RESUMO
<p><b>OBJECTIVE</b>To construct the recombinant fowlpox virus (rFPV) coexpressing HIV-1 gag-gp120 and hIL-6.</p><p><b>METHODS</b>The recombinant expressing plasmid pUTA-GE-IL6 was successfully constructed by inserting gag-gp120 gene and hIL-6 gene into the downstream of the combined promoter ATI-p7.5 and p7.5 tandem promoter respectively. After transfecting the plasmid into chicken embryonic fibroblast (CEF) cells preinfected with FPV 282E4 strain and selecting the recombinant virus under the pressure of BUdR. The recombinant virus was analyzed by nucleic acid probe hybridization and immunoblotting. In addition, the formation of virus-like particle and the expression of interested proteins in the recombinant virus-infected p815 cells were observed, and the immunogenicity of the recombinant virus was also analyzed.</p><p><b>RESULTS</b>There was colorable dot for the positive recombinant virus, immunoblotting analysis showed that the recombinant virus could expressed both gag-gp120 and IL-6. Virus-like particles (VLP) were formed in virus-infected cells, and the interested proteins could be expressed in mammalian cells infected by the recombinant virus. The immunity index from the immunized mice showed that the recombinant virus had good immunogenicity.</p><p><b>CONCLUSION</b>The recombinant fowlpox virus coexpressing gag-gp120 and IL-6 was successfully constructed, which may provide basis for the preparation of live vector genetic engineering vaccine and macromolecule particle vaccine against HIV-1.</p>
Assuntos
Animais , Embrião de Galinha , Camundongos , Anticorpos Antivirais , Sangue , Western Blotting , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Fibroblastos , Biologia Celular , Metabolismo , Varíola Aviária , Sangue , Alergia e Imunologia , Virologia , Vírus da Varíola das Aves Domésticas , Genética , Alergia e Imunologia , Produtos do Gene gag , Genética , Metabolismo , Vetores Genéticos , Genética , Proteína gp120 do Envelope de HIV , Genética , Metabolismo , HIV-1 , Genética , Metabolismo , Imunização , Métodos , Interleucina-6 , Genética , Metabolismo , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Plasmídeos , Genética , Proteínas Recombinantes de Fusão , Genética , Alergia e Imunologia , Metabolismo , Transfecção , Vacinas Virais , Genética , Alergia e Imunologia , MetabolismoRESUMO
To screening out Chinese vaccine candidate against HIV-1, Chinese vaccine strain 282E4 of fowlpox virus was used as the vector to construct the recombinant fowlpox virus (rFPV) coexpressing gp120 of Chinese HIV-1 strain and IL-18, and the recombinant virus was indentified by PCR and Western blot. The specific DNA fragment could be amplified by PCR from the genome of rFPV. Western blot analysis showed that gp120 and IL-18 could be expressed not only in chicken embryo fibroblast (CEF) cells infected by rFPV, but also in mammalian cells infected by rFPV. After the recombinant fowlpox virus was inoculated into BALB/c mice, the spleen specific CTL activities and serum antibodies in the immunized mice were detected, which demonstrated that the rFPV had good immunogenicity and could induce BALB/c mice to produce specific humoral and cellular immunity. IL-18 palyed the role of immunoadjuvant. The study lays the basis on the preparation of genetic engineering live vector vaccine against HIV-1.