[Expression of HCV NS5A gene in HepG2 cell and the effect of the NS5A expression on PI3K in vitro].

AIM: To reveal the influence of HCV-NS5A on PI3K, we probe into the relationship between NS5A and PI3K in vitro. METHODS: Full length NS5A gene of HCV was amplified by PCR, using the plasmid containing HCV full-length open reading frame (ORF) as template, and cloned into the eukaryotic expressing plasmid pcDNA3.0(-) by DNA recombination technique. The recombinant vector was identified by digestion with restriction enzymes and polymerase chain reaction and by directly sequencing. Then both the recombinant vector pcDNA3.0(-)-NS5A and the control vector pcDNA3.0(-) were transfected HepG2 cell using Lipofectamin2000. RESULTS: Expressing NS5A was proven by RT-PCR and Western blot analysis in transfected HepG2 cell. Further more we examined the PI3K protein expressed in the HepG2 cell expressing recombinant NS5A. CONCLUSION: NS5A can activate PI3K in vitro and its signal cascade pathway.

[Study on relationship between HBV post-transcriptional regulatory element and response to IFN-alpha by using a reporter gene luciferase].

AIM: To identify the function of HBV post-transcriptional regulatory element(HPRE) in response to IFN-alpha by using the reporter gene luciferase (LUC). METHODS: The gene encoding LUC was obtained by PCR from the vector pGEM-luc and then cloned into the eukaryotic expression vector pcDNA3.0. The HPRE, HPREalphabeta(1) and HPREbeta(1)beta(2) fragments were amplified by PCR from HBV genome and then cloned into the recombinant vector pcDNA3.0-luc. These constructed plasmids were transfected into the human hepatoma cell line HepG2. The expression of LUC before and after the addition of IFN-alpha was detected by LUC assay system. RESULTS: The DNA sequencing analysis showed that the recombinant plasmids pcDNA3.0-luc, pcDNA3.0-luc-HPRE, pcDNA3.0-luc-HP-REalphabeta(1) and pcDNA3.0-luc-HPREbeta(1)beta(2) were successfully constructed. The results of LUC detection assay showed that HPRE, HPREalphabeta(1) and HPREbeta(1)beta(2) could enhance the expression of LUC before the addition of IFN-alpha. When IFN-alpha was added, only the fragment of HPRE, HPREbeta(1)beta(2) could significantly reduce the expression of LUC, but the expression of LUC was not influenced by HPREalphabeta(1). CONCLUSION: The functional element beta(2) of HPRE plays a more important part in response to IFN-alpha than the element alpha and beta(1), which may be valuable for further research on mechanisms of IFN-alpha therapy in HBV infection and function of HPRE binding protein.