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Volume 22 Issue 1
February 2007

    Hong-bo FAN, Jun-wei LI, Zhi-lin LI, Wei ZHENG, Po Tien and De-yin GUO. Cloning of M and NP Gene of H5N1 Avian Influenza Virus and Immune Efficacy of their DNA Vaccines[J]. Virologica Sinica, 2007, 22(1): 46-52.
    Citation: Hong-bo FAN, Jun-wei LI, Zhi-lin LI, Wei ZHENG, Po Tien, De-yin GUO. Cloning of M and NP Gene of H5N1 Avian Influenza Virus and Immune Efficacy of their DNA Vaccines .VIROLOGICA SINICA, 2007, 22(1) : 46-52.
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    H5N1 亚型禽流感病毒M基因NP基因的克隆 及其DNA疫苗的免疫保护效力

    • 武汉大学生命科学学院，现代病毒学中心，病毒学国家重点实验室，中国武汉 430072

    • 收稿日期： 2006-08-30
      录用日期： 2006-10-23
    • 摘要：应用禽流感病毒反转录通用引物和H5N1亚型禽流感M基因、NP基因的型特异性引物，采用RT-PCR方法扩增出H5N1亚型禽流感病毒A/chicken/Hubei/489/2004株的M基因和NP基因，并将M基因和NP基因成功克隆到pMD18-T载体中。以pHM6为载体分别构建了表达M基因和NP基因的真核表达质粒pHM6-m 和pHM6-np，测序鉴定。采用肌肉注射法分别以30μg pHM6-m质粒，30μg pHM6-np质粒，pHM6-m和pHM6-np各15μg混合质粒一次免疫6周龄BALB/c小鼠，2周后用同源病毒以鼻腔接种途径进行攻毒，接下来12天内观察小鼠发病死亡情况。攻毒后pHM6-m质粒组免疫保护率为62.5%，pHM6-np质粒组免疫保护率为25.0%，pHM6-m和pHM6-np混合质粒组免疫保护率为50.0%。表明所构建的H5N1亚型禽流感病毒M基因、NP基因的真核表达质粒均可作为DNA疫苗诱导有效的免疫保护，且pHM6-m质粒免疫保护效果优于pHM6-np质粒。

    Cloning of M and NP Gene of H5N1 Avian Influenza Virus and Immune Efficacy of their DNA Vaccines

    • Modern Virology Center,State Key Laboratory of Virology,College of Life Sciences,Wuhan University,Wuhan 430072,China

    • Corresponding author: De-yin GUO, dguo@whu.edu.cn
    • Received Date: 30 August 2006
      Accepted Date: 23 October 2006

      Fund Project: National Basic Science Talents Training Grant NSFC J0630648

    • The M and NP gene of H5N1 avian influenza virus (A/chicken/Hubei/489/2004) were amplified by RT-PCR from viral RNA, and cloned into pMD18-T vector respectively. Then the expression plasmid containing M gene (pHM6-m) or NP gene (pHM6-np) was constructed by inserting M or NP gene into the pHM6 eukaryote expression vector, and sequenced. 32 BALB/c mice (6-week-old) were divided into four groups at random. Three groups of BALB/c mice were inoculated with 30 μg of plasmid pHM6-m, 30 μg of plasmid pHM6-np or the mixture of plasmid pHM6-m (15 μg ) and pHM6-np(15 μg) once via the intramuscular route respectively. A group of mice were injected with 100 μl PBS as controls. Two weeks later, all mice were challenged with homologous H5N1 avian influenza virus, and observed in the following 12 days. The survival rates of mice in pHM6-m group, pHM6-np group and mixed plasmids group were 62.5%, 25.0% and 50.0%, respectively. Results showed that effective protection could be provided by either pHM6-m or pHM6-np, but pHM6-m provided a better protective effect than pHM6-np.
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      2. He H X,Qin X M,Zhang Q Z,et al. 2004. Cloning of HA Gene of H9N2 Subtype Avian Influenza Virus and Immune Protection Test of Its DNA Vaccine[J]. Prog Biochemi Biophy,31(2): 163-166.(in Chinese)

