蛋白样品；用鼠抗β-actin单克隆抗体能检测到约40kDa大小的蛋白，该蛋白与actin蛋白的大小一致。
参考图22-23，图22为重组病毒的β-actin蛋白检测结果，其中，M：蛋白分子量；1：阴性对照；2：转染pXJ40flag-HA质粒的细胞蛋白样品；3：感染rDHN3mF鸡胚尿囊液细胞蛋白样品；4：感染rDHN3mF-HA尿囊液细胞蛋白样品；5：感染rDHN3mF-2HA尿囊液细胞蛋白样品；
图23为等比例放大图21、22图的WB条带图。
详细方法如下：
检测NDV蛋白：
将BHK-21细胞置6孔板中培养至90％，用PBS洗两遍，分别加入2ml0.1％trypsin-DMEM孵育液，用2MOI DHN3鸡胚尿囊液，或2MOI rDHN3mF-HA或2MOI rDHN3mF-2HA鸡胚尿囊液感染BHK-21细胞。阴性对照加200μl SPF鸡胚尿囊液。24h后收集细胞，分别加入200μl的RIPA强裂解液(含1mM PMSF)，95℃干浴裂解10min后，12 000×g离心10min，取上清20μl进行10％ SDS-PAGE(Bio-Rad)。后将蛋白电转移到NC膜上。5％脱脂乳封闭2h，TBST(0.05％Tween20)洗涤四次，每次5min。加入1：1000稀释的鸡NDV高免血清4℃孵育过夜。TBST洗涤四次，每次5min。分别加1﹕10000倍TBST稀释辣根过氧化物酶(HRP)标记兔抗鸡Ig G(LIC)室温孵育1h。TBST洗涤四次，每次5min。向加入超敏ECL化学发光显色液。用Azure BiosystemsC600多功能分子成像系统成像。
检测HA蛋白与β-actin蛋白：
将BHK-21细胞置直径3.5cm培养皿培养至60％，用PBS洗两遍后，用LipofectamineLTX DNA Transfection Reagents转染试剂盒将pXJ40flag-HA质粒转染入BHK-21细胞中。将BHK-21细胞置6孔板中培养至90％，用PBS洗两遍，分别加入2ml 0.1％trypsin-DMEM孵育液，用2MOI DHN3鸡胚尿囊液，或2MOI rDHN3mF-HA或2MOI rDHN3mF-2HA鸡胚尿囊液感染BHK-21细胞。阴性对照加200μl SPF鸡胚尿囊液。24h后收集细胞，分别加入200μl的RIPA强裂解液(含1mM PMSF)，95℃干浴裂解10min后，12 000×g离心10min，取上清20μl进行10％SDS-PAGE(Bio-Rad)。后将蛋白电转移到NC膜上。5％脱脂乳封闭2h，TBST(0.05％Tween20)洗涤四次，每次5min。分别加入1：2000稀释的兔HA多克隆抗体、鼠β-actin单克隆抗体，4℃孵育过夜。TBST洗涤四次，每次5min。分别加1﹕10000倍TBST稀释、山羊抗兔荧光二抗、山羊抗鼠荧光二抗室温孵育1h。TBST洗涤四次，每次5min。用Azure Biosystems C600多功能分子成像系统成像。
5.重组病毒的生物学特性分析
(1)重组病毒的HA测定
在96孔微量反应孔中加入0.025mL/孔的生理盐水，再向每排的第一孔中加入待检病毒0.025mL/孔，并按顺序做倍比稀释，稀释至第11孔弃掉0.025mL，将第12孔做为阴性对照，最后向每孔加入0.025mL的1％鸡红细胞悬浮液，用微量振荡器混合均匀，室温静置20min～25min，判定血凝效价(HA)，为减少实验误差，每个样品重复测三次。
(2)重组病毒的TCID50测定
将BHK-21细胞提前一天铺于96孔板中，将细胞培养至90％后开始下一步实验，弃去96孔中的旧培养基，用PBS将细胞清洗2次。用0.1％trypsin-DMEM孵育液将重组病毒rDHN3mF-HA或rDHN3mF-2HA按顺序做倍比稀释，稀释梯度为10 -1～10 -10。将不同稀释度的病毒液分别以100μL感染细胞，做好标记，每个稀释度做三个平行。5d～7d后在倒置显微镜下观察病变，为减少误差，病毒样品重复测三次。使用Reed-Muench法计算TCID50。
(3)重组病毒的EID50测定
将重组毒株rDHN3mF-HA或rDHN3mF-2HA尿囊液用生理盐水按顺序作10倍倍比稀释，稀释梯度为10 -1～10 -10，将10 -5～10 -10五个稀释梯度分别接种5枚9日～11日龄的SPF鸡胚，每枚接种100μL，置于37℃恒温培养箱。观察24h内的死胚并弃去；每日早中晚照胚三次，及时冻存死胚，连续观察7d。经过7d后未死的胚在4℃冰箱静置4h以上收取尿囊液测定HA，阳性为感染，使用Reed-Muench的方法计算鸡胚半数感染量EID50。
测试结果可见表8；
表8测试结果
四.结论
我们对H9N2亚型禽流感HA基因经过跨膜区分析预测，将全长膜结合HA和另外一个带有截短跨膜结构域的可溶性HA蛋白通过DNA重组的方法一同插进新城疫基因VII型DHN3mF弱毒株的M和P基因之间，分别构建了感染性cDNA克隆pBR322-DHN3mF-HA与pBR322-DHN3mF-2HA质粒。将二质粒分别与pXJ40-NP，pXJ40-P，pXJ40-L和pXJ40-DE3质粒混合，共转染BHK-21细胞，成功获得两株稳定的能有效表达H9N2亚型禽流感HA基因和NDV抗原蛋白的重组病毒rDHNmF-HA与rDHNmF-2HA。根据WB结果显示，增加带有截短跨膜结构域的可溶性HA蛋白表达后HA蛋白的表达效率比原有HA基因表达效率更高。经测定，插入外源基因后的重组新城疫病毒的生物学特性与亲本毒差异性小。该重组病毒有望进一步开发成抗禽流感病毒和抗新城疫病毒的二联疫苗，为控制禽流感病毒和新城疫病毒感染提供新的防御手段。
Claims (5)
1.一种表达H9亚型禽流感双拷贝HA基因的Ⅶ型致弱新城疫重组病毒，其特征在于，所述病毒为rDHN3mF-2HA、rDHN3mF-HA，rDHN3mF-2HA的序列如序列表SEQ ID NO：1所示；rDHN3mF-HA的序列如序列表SEQ ID NO：2所示。
2.根据权利要求1所述的表达H9亚型禽流感双拷贝HA基因的Ⅶ型致弱新城疫重组病毒，其特征在于，采用质粒pBR322-DHN3mF-HA或质粒pBR322-DHN3mF-2HA转染细胞，得到表达H9亚型禽流感双拷贝HA基因的Ⅶ型致弱新城疫重组病毒；
所述质粒pBR322-DHN3mF-2HA的制备方法如下：
将F1片段、F2片段、2HA片段、F4片段同源重组得到质粒pBR322-DHN3mF-2HA；
2HA片段的序列如SEQ ID NO：20所示。
3.根据权利要求2所述的表达H9亚型禽流感双拷贝HA基因的Ⅶ型致弱新城疫重组病毒，其特征在于，所述细胞为BHK-21细胞。
4.一种如权利要求1-3任一所述的表达H9亚型禽流感双拷贝HA基因的Ⅶ型致弱新城疫重组病毒的制备方法，其特征在于，将BHK-21细胞用Lipofectamine LTX DNATransfection Reagents转染试剂盒转染；
转染试剂为：
A液：Opti-MEM Medium 150μl、Lipofectamine LTX Reagent 15μl；
B液：
Opti-MEM DNA 175μl、质粒pBR322-DHN3mF-HA或质粒pBR322-DHN3mF-2HA 4μg、质粒pXJ40-NP 2.5μg、质粒pXJ40-P 1.25μg、质粒pXJ40-L 1.25μg、质粒pXJ40-DE33μg、PLUSReagent 3.5μl。
5.如权利要求1-3任一所述的表达H9亚型禽流感双拷贝HA基因的Ⅶ型致弱新城疫重组病毒作为疫苗的用途。
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| CN101376027A (zh) * | 2008-09-24 | 2009-03-04 | 中国农业科学院哈尔滨兽医研究所 | 表达禽流感病毒H9亚型HA蛋白的重组新城疫病毒LaSota弱毒疫苗株 |
| CN114703207A (zh) * | 2022-04-07 | 2022-07-05 | 华南农业大学 | 重组质粒的制备方法和重组病毒 |
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| CN101376027A (zh) * | 2008-09-24 | 2009-03-04 | 中国农业科学院哈尔滨兽医研究所 | 表达禽流感病毒H9亚型HA蛋白的重组新城疫病毒LaSota弱毒疫苗株 |
| CN114703207A (zh) * | 2022-04-07 | 2022-07-05 | 华南农业大学 | 重组质粒的制备方法和重组病毒 |
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