This study aimed to construct the dual-gene expression vector p Hsa-miR16-siRNA which can express human miR-16 and HBV X siRNA, and examine its regulatory effect on HBV gene expression in the HepG2.2.15 cell line. The...This study aimed to construct the dual-gene expression vector p Hsa-miR16-siRNA which can express human miR-16 and HBV X siRNA, and examine its regulatory effect on HBV gene expression in the HepG2.2.15 cell line. The expression vectors siR-1583 and pHsa-miR16-siRNA were designed and constructed. Hep G2.2.15 cells were transfected with the empty vector, siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively. ELISA was performed to measure the expression of HBsAg and HBeAg in the culture supernatant 48 and72 h post transfection. Fluorescence quantitative PCR was used to measure the HBV mRNA degradation efficiency and HBV DNA copy number. The results showed that the expression of HBV genes was significantly inhibited in Hep G2.2.15 cells transfected with siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively, when compared with that in cells transfected with the empty vectors, with the inhibitory effect of pHsa-miR16-siRNA being the most significant. ELISA showed that the inhibitory rates of HBs Ag and HBeAg in pHsa-miR16-siRNA transfected cells were correspondingly 87.3% and 85.0% at 48 h, and 88.6% and 86.5% at 72 h post transfection（P〈0.01 vs. control group）. RT-PCR showed that the level of HBV mRNA decreased by 80.2%（t=–99.22, P〈0.01）, the genomic HBV DNA by 92.8%（t=–73.06, P〈0.01）, and the supernatant of HBV DNA copy number by 89.8%（t=–47.13, P〈0.01） in pHsa-miR16-siRNA transfected group. It was suggested that the dual-gene expression vector pHsa-miR16-siRNA can inhibit the replication of HBV more efficiently than a single-gene expression vector.展开更多
Objective： To facilitate manipulation of gene expression in different host cells, we used pEGFP-N1 as backbone to construct a versatile vector that can drive foreign gene expression in prokaryotic and eukaryotic cell...Objective： To facilitate manipulation of gene expression in different host cells, we used pEGFP-N1 as backbone to construct a versatile vector that can drive foreign gene expression in prokaryotic and eukaryotic cells. Methods： A cloning and expression vector, pEGFP-NI-lac, was constructed by inserting the prokaryotic lac promoter of pUC 19 into the eukaryotic expression vector, pEGFP-N1, between the eukaryotic PCMV promoter and enhanced green fluorescent protein （EGFP） open reading frames. To assess the function of pEGFP-NI-lac, the nucleotide sequence encoding the hepatitis C virus （HCV） core protein was cloned into the multiple cloning sites. Western blotting analysis was used to detect the expression of the HCV core protein in Escherichia coli DH5a and HepG2 cells. Results： Restriction enzyme digestion and sequence analysis indicated that pEGFP-NI-lac was successfully constructed and the HCV core gene was cloned into this vector. The Western blotting results showed that pEGFP-NI-lac promoted expression of HCV core gene in prokaryotic E. coli DH5a and eukaryotic HepG2 cells. Conclusion： The pEGFP-NI-lac vector has been successfully constructed and functions in both prokaryotic and eukaryotic cells. The EGFP reporter can be used as an insert-inactivation marker for clone selection or as an expression tag. This vector can be used for cloning and expression of genes in both prokaryotic and eukaryotic cells, making gene cloning, expression and functional studies convenient as well as time- and labor-efficient展开更多
Objective To observe the efficiency and time course of gene expression and the safety of adenoviral vector mediated gene transfer in vivo.Methods After soaking soluble stents in a high concentration of glucose solut...Objective To observe the efficiency and time course of gene expression and the safety of adenoviral vector mediated gene transfer in vivo.Methods After soaking soluble stents in a high concentration of glucose solution containing Adv5-CMV (cytomegalovirus) (control group) or Adv5-CMV/LacZ (treatment group) for 30 minutes, the stents were inserted into the lumina of cut rat carotid arteries and end-to-end anastomoses of the cut carotid were performed with standard microvascular surgical techniques. On days 2, 7, 14, 28, 60 and 90 after gene transfer, anastomotic arteries of the two groups were observed. On days 7 and 14, the ascending aortas, hearts, brains, livers, lungs, spleens and kidneys of the treatment group were observed. All samples were analyzed for the presence of β-galactosidase activity and histochemical staining.Results β-galactosidase activity was not detected in the carotid arteries of the control group and organs not directly exposed to adenoviral vector of the treatment group. The amount of β-galactosidase activity (×10-3?U/g tissue) in the treatment group on the 2nd, 7th, 14th, 28th, 60th and 90th day after gene transfer was 3.87, 11.38, 9.8, 6.43, 3.18 and 2.43, respectively. Microscopic examination of sections from vessels of the control group and from the aortas, hearts, brains, livers, lungs, spleens or kidneys of the treatment group revealed no X-gal staining. Microscopic examination of carotid arteries of the treatment group revealed blue-staining in all anastomotic arteries and in all layers of the arterial wall observed on days 7 and 14 after gene transfer.Conclusion Adenoviral vector can effectively infect blood vessels in vivo. After adenoviral vector mediated direct gene transfer into anastomotic rat carotid arteries, recombinant gene expression began on day 2, peaked between days 7 and 14, prominently declined after day 28, and persisted at low levels more than three months. A recombinant gene could be delivered to a specific site by direct gene transfer in vivo by adenoviral vector infection.展开更多
Abstract:Objective To develop a retroviral-mediated high efficient expression system of human coagulation factor Ⅷ. Methods The LNC-FⅧBD retroviral vector was generated by cloning a human B-domain-deleted (760aa～16...Abstract:Objective To develop a retroviral-mediated high efficient expression system of human coagulation factor Ⅷ. Methods The LNC-FⅧBD retroviral vector was generated by cloning a human B-domain-deleted (760aa～1639aa) Factor Ⅷ (FⅧ) cDNA (FⅧ cDNA BD) into the retroviral vector pLNCX. Several mammalian cell lines, including NIH3T3, CHO, Cos-7 and human hepatic cell line, L-02, were transduced with viral supernatant from the highest virus-producing PA317 clone. Antigen and coagulant activity of human FⅧ in cell culture medium were measured by ELISA and a one-stage method, respectively. RT-PCR was performed for the detection of FⅧBD mRNA. Results Human FⅧ was expressed in all four target cells, with the highest FⅧ expression observed in NIH3T3. The coagulant activity of secreted FⅧ was up to 1.6U/106 cells*24?hrs-1, and the FⅧ antigen was 500?ng/106 cells*24?hrs-1. FⅧ coagulant activity and antigen expressed by transduced CHO cells were 0.12?U/106 cells*24?hrs-1 and 62.4?ng/106 cells*24?hrs-1, respectively. Human FⅧ expression was relatively low in Cos-7 and L-02 cells. RT-PCR results demonstrated transcription of FⅧcDNA BD in the target cells.Conclusions The constructed retroviral vector was able to direct high level expression of human FⅧ in various mammalian cell lines. It has potential utility in the future gene therapy for Hemophilia A.展开更多
基金supported by grants from the Independent Innovation Research Fund of Huazhong University of Science and Technology(No.2016YXMS200)Natural Science Foundation of the Science and Technology Department of Hubei Province(No.ZRMS2017000406)
文摘This study aimed to construct the dual-gene expression vector p Hsa-miR16-siRNA which can express human miR-16 and HBV X siRNA, and examine its regulatory effect on HBV gene expression in the HepG2.2.15 cell line. The expression vectors siR-1583 and pHsa-miR16-siRNA were designed and constructed. Hep G2.2.15 cells were transfected with the empty vector, siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively. ELISA was performed to measure the expression of HBsAg and HBeAg in the culture supernatant 48 and72 h post transfection. Fluorescence quantitative PCR was used to measure the HBV mRNA degradation efficiency and HBV DNA copy number. The results showed that the expression of HBV genes was significantly inhibited in Hep G2.2.15 cells transfected with siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively, when compared with that in cells transfected with the empty vectors, with the inhibitory effect of pHsa-miR16-siRNA being the most significant. ELISA showed that the inhibitory rates of HBs Ag and HBeAg in pHsa-miR16-siRNA transfected cells were correspondingly 87.3% and 85.0% at 48 h, and 88.6% and 86.5% at 72 h post transfection（P〈0.01 vs. control group）. RT-PCR showed that the level of HBV mRNA decreased by 80.2%（t=–99.22, P〈0.01）, the genomic HBV DNA by 92.8%（t=–73.06, P〈0.01）, and the supernatant of HBV DNA copy number by 89.8%（t=–47.13, P〈0.01） in pHsa-miR16-siRNA transfected group. It was suggested that the dual-gene expression vector pHsa-miR16-siRNA can inhibit the replication of HBV more efficiently than a single-gene expression vector.
基金Supported by the National High Technology Research and Development Program of China (863 Program, 2009AA02Z111)the National Natural Science Foundation of China (30872223)the Funds of the State Key Laboratory of Pathogen and Biosecurity
文摘Objective： To facilitate manipulation of gene expression in different host cells, we used pEGFP-N1 as backbone to construct a versatile vector that can drive foreign gene expression in prokaryotic and eukaryotic cells. Methods： A cloning and expression vector, pEGFP-NI-lac, was constructed by inserting the prokaryotic lac promoter of pUC 19 into the eukaryotic expression vector, pEGFP-N1, between the eukaryotic PCMV promoter and enhanced green fluorescent protein （EGFP） open reading frames. To assess the function of pEGFP-NI-lac, the nucleotide sequence encoding the hepatitis C virus （HCV） core protein was cloned into the multiple cloning sites. Western blotting analysis was used to detect the expression of the HCV core protein in Escherichia coli DH5a and HepG2 cells. Results： Restriction enzyme digestion and sequence analysis indicated that pEGFP-NI-lac was successfully constructed and the HCV core gene was cloned into this vector. The Western blotting results showed that pEGFP-NI-lac promoted expression of HCV core gene in prokaryotic E. coli DH5a and eukaryotic HepG2 cells. Conclusion： The pEGFP-NI-lac vector has been successfully constructed and functions in both prokaryotic and eukaryotic cells. The EGFP reporter can be used as an insert-inactivation marker for clone selection or as an expression tag. This vector can be used for cloning and expression of genes in both prokaryotic and eukaryotic cells, making gene cloning, expression and functional studies convenient as well as time- and labor-efficient
文摘Objective To observe the efficiency and time course of gene expression and the safety of adenoviral vector mediated gene transfer in vivo.Methods After soaking soluble stents in a high concentration of glucose solution containing Adv5-CMV (cytomegalovirus) (control group) or Adv5-CMV/LacZ (treatment group) for 30 minutes, the stents were inserted into the lumina of cut rat carotid arteries and end-to-end anastomoses of the cut carotid were performed with standard microvascular surgical techniques. On days 2, 7, 14, 28, 60 and 90 after gene transfer, anastomotic arteries of the two groups were observed. On days 7 and 14, the ascending aortas, hearts, brains, livers, lungs, spleens and kidneys of the treatment group were observed. All samples were analyzed for the presence of β-galactosidase activity and histochemical staining.Results β-galactosidase activity was not detected in the carotid arteries of the control group and organs not directly exposed to adenoviral vector of the treatment group. The amount of β-galactosidase activity (×10-3?U/g tissue) in the treatment group on the 2nd, 7th, 14th, 28th, 60th and 90th day after gene transfer was 3.87, 11.38, 9.8, 6.43, 3.18 and 2.43, respectively. Microscopic examination of sections from vessels of the control group and from the aortas, hearts, brains, livers, lungs, spleens or kidneys of the treatment group revealed no X-gal staining. Microscopic examination of carotid arteries of the treatment group revealed blue-staining in all anastomotic arteries and in all layers of the arterial wall observed on days 7 and 14 after gene transfer.Conclusion Adenoviral vector can effectively infect blood vessels in vivo. After adenoviral vector mediated direct gene transfer into anastomotic rat carotid arteries, recombinant gene expression began on day 2, peaked between days 7 and 14, prominently declined after day 28, and persisted at low levels more than three months. A recombinant gene could be delivered to a specific site by direct gene transfer in vivo by adenoviral vector infection.
基金ThisworkwassupportedbytheDrug developmentFoundationof ShanghaiScienceandTechnicalCommittee (No .975 4190 0 1)andDr .ClydeWuFoundationofShanghaiInstituteofHematology .
文摘Abstract:Objective To develop a retroviral-mediated high efficient expression system of human coagulation factor Ⅷ. Methods The LNC-FⅧBD retroviral vector was generated by cloning a human B-domain-deleted (760aa～1639aa) Factor Ⅷ (FⅧ) cDNA (FⅧ cDNA BD) into the retroviral vector pLNCX. Several mammalian cell lines, including NIH3T3, CHO, Cos-7 and human hepatic cell line, L-02, were transduced with viral supernatant from the highest virus-producing PA317 clone. Antigen and coagulant activity of human FⅧ in cell culture medium were measured by ELISA and a one-stage method, respectively. RT-PCR was performed for the detection of FⅧBD mRNA. Results Human FⅧ was expressed in all four target cells, with the highest FⅧ expression observed in NIH3T3. The coagulant activity of secreted FⅧ was up to 1.6U/106 cells*24?hrs-1, and the FⅧ antigen was 500?ng/106 cells*24?hrs-1. FⅧ coagulant activity and antigen expressed by transduced CHO cells were 0.12?U/106 cells*24?hrs-1 and 62.4?ng/106 cells*24?hrs-1, respectively. Human FⅧ expression was relatively low in Cos-7 and L-02 cells. RT-PCR results demonstrated transcription of FⅧcDNA BD in the target cells.Conclusions The constructed retroviral vector was able to direct high level expression of human FⅧ in various mammalian cell lines. It has potential utility in the future gene therapy for Hemophilia A.
