[Screening and cloning of hepatitis C virus non-structural protein 4A interacting protein gene in hepatocytes].
OBJECTIVE
To investigate biological functions of hepatitis C virus (HCV) non-structural protein 4A (NS4A).


METHODS
Yeast-two hybrid technique was performed to seek proteins in hepatocytes interacting with HCV NS4A. HCV NS4A bait plasmid was constructed by ligating the NS4A gene with carrier plasmid pGBKT7, then it was transformed into yeast AH109 (alpha type). The transformed yeast cells were amplified and mated with yeast cells Y187 (alpha type) containing liver cDNA library plasmid pACT2 in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for selection two times. After extracting plasmid from blue colonies, plasmid DNA was transformed into competent E.coli and analyzed by DNA sequencing and bioinformatics methods.


RESULTS
Among twenty-two positive colonies there were eleven positive for metallothionein 2A, three for eukaryotic translation elongation factor 1 alpha 1, two for albumin, two for RNA binding motif protein 21, two for myomesin, one for cytochrome C oxidase II, and one for ATPase.


CONCLUSIONS
Genes of HCV NS4A interacting proteins in hepatocytes were successfully cloned and the results pave the way for studying the biological functions of NS4A and associated proteins.
Liu
Yan
Y
Gene Therapy Research Center, Institute of Infectious Diseases, 302 Hospital of PLA, Beijing 100039, China. ly@genetherapy.com.cn
Bai
Gui-qin
GQ
Cheng
Jun
J
Wu
Shun-hua
SH
Wang
Lin
L
Yan
Fu-ming
FM
Zhang
Ling-xia
LX
Cui
Yu-fang
YF
D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D016174:Hepacivirus / Q000235:genetics*; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins / Q000235:genetics*
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C455041:NS4A cofactor peptide, Hepatitis C virus; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins