AIM: To study interactions between hepatitis B virus (HBV) and interferon-a in liver- derived cells. METHODS. mRNAs were separately isolated from an HBV-transfected cell line (HepG2 2.2.15) and its parental cell line ...AIM: To study interactions between hepatitis B virus (HBV) and interferon-a in liver- derived cells. METHODS. mRNAs were separately isolated from an HBV-transfected cell line (HepG2 2.2.15) and its parental cell line (HePG2) pre- and post-interferon-a (IFN-a) treatment at 6, 24 and 48 h, followed by hybridization with a cDNA microarray filter dotted with 14 000 human genes. After hybridization and scanning of the arrays, the data were analyzed using ArrayGauge software. The microarray data were further verified by Northern blot analysis. RESULTS: Compared to HepG2 cells, 14 genes with known functions were down-regulated 3 to 12- magnitudes, while 7 genes were up-regulated 3-13 magnitudes in HepG2 2.2.15 cells prior to IFN-a treatment. After interferon-a treatment, the expression of four genes (vascular endothelial growth factor, tyrosine phosphate 1E, serine protein with IGF-binding motif and one gene of clathrin light chain) in HepG2 2.2.15 were up-regulated, while one gene encoding a GTP-binding protein, two genes of interferon-induced kinases and two proto-oncogenes were further down- regulated. Interestingly, under IFN-a treatment, a number of differentially regulated genes were new ESTs or genes with unknown functions. CONCLUSION: The up-regulated genes in HepG2 2.2.15 cell line suggested that under IFN-a treatment, these repressed cellular genes in HBV infected hepatooltes could be partially restored, while the down- regulated genes were most likely the cellular genes which could not be restored under interferon treatment. These down-regulated genes identified by microarray analysis could serve as new targets for anti-HBV drug development or for novel therapies.展开更多
Objective To construct a recombinant lactobacillus (Lb.) strain excreting the human chorionic gonadotropin beta-subunit (hCGβ) Methods The hCGβ cDNA was ligated to the signal peptide sequence of S-layer protein fr...Objective To construct a recombinant lactobacillus (Lb.) strain excreting the human chorionic gonadotropin beta-subunit (hCGβ) Methods The hCGβ cDNA was ligated to the signal peptide sequence of S-layer protein from Lb. brevis and then cloned into down-stream of lactose-inducible promoter of an integrative plasmid, pIlac. After electroporation into Lb. casei CECT5276, PCR using the genomic DNA of the recombinant lactobacillus as template was performed to confirm whether the hCGβ gene had been integrated into the genome. Radioimmunoassay (RIA) was used to determine the level of hCGβ in the supernatant and the cell lysate. Results The hCGβ was integrated into the genome of Lb. casei CECT5276. The highest concentration of hCGβ in the culture supernatant amounted to 440 mIU/mL 21 h after lactose induction. About 2/3 of the abstract Objective proteins were excreted into the supernatant. Conclusion We have obtained stable and efficient hCGβ excretion in Lb. casei, which was inducible by lactose.展开更多
基金Supported by the Chinese State Basic Science Foundation,No.1999054105 and Med-X Foundation of Fudan University
文摘AIM: To study interactions between hepatitis B virus (HBV) and interferon-a in liver- derived cells. METHODS. mRNAs were separately isolated from an HBV-transfected cell line (HepG2 2.2.15) and its parental cell line (HePG2) pre- and post-interferon-a (IFN-a) treatment at 6, 24 and 48 h, followed by hybridization with a cDNA microarray filter dotted with 14 000 human genes. After hybridization and scanning of the arrays, the data were analyzed using ArrayGauge software. The microarray data were further verified by Northern blot analysis. RESULTS: Compared to HepG2 cells, 14 genes with known functions were down-regulated 3 to 12- magnitudes, while 7 genes were up-regulated 3-13 magnitudes in HepG2 2.2.15 cells prior to IFN-a treatment. After interferon-a treatment, the expression of four genes (vascular endothelial growth factor, tyrosine phosphate 1E, serine protein with IGF-binding motif and one gene of clathrin light chain) in HepG2 2.2.15 were up-regulated, while one gene encoding a GTP-binding protein, two genes of interferon-induced kinases and two proto-oncogenes were further down- regulated. Interestingly, under IFN-a treatment, a number of differentially regulated genes were new ESTs or genes with unknown functions. CONCLUSION: The up-regulated genes in HepG2 2.2.15 cell line suggested that under IFN-a treatment, these repressed cellular genes in HBV infected hepatooltes could be partially restored, while the down- regulated genes were most likely the cellular genes which could not be restored under interferon treatment. These down-regulated genes identified by microarray analysis could serve as new targets for anti-HBV drug development or for novel therapies.
文摘Objective To construct a recombinant lactobacillus (Lb.) strain excreting the human chorionic gonadotropin beta-subunit (hCGβ) Methods The hCGβ cDNA was ligated to the signal peptide sequence of S-layer protein from Lb. brevis and then cloned into down-stream of lactose-inducible promoter of an integrative plasmid, pIlac. After electroporation into Lb. casei CECT5276, PCR using the genomic DNA of the recombinant lactobacillus as template was performed to confirm whether the hCGβ gene had been integrated into the genome. Radioimmunoassay (RIA) was used to determine the level of hCGβ in the supernatant and the cell lysate. Results The hCGβ was integrated into the genome of Lb. casei CECT5276. The highest concentration of hCGβ in the culture supernatant amounted to 440 mIU/mL 21 h after lactose induction. About 2/3 of the abstract Objective proteins were excreted into the supernatant. Conclusion We have obtained stable and efficient hCGβ excretion in Lb. casei, which was inducible by lactose.
