Construction of point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal and their expressions in Huh 7 cells.
- Author:
Xue-ping WANG
1
;
Fu-jun LI
;
Nagano-fujii MOTOKO
;
Kitayama KIKUMI
;
Hak HOTTA
Author Information
- Publication Type:Journal Article
- MeSH: Amino Acid Sequence; Cell Line; Gene Expression; Genetic Engineering; methods; Hepacivirus; enzymology; Immunohistochemistry; Intracellular Space; metabolism; Molecular Sequence Data; Plasmids; genetics; Point Mutation; Protein Structure, Secondary; Protein Transport; Viral Nonstructural Proteins; chemistry; genetics; metabolism
- From: Journal of Southern Medical University 2009;29(4):720-723
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal, and express the constructs in Huh 7 cells.
METHODSUsing pSG5/M-H05-5/4A as the template (A1-1) and primers designed according to the typing criteria, 4 single point mutation plasmids, namely pSG5/M-H05-5(A1-2)/4A(A1-2) (Y56F), pSG5/M-H05-5(B1-1)/4A(B1-1) (L80Q), pSG5/M-H05-5(B2-1)/4A(B2-1) (V51A), and pSG5/M-H05-5(B2-2)/4A(B2-2) (S61A), were constructed. With A1-2, B2-1, and B2-2 as the templates, the leucine to glutamine mutation at position 80 (L80Q) was induced to construct another 3 double point mutation plasmids pSG5/M-H05-5(B1-2)/4A(B1-2), pSG5/M-H05-5(A2-1)/4A(A2-1), and pSG5/M-H05-5(A2-2)/4A(A2-2), respectively. DNA sequencing was performed for confirmation of the mutations. Huh 7 cells were transfected with the constructs using FuGene 6 transfection reagents. Indirect immunofluorescence staining and Western blotting were used to detect the expression of the constructs.
RESULTSIndirect immunofluorescence assay revealed 4 subcellular localization patterns of NS3 protein, including dot-like staining, diffuse staining, doughnut-like staining, and rod-shape staining. Western blotting also demonstrated successful expression of the constructs and weak in cis and in trans NS3 serine protease activities of subtypes A2-1 and B2-1 in comparison with other subtypes.
CONCLUSIONThe point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal are constructed successfully, which provides the basis for further study of different subtypes of HCV.
