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


CONCLUSIONS
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.
Yao
Wen-juan
WJ
Life Sciences College, Nanjing Normal University, Nanjing 210097, China. njsdyd@163.com
Diao
Zhen-yu
ZY
Deng
Xiao-zhao
XZ
Kong
Jing
J
Zhou
Zong-an
ZA
Gao
Jian
J
Zhang
Yun
Y
D000818:Animals; D045744:Cell Line, Tumor; D004262:DNA Restriction Enzymes / Q000378:metabolism; D017930:Genes, Reporter; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D050436:Regulatory Elements, Transcriptional / Q000235:genetics*; D017422:Sequence Analysis, DNA
D016898:Interferon-alpha; D008156:Luciferases; D004262:DNA Restriction Enzymes