OBJECTIVE: To construct the recombinant baculovirus Ac-cytomegalovirus (CMV)-hSox9 for gene therapy of intervertebral disc degeneration. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
OBJECTIVE: To construct the recombinant baculovirus Ac-cytomegalovirus (CMV)-hSox9 for gene therapy of intervertebral disc degeneration. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.