Construction of point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal and their expressions in Huh 7 cells / 南方医科大学学报
Journal of Southern Medical University
;
(12): 720-723, 2009.
Article
Dans Chinois
| WPRIM
| ID: wpr-233700
ABSTRACT
<p><b>OBJECTIVE</b>To construct point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal, and express the constructs in Huh 7 cells.</p><p><b>METHODS</b>Using 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.</p><p><b>RESULTS</b>Indirect 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.</p><p><b>CONCLUSION</b>The 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.</p>
Texte intégral:
Disponible
Indice:
WPRIM (Pacifique occidental)
Sujet Principal:
Plasmides
/
Immunohistochimie
/
Données de séquences moléculaires
/
Génie génétique
/
Expression des gènes
/
Lignée cellulaire
/
Chimