RESUMO
Baculovirus has many advantages as vectors for gene transfer. We demonstrated that recombinant baculovirus vectors expressing p35 (Ac-CMV-p35) and eGFP (Ac-CMV-GFP) could be transduced into human kidney 293 cells efficiently. The level of transgene expression was viral dose dependent and high-level expression of the target gene could be achieved under the heterogonous promoter. MTT assay suggested that both Ac-CMV-p35 and Ac-CMV-GFP did not have cytotoxic effect on human embryo kidney 293 cells. Cell growth curve showed the Ac-CMV-p35 and Ac- CMV-GFP transduced and non-transduced cells had similar proliferation rate, so baculovirus-mediated p35expression had no adverse effect on cell proliferation. In addition, baculovirus-mediated p35 gene expression protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D, UV or serum-free media. These results suggested that the baculovirus vector mediated p35 gene expression was functional and it could be widely used in molecular research and even gene therapy.
RESUMO
<p><b>OBJECTIVE</b>To construct the recombinant baculovirus Ac-cytomegalovirus (CMV)-hSox9 for gene therapy of intervertebral disc degeneration.</p><p><b>METHODS</b>Bac-to-Bac system was used for the construction of baculovirus Ac-CMV-hSox9. The cDNA of hSox9 was first cloned into a plasmid vector under the control of CMV promotor to generate the donor plasmid pFastBacDuljgreen fluorescene protein (GFP)-CMV (pFGC)-hSox9. The resultant plasmid was transformed into DH10Bac cells and then the transformation mixture was spread on Luria-Bertani (LB) agarose culture medium containing isopropyl-beta-D-thiogalactoside (IPTG), X-gal, gentamicin, kanamycin and tetracycline. The white colonies were selected and cultured for amplification, and the hSox9Bacmid DNA was extracted. After verification, recombinant baculovirus Ac-CMV-hSox9 was obtained through transfecting Sf 21 cells. The expression of hSox9 gene in the intervertebral disc cells in rabbits was determined by Western blotting and immunohistochemical staining.</p><p><b>RESULTS</b>Polymerase chain reaction (PCR) confirmed the presence of hSox9 gene in the recombinant baculovirus and the Sf 21 cells transfected by the baculovirus showed the expression of fluorescence protein. Western blotting and immunohistochemical staining analysis indicated that exogenous hSox9 gene was expressed in the disc cells.</p><p><b>CONCLUSIONS</b>The successful construction of the recombinant baculovirus Ac-CMV-hSox9 and the confirmation of the target gene expression provides a novel expression vector system for basic research and clinical treatment of intervertebral degenerative disc disease.</p>
Assuntos
Animais , Humanos , Coelhos , Baculoviridae , Genética , Citomegalovirus , Genética , Expressão Gênica , Terapia Genética , Vetores Genéticos , Proteínas de Grupo de Alta Mobilidade , Genética , Metabolismo , Imuno-Histoquímica , Disco Intervertebral , Biologia Celular , Metabolismo , Patologia , Vértebras Lombares , Plasmídeos , Proteínas Recombinantes , Fatores de Transcrição SOX9 , Doenças da Coluna Vertebral , Terapêutica , Fatores de Transcrição , Genética , Metabolismo , TransfecçãoRESUMO
The Baculoviridae are a large family of enveloped DNA viruses exclusively pathogenic to arthropods. Baculoviruses have been extensively used in insect cell-based recombinant protein expression system and as biological pesticides. They have been deomostrated to be safe to mammals, birds and fish. Recently, baculoviruses has been shown to transduce different mammalian cells in spite of the fact that they cannot replicate in mammalian cells (11, 73, 76). This has resulted in the development of baculoviruses as mammalian expression systems and even as vestors for gene therapy.