Screening and identification of proteins interacting with HCV NS4A via yeast double hybridization in leukocytes and gene cloning of the interacting protein.
- Author:
Yong-qian CHENG
1
;
Lin WANG
;
Jun CHENG
;
Yan LIU
;
Dong-ping XU
;
Yan-wei ZHONG
;
Jian-hui QU
;
Jiang-ke TIAN
;
Jiu-zeng DAI
;
Xiao-dong LI
Author Information
- Publication Type:Journal Article
- MeSH: Adaptor Proteins, Signal Transducing; genetics; isolation & purification; metabolism; Carrier Proteins; genetics; metabolism; Cloning, Molecular; Gene Library; Humans; Leukocytes; cytology; metabolism; Protein Binding; Transformation, Genetic; Two-Hybrid System Techniques; Viral Nonstructural Proteins; Viral Proteins; genetics; metabolism
- From: Chinese Journal of Experimental and Clinical Virology 2007;21(1):47-49
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo screen proteins interacting with HCV NS4A protein in leukocytes by yeast-double hybridization.
METHODSThe 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.
RESULTSForty-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.
CONCLUSIONSeven kinds of proteins interacting with NS4A in leukocytes were successfully screened and the results brought some new clues for studying the pathogenesis of HCV.