[Screening and identification of proteins interacting with HCV NS4A via yeast double hybridization in leukocytes and gene cloning of the interacting protein].
OBJECTIVE
To screen proteins interacting with HCV NS4A protein in leukocytes by yeast-double hybridization.


METHODS
The bait plasmid pGBKT7-NS4A was transformed into yeast AH109 was transformed, and the expressing of the fusion protein was identified by SDS-page. The transformed yeast was mated with yeast Y187 containing leukocytes cDNA library plasmid in 2xYPDA medium. Diploid yeast was 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 selecting two times and screening. After extracting and sequencing of plasmid from blue colonies, analysis was conducted by bioinformatics. And, the gene encoding the interesting protein was cloned, and back-cross was performed.


RESULTS
Forty-five colonies were sequenced, among them, 29 colonies were human calcium modulating cyclophilin ligand (CAML). The gene encoding CAML was cloned, and the interaction between NS4A and CAML was ensured.


CONCLUSIONS
Seven kinds of proteins interacting with NS4A in leukocytes were successfully screened and the results brought some new clues for studying the pathogenesis of HCV.
Cheng
Yong-qian
YQ
Viral Hepatitis Research Center, The No.302 Hospital of People's Liberation Army, Beijing 100039, China.
Wang
Lin
L
Cheng
Jun
J
Liu
Yan
Y
Xu
Dong-ping
DP
Zhong
Yan-wei
YW
Qu
Jian-hui
JH
Tian
Jiang-ke
JK
Dai
Jiu-zeng
JZ
Li
Xiao-dong
XD
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D015723:Gene Library; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007962:Leukocytes / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D014170:Transformation, Genetic; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C089404:CAMLG protein, human; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C455041:NS4A cofactor peptide, Hepatitis C virus; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins