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Citation: Lan LI, Yi-shu YANG, Ze-lin LI, Yi ZENG. Prokaryotic Expression and Purification of HIV-1 Vif and hAPOBEC3G, Preparation of Polyclonal Antibodies [J].VIROLOGICA SINICA, 2008, 23(3) : 173-182.  http://dx.doi.org/10.1007/s12250-008-2909-z

Prokaryotic Expression and Purification of HIV-1 Vif and hAPOBEC3G, Preparation of Polyclonal Antibodies

  • Corresponding author: Yi ZENG, Zengy@public.bta.net.cn
  • Received Date: 10 September 2007
    Accepted Date: 19 February 2008
    Available online: 01 June 2008

    Fund Project: National Natural Science Foundation of China 30400368The Natural Science foundation of Beijing 5072003Beijing Natural Science foundation Program and Scientific Research Key Program of Beijing Municipal commission of Education KZ20051005001

  • To prepare HIV-1 Vif and hAPOBEC3G and to produce their antibodies, the full length gene fragment of HIV-1 vif was amplified by PCR from plasmid of HIV-1 NL4.3 cDNA, and APOBEC3G gene was achieved by RT-PCR from the total RNA of H9 cells. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-32a). Recombinant pET-vif and pET-APOBEC3G were expressed respectively in Eserichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine tag at the C-terminus for convenient purification and detection. To express and purify the HIV-1 Vif and hAPOBEC3G in E.coli cells, the accuracy of inserted gene and specificity of proteins were detected by two enzymes digestion technology, SDS-PAGE, and Western blotting. Rabbits were immuzied by Vif or APOBEC3G protein. Serum samples were tested by indirect ELISA to determine the level of antibodies. Immunoenzyme and immunofluorescence assay were performed to identify the specificity of polyclonal antibodies. The titer of the anti-Vif antibodies was 1:204800, and that of anti-APOBEC3G antibodies was 1:102400. The antibidies could detect the antigen expressing in the cells. These fusion proteins with high purity and their corresponding polyclonal antibodies with high titer and specificity were achieved. These results ensure the immunogenicity and antigenicity of the purified recombinant proteins.

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    1. Doehle B P, Schafer A, Cullen B R. 2005. Human APOBEC3B is a potent inhibiror of HIV-1 infectivity and is rexistant to HIV-1Vif. Virology, 339(2): 281-288.
        doi: 10.1016/j.virol.2005.06.005

    2. Dussart S, Douaisi M, Courcoul M, et al. 2005. APOBEC3G ubiquitination by Nedd4-1 favors its packaging into HIV-l particles. J Mole Biol, 345(3): 547-558.
        doi: 10.1016/j.jmb.2004.10.067

    3. Fisher A G, Ensoli B, Ivanoff L, et la.1987. The sor gene of HIV-l is required for efficient virus transmissino in vitro. Science, 237: 888-893.
        doi: 10.1126/science.3497453

    4. Fujita M, Akari H, Sakurai A, et al. 2004. Expression of HIV-l accessory protein Vif is controlled uniquely to be low and optimal by proteasome degradation. Microbes Infect, 6: 791-798.
        doi: 10.1016/j.micinf.2004.04.011

    5. Gabuzda D H, Lawrence K, Langhoff E, el al. 1992. Role of vif in replication of human immunodeficiency virus type l in CD4+T lymphocytes. J Virol, 66: 6489-6495.

    6. Lee S K, Dykxhoorn D M, Kumar P, el al. 2005. Lentiviral delivery of short hairpin RNAs protects CD4 T cells from multiple clades and primary isolates of HIV. Blood, 106(3): 818-826.
        doi: 10.1182/blood-2004-10-3959

    7. Luo K, Liu B, XiaoZ, et al. 2004. Amino-terminal region of the human immunodeficiency virus type l nucleocapsid is required for human APOBEC3G packaging. J Virol, 78 (21): 11841-11852.
        doi: 10.1128/JVI.78.21.11841-11852.2004

    8. Rose K M, Marin M, Kozak S L, et al. 2004. The Viral infectivity factou (Vif) of HIV-1 unveiled. Trends Mol Med, 10(6): 291-297.
        doi: 10.1016/j.molmed.2004.04.008

    9. Sheehy A M, Gaddis N C, Choi J D, et al. 2002. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein.Nature, 418: 646-650.
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    10. Strebel K, Daugherty D, Clouse K, e tal. 1987. The HIV'A'(sor) gene product is essential for vius infectivity. Nature, 328: 728-730.
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    11. Von S U, Song J, Aiken C, et al. 1993. Vif is crucial for human immunodeficiency virus type I proviral DNA synthesis in infected cells. J Virol, 67: 4945-4955.

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    Prokaryotic Expression and Purification of HIV-1 Vif and hAPOBEC3G, Preparation of Polyclonal Antibodies

      Corresponding author: Yi ZENG, Zengy@public.bta.net.cn
    • College of Life Science & Bio-engineering, Beijing University of Technology, Beijing 100124, China
    Fund Project:  National Natural Science Foundation of China 30400368The Natural Science foundation of Beijing 5072003Beijing Natural Science foundation Program and Scientific Research Key Program of Beijing Municipal commission of Education KZ20051005001

    Abstract: To prepare HIV-1 Vif and hAPOBEC3G and to produce their antibodies, the full length gene fragment of HIV-1 vif was amplified by PCR from plasmid of HIV-1 NL4.3 cDNA, and APOBEC3G gene was achieved by RT-PCR from the total RNA of H9 cells. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-32a). Recombinant pET-vif and pET-APOBEC3G were expressed respectively in Eserichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine tag at the C-terminus for convenient purification and detection. To express and purify the HIV-1 Vif and hAPOBEC3G in E.coli cells, the accuracy of inserted gene and specificity of proteins were detected by two enzymes digestion technology, SDS-PAGE, and Western blotting. Rabbits were immuzied by Vif or APOBEC3G protein. Serum samples were tested by indirect ELISA to determine the level of antibodies. Immunoenzyme and immunofluorescence assay were performed to identify the specificity of polyclonal antibodies. The titer of the anti-Vif antibodies was 1:204800, and that of anti-APOBEC3G antibodies was 1:102400. The antibidies could detect the antigen expressing in the cells. These fusion proteins with high purity and their corresponding polyclonal antibodies with high titer and specificity were achieved. These results ensure the immunogenicity and antigenicity of the purified recombinant proteins.