[Construction of eukaryotic expression vector expressing hepatitis C virus NS5B and EGFP fusion protein and establishment of stable transfected HepG2 cell line].
OBJECTIVE
To construct a eukaryotic expression vector for expressing hepatitis C virus (HCV) recombinant NS5B-EGFP fusion protein and obtain a stable transfected HepG2 cell line.


METHODS
The coding region of NS5B gene of HCV was amplified by PCR and was digested by Xho I/Kpn I. This fragment was inserted into pEGFPN3 with T4 ligase and transformed E. coli TG1. The positive recombinant plasmid was selected, then the recombinant plasmid was transfected into HepG2 cell by Lipofectin AMINE 2000. Cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. The stable transfected cell line expressing high level NS5B-EGFP fusion protein was obtained.


RESULTS
The eukaryotic expression vector named pEGFPN3-ns5b was successfully constructed and the stable transfected HepG2 cell line expressing NS5B-EGFP fusion protein was obtained.


CONCLUSIONS
The stable transfected HepG2 cell line could express NS5B-EGFP fusion protein, could be used for anti-HCV infection with ns5b gene as the target.
Lei
Ying-feng
YF
Department of Microbiology, The Fourth Military Medical University, Xi' an, China.
Xue
Xiao-ping
XP
Yin
Wen
W
Lü
Xin
X
Yang
Jing
J
D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
D049452:Green Fluorescent Proteins; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C485184:enhanced green fluorescent protein; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase