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-C...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 pFastBacDul-green fluorescene protein （ GFP ）-CMV （pgGC）-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-β-D-thiogalactoside （IPTG）, X-gal, gentamicin, kanamycin and tetracycline. The white colonies were selected and cultured for amplification, and the hSox9 Bacmid DNA was extracted. After verification, recombinant baculovirns 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 indicated that exogenous hSox9 disc cells. staining analysis 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.展开更多
基金This work was supported by the National Natural Science Foundation of China （No. 30672130 ）, China Postdoctoral Foundation （No. 20060390249 ） and National Basic Research Priorities Program of China （No. 2003CB1140）.
文摘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 pFastBacDul-green fluorescene protein （ GFP ）-CMV （pgGC）-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-β-D-thiogalactoside （IPTG）, X-gal, gentamicin, kanamycin and tetracycline. The white colonies were selected and cultured for amplification, and the hSox9 Bacmid DNA was extracted. After verification, recombinant baculovirns 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 indicated that exogenous hSox9 disc cells. staining analysis 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.