      3. Hoffmann E,Stech J,Guan Y,et al. 2001. Universal primer set for the full-length amplification of all influenza A viruses[J]. Arch Virol,146(12): 2275-2289.
          doi: 10.1007/s007050170002

      4. Jiang Y P,Yu K Z,Deng G H,et al. 2004. Protection Effi-cacity of DNA Vaccine Encoding Hemagglutinin of H5 Subtype Avian Influenza Virus [J].Agricult Sci in China,37(7): 1071-1075.(in Chinese)

      5. Jiang Y P,Zhang H B,LI C J,et al. 2005. Immunity Protection of H5 Subtype Avian Influenza DNA Vaccine pCAGGoptiHA5 from HPAIV Lethal Challenge[J]. Chin J Anim Veter Sci,36(11): 1178-1182.(in Chinese)

      6. Lei C H,Chen J,Cui S J,et al. 2005. Cloning and sequence analysis of NS gene of H5N1 avian influenza virus strains from Xinjiang[J]. Chin J Veter Sci Technol,35(10): 779-785.(in Chinese)

      7. Long J X,Wang Q Z,LU J H,et al. 2005. Cloning of full-length genes of H5N1 subtype Avian influenza virus strain A/duck/Shangdong/093/2004 and analysis of the sequences[J]. Acta Microbiologica Sinica,45(5): 690-696.(in Chinese)

      8. Okuda K,Ihata A,Watabe S,et al. 2001. Protective immunity against influenza A virus induced by immunization with DNA plasmid containing influenza M gene[J]. Vaccine,19(27): 3681-3691.
          doi: 10.1016/S0264-410X(01)00078-0

      9. Zhang Q Z,Qin X M,Dong H L,et al. 2005. Efficient protection of H5N1 Influenza Virus DNA Vaccine Delivering by Electroporation in Mammalian and Avian System[J]. Prog Biochem Biophy,32(8): 726-733

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      11. Zhao X M,Zhang Q Z,Duan M X,et al. 2005. Construction and Immune Assay of HA DNA Vaccine of H5N1 Subtype Avian Influenza Viruses[J]. Chin Agricult Sci Bull,21(5): 70-80.(in Chinese)

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      Cloning of M and NP Gene of H5N1 Avian Influenza Virus and Immune Efficacy of their DNA Vaccines

        Corresponding author: De-yin GUO, dguo@whu.edu.cn
      • Modern Virology Center,State Key Laboratory of Virology,College of Life Sciences,Wuhan University,Wuhan 430072,China
      • Received Date: 30 August 2006
      • Accepted Date: 23 October 2006
      Fund Project:  National Basic Science Talents Training Grant NSFC J0630648

      Abstract: The M and NP gene of H5N1 avian influenza virus (A/chicken/Hubei/489/2004) were amplified by RT-PCR from viral RNA, and cloned into pMD18-T vector respectively. Then the expression plasmid containing M gene (pHM6-m) or NP gene (pHM6-np) was constructed by inserting M or NP gene into the pHM6 eukaryote expression vector, and sequenced. 32 BALB/c mice (6-week-old) were divided into four groups at random. Three groups of BALB/c mice were inoculated with 30 μg of plasmid pHM6-m, 30 μg of plasmid pHM6-np or the mixture of plasmid pHM6-m (15 μg ) and pHM6-np(15 μg) once via the intramuscular route respectively. A group of mice were injected with 100 μl PBS as controls. Two weeks later, all mice were challenged with homologous H5N1 avian influenza virus, and observed in the following 12 days. The survival rates of mice in pHM6-m group, pHM6-np group and mixed plasmids group were 62.5%, 25.0% and 50.0%, respectively. Results showed that effective protection could be provided by either pHM6-m or pHM6-np, but pHM6-m provided a better protective effect than pHM6-np.

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