[Cellular localization of HCBP1 and its interaction with HCV core protein in vivo].
OBJECTIVE
To analyze the interaction of hepatitis C virus (HCV) core protein with HCBP1 and observe the expression and cellular localization of HCBP1.


METHODS
The cDNA fragments encoding HCV core protein and HCBP1 were amplified by PCR and subsequently cloned into pGEM T vector, respectively. After sequence verification, the two recombined vectors were respectively subcloned into two hybrid plasmids, pM and pVP16. pM-core, pVP16- HCBP1 and the reporter vector pG5CAT were co-transfected into COS-7 cells, and the interaction between HCV core protein and HCBP1 was assayed by detecting CAT gene expression after 48 h. The expression and subcellular localization of the fusion protein in the transfected COS-7 cells were analyzed by Western blotting and fluorescence microscopy, respectively.


RESULTS
CAT-ELISA showed that the absorbance of the co-transfection group was significantly higher than that o f the negative control groups but lower than that of the positive control group. Western blotting confirmed the expression of fusion protein in the transfected COS-7 cells. Fluorescence microscopy showed that the fusion protein was distributed mainly in the cytoplasm, and in contrast, diffuse EGFP expression was detected in COS-7 cells transfected with the empty vector.


CONCLUSIONS
Mammalian two-hybrid assay confirms the capacity of HCBP1 to bind HCV core protein, and the expression vector for HCBP1-EGFP fusion gene has been constructed successfully and expressed in COS-7 cells.
Chen
Tian-yan
TY
Department of Infectious Diseases, First Affiliated Hospital, Xi'an Jiaotong University College of Medicine, Xi'an 710061, China. chentianyan@126.com
Liu
Min
M
Chen
Yun-ru
YR
Lin
Shu-mei
SM
Ye
Feng
F
Zhang
Xi
X
Liu
Jin-feng
JF
Zhao
Ying-ren
YR
Zhang
Shu-lin
SL
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D011485:Protein Binding; D011991:Receptors, Virus / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014758:Viral Core Proteins / Q000378:metabolism*
D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus