INTRODUCTIONHepatitis B virus (HBV) is regarded as one of themain etiologic factors involved in the developmentof human hepatocellular carcinoma (HCC).The open reading frame (orf)of X gene of HBVencoded a transactivat...INTRODUCTIONHepatitis B virus (HBV) is regarded as one of themain etiologic factors involved in the developmentof human hepatocellular carcinoma (HCC).The open reading frame (orf)of X gene of HBVencoded a transactivating factor is the evidence thatstrongly supported the notion that the X gene ofHBV DNA integrated in HCC genomic DNA couldcontribute to the carcinogenesis of liver cells byactivation of some related cellular genes展开更多
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
AIM To construct the recombinant of HDVcDNA and HBV-specific ribozyme gene byrecombinant PCR in order to use HDV as atransporting vector carrying HBV-specificribozyme into liver cells for inhibiting thereplication of ...AIM To construct the recombinant of HDVcDNA and HBV-specific ribozyme gene byrecombinant PCR in order to use HDV as atransporting vector carrying HBV-specificribozyme into liver cells for inhibiting thereplication of HBV.METHODS We separately cloned the ribozyme（RZ）gene and recombinant DVRZ（comprisingHDV cDNA and HBV-specific ribozyme gene）intothe downstream of T7 promoter of pTAdv-Tvector and studied the in vitro cleavage activityof their transcripts（rRZ,rDVRZ）on target RNA（rBVCF）from in vitro transcription of HBV Cgene fragment（BVCF）.RESULTS Both the simple（rRZ）and therecombinant ribozyme rDVRZ could efficientlycatalyze the cleavage of target RNA（rBVCF）under different temperatures（37℃,42℃ and55℃）and Mg<sup>2+</sup>concentrations（10 mmol/L,15 mmol/L and 20 mmol/L）and their catalyticactivity tended to increase as the temperaturewas rising.But the activity of rRZ was evidentlyhigher than that of rDVRZ.CONCLUSION The recombinant of HDV cDNAand ribozyme gene had the potential of beingfurther explored and used in gene therapy of HBVinfection.展开更多
基金Projects of the Science Development Foundation of Shanghai (994919033)Tackling Key Problems in Science and Technology from the State Science and Technology Ministry(TJ99LA01)
文摘INTRODUCTIONHepatitis B virus (HBV) is regarded as one of themain etiologic factors involved in the developmentof human hepatocellular carcinoma (HCC).The open reading frame (orf)of X gene of HBVencoded a transactivating factor is the evidence thatstrongly supported the notion that the X gene ofHBV DNA integrated in HCC genomic DNA couldcontribute to the carcinogenesis of liver cells byactivation of some related cellular genes
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.
基金Natural Science Foundation of Guangdong Province,No.940311.
文摘AIM To construct the recombinant of HDVcDNA and HBV-specific ribozyme gene byrecombinant PCR in order to use HDV as atransporting vector carrying HBV-specificribozyme into liver cells for inhibiting thereplication of HBV.METHODS We separately cloned the ribozyme（RZ）gene and recombinant DVRZ（comprisingHDV cDNA and HBV-specific ribozyme gene）intothe downstream of T7 promoter of pTAdv-Tvector and studied the in vitro cleavage activityof their transcripts（rRZ,rDVRZ）on target RNA（rBVCF）from in vitro transcription of HBV Cgene fragment（BVCF）.RESULTS Both the simple（rRZ）and therecombinant ribozyme rDVRZ could efficientlycatalyze the cleavage of target RNA（rBVCF）under different temperatures（37℃,42℃ and55℃）and Mg<sup>2+</sup>concentrations（10 mmol/L,15 mmol/L and 20 mmol/L）and their catalyticactivity tended to increase as the temperaturewas rising.But the activity of rRZ was evidentlyhigher than that of rDVRZ.CONCLUSION The recombinant of HDV cDNAand ribozyme gene had the potential of beingfurther explored and used in gene therapy of HBVinfection.
