Stable Huh-7 cell lines expressing non-structural proteins of genotype 1a of hepatitis C virus.
Hepatitis C virus (HCV) infection has infected approximately 3% of the world population. HCV genotype 1a is distributed throughout the world, and along with genotype 1b, is relatively resistant to current standards of therapy compared to other HCV genotypes. The present study was designed to produce stable Huh-7 cell lines expressing non-structural proteins of HCV genotype la, representing an in vitro system to facilitate the development of new antiviral drugs against chronic HCV infection. The non-structural genes of HCV genotype 1a were amplified and cloned in a mammalian expression vector pCR 3.1/FIagTag. Huh-7 cells were transfected with one of two expression plasmids, the first containing the NS2, NS3, and NS4a cassette, and second containing the NS5a and NS5b genes. Stable cell lines were produced under the selection of gentamycin (G418). mRNA and protein expression analysis was performed by RT-PCR and Western blotting. The RT-PCR and Western blot results confirmed the stable expression of each of the gene products. Stable Huh-7 cell lines with HCV la non-structural proteins may be helpful for evaluating the role of HCV proteins in molecular pathogenesis, and could facilitate the development of new therapeutic drugs.
Applied and Functional Genomics Laboratory, Centre of Excellence in Molecular Biology, 87-West Canal Bank Road, 53700 Lahore, Pakistan. imran_shahid34@yahoo.com
Imran
I
Shahid
Sana
S
Gull
Bushra
B
Ijaz
Waqar
W
Ahmad
Muhammad
M
Ansar
Sultan
S
Asad
Humera
H
Kausar
Muhammad Tahir
MT
Sarwar
Muhammad Kazim
MK
Khan
Sajida
S
Hassan
D000998:Antiviral Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D016174:Hepacivirus / Q000145:classification / Q000187:drug effects / Q000235:genetics*; D006526:Hepatitis C / Q000188:drug therapy; D006801:Humans; D010957:Plasmids / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
D000998:Antiviral Agents; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C097010:NS2 protein, Hepatitis C virus; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase