[Construction of pSG5/NS5A5BdeltaC and its expression in Huh7 cells].
OBJECTIVE
To construct the plasmid pSG5/NS5A5BdeltaC, and to investigate its expression in Huh7 cells.


METHODS
The plasmidpTM1-NS2NS5A5BdeltaC was taken as the template. The primers were designed according to the characteristics of the sequence and endoenzyme cleavage sites in HCV NS5A5BdeltaC and vector pTM1, pSG5. The HCV NS5A5BdeltaC gene fragment was amplified by PCR from pTM1-NS2NS5A5BdeltaC and inserted into vector pSG5. The positive clones were screened by Ampicillin and identified by the restriction endoenzyme Sac I and Bgl II digestion and agarose gel electrophoresis. The constructs were transfected into Huh7 cells with FuGene 6 reagents. Immunofluorescence and Western blot were performed to detect the expression of the constructs in Huh7 cells.


RESULTS
Endoenzyme digestion analysis showed that the size and the inserting orientation of the fragment met the design expectation. Immunofluorescence staining displayed the expression of HCV NS5A5BdeltaC protein, which was located in the cytoplasm, and the expression rate reached as high as 40%. SDS-PAGE analysis showed that the relative molecular mass of the expressed product by pSG5/NS5A5BdeltaC was about 82 ku, which was consistent with the theoretical value.


CONCLUSIONS
pSG5/NS5A5BdeltaC is successfully constructed, and it can be expressed transiently in Huh7 cells, which would lay a foundation for the further study on function of HCV polyprotein.
Wang
Xue-ping
XP
Editorial Department of Journal of Fourth Military Medical University, Xi'an, China. wangxp@fmmu.edu.cn
Li
Fu-jun
FJ
Deng
Lin
L
Nagano-Fujii
Motoko
M
Kitayama
Kikumi
K
Hotta
Hak
H
D015153:Blotting, Western; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase
D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase