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1.
Acta Academiae Medicinae Sinicae ; (6): 13-18, 2013.
Artículo en Chino | WPRIM | ID: wpr-284312

RESUMEN

<p><b>OBJECTIVE</b>To establish a stable cell line that can replicate hepatitis B virus (HBV) DNA carrying the reverse transcriptase sequence derived from a clinical isolate.</p><p><b>METHODS</b>Nested PCR was used to amplify the HBV DNA fragment from the serum. The fragment was cloned into a plasmid that can support HBV replication in vitro by fragment substitution reaction (FSR), followed by the cloning of the neomycin expressing fragment downstream from HBV DNA. G418 selection was conducted after the transfection of HepG2 cells with the recombinant DNA. Real-time PCR and enzyme linked immunosorbent assay (ELISA) were used to screen stable cell lines that can replicate HBV DNA, and the replication of HBV DNA by the cell line was confirmed by using Southern blot analysis.</p><p><b>RESULTS</b>Fragment nt55-1654 amplified from the serum DNA was substituted to the plasmid pLL, generating the plasmid p11. The neomycin fragment was cloned into p11, leading to the plasmid p11-neo, and p11-neo was confirmed to be HBV-replication-competent. A stable cell line named 3-10 that can replicate HBV DNA was obtained.</p><p><b>CONCLUSIONS</b>A stable cell line was established that can replicate HBV DNA carrying the reverse transcriptase sequence derived from a clinical isolate. Real-time PCR plus ELISA may help to rapidly screen out stable cell lines replicating HBV DNA.</p>


Asunto(s)
Humanos , Línea Celular , Clonación Molecular , Replicación del ADN , ADN Viral , Vectores Genéticos , Células Hep G2 , Virus de la Hepatitis B , Genética , Hepatocitos , Biología Celular , Virología , Plásmidos , ADN Polimerasa Dirigida por ARN , Genética , Replicación Viral , Genética
2.
Chinese Journal of Hepatology ; (12): 565-569, 2013.
Artículo en Chino | WPRIM | ID: wpr-278039

RESUMEN

<p><b>OBJECTIVE</b>To investigate the biological role of auto-induced expression of hepatitis C virus (HCV) core protein (protein C) using a recombinant protein in an in vitro cell-based system.</p><p><b>METHODS</b>The PCR-amplified full-length HCV protein C gene (573 bp) was inserted into the pET28a prokaryotic expression vector. The recombinant plasmid was transformed into BL21(DE3)pLysS E. coli to achieve high-concentration expression of the recombinant C protein by auto-induction. The recombinant protein C was purified by Ni-NTA affinity chromatography, and tested in a protein binding assay for its ability to bind the HCV NS3 protein.</p><p><b>RESULTS</b>The transformed E. coli produced a large amount of recombinant protein C, as detected in the sonicated supernatant of the bacteria culture. The antigenic reactivity of the recombinant protein C was confirmed by western blotting. However, the recombinant protein C could not be purified by Ni-NTA affinity chromatography, but co-precipitated with the HCV NS3 protein.</p><p><b>CONCLUSION</b>Soluble recombinant protein C was successfully expressed by auto-induction, and shown to interact with the HCV NS3 protein, which provides a novel insight into the putative biological activity of this factor in HCV-related molecular processes. Future studies of this recombinant HCV protein C's crystal structure and antigenicity may provide further clues to its biological function(s) and potential for clinical applications.</p>


Asunto(s)
Escherichia coli , Metabolismo , Vectores Genéticos , Hepacivirus , Proteínas Recombinantes , Genética , Metabolismo , Proteínas del Núcleo Viral , Genética , Metabolismo , Proteínas no Estructurales Virales , Metabolismo
3.
Chinese Journal of Hepatology ; (12): 692-695, 2011.
Artículo en Chino | WPRIM | ID: wpr-330660

RESUMEN

<p><b>OBJECTIVE</b>To search for the optimal approach for hepatocyte-directed differentiation of hepatic progenitor cells and investigate the molecular mechanism of the hepatic differentiation.</p><p><b>METHODS</b>Hepatic progenitor cells were infected with recombinant adenovirus which containing human LIF, BMP2 or BMP9 gene. The maturation and differentiation of progenitor cells were examined by PAS staining and ICG uptake methods at 4, 7 and 10 days post infection. The production of Albumin (Alb) was measured by luciferase activity at day 4, 7, 10 and 14.</p><p><b>RESULTS</b>PAS staining assay revealed that BMP2 and BMP9 enhanced glycogen storage in hepatic progenitor cells most obviously at day 7. The percentages of positive cells were 30% and 45% respectively at 7 days post-infection. Meanwhile, 40% and 30% cells were positive by ICG uptake assay after BMP2 and BMP9 induction. Luciferase activity indicated that BMP9 induced ALB-Luc activity most significantly at day 7. However, less inductive activity was found in LIF-treated group.</p><p><b>CONCLUSION</b>These results indicated tuat hepatic progenitor cells were differentiated into hepatocyte-like cells by BMPs and LIF induction.</p>


Asunto(s)
Humanos , Adenoviridae , Proteínas Morfogenéticas Óseas , Farmacología , Diferenciación Celular , Células Cultivadas , Hepatocitos , Biología Celular , Metabolismo , Virología , Factor Inhibidor de Leucemia , Farmacología , Células Madre , Biología Celular , Metabolismo , Virología
4.
China Biotechnology ; (12)2006.
Artículo en Chino | WPRIM | ID: wpr-686173

RESUMEN

In order to demonstrate PTB bind to HPRE,reverse transcription,PCR-mediated detection,were used.HepG2.2.15 cell line and HBs-HPRE transient expression cells were adopted to identify PTB function in HBV life cycle.The results showed that PTB could directly bind to HPRE RNA.Functional analysis indicated that PTB could inhibit the expression of HBs antigen and this inhibition was in a dose-dependent manner in HepG2.2.15 cells.Higher expression of HBs in cells transfected pcDNA3-HBs-HPRE comparing with pcDNA3-HBs,and this high expression could also be inhibited by PTB.The data demonstrated that PTB inhibits HBs expression by interacting with HPRE.

5.
Chinese Journal of Hepatology ; (12): 561-564, 2006.
Artículo en Chino | WPRIM | ID: wpr-341301

RESUMEN

<p><b>OBJECTIVE</b>To screen and identify proteins that interact with hepatitis C virus NS3 by means of T7-phage display system.</p><p><b>METHODS</b>Hepatitis C virus NS3 was expressed by prokaryotic expression and used as a selected molecule to biopan the T7 select human liver cDNA library; the selected positive clones were identified using DNA sequencing and analyzed with BLAST program in GenBank.</p><p><b>RESULTS</b>After BLAST analysis in all the positive clones, the proteins which interacted with the hepatitis C virus NS3 were found to be serpin peptidase inhibitor, clade A, member 1 (SERPINA1) and cyclophilin-LC.</p><p><b>CONCLUSION</b>T7-phage display system is a convenient, rapid and effective method for screening interacting proteins. The proteins thus selected will provide an important means for studying the pathogenesis and carcinogenesis of HCV.</p>


Asunto(s)
Humanos , Línea Celular , Biblioteca de Genes , Hepacivirus , Metabolismo , Biblioteca de Péptidos , Mapeo de Interacción de Proteínas , Métodos , Proteínas Virales de Fusión , Genética , Metabolismo , Proteínas no Estructurales Virales , Genética , Metabolismo
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