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Virologica Sinica, 23 (3) : 173-182, 2008
Review
Prokaryotic Expression and Purification of HIV-1 Vif and hAPOBEC3G, Preparation of Polyclonal Antibodies
College of Life Science & Bio-engineering, Beijing University of Technology
 Correspondence:
(405.12KB)  
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.
  Published online: 5 Jun 2008
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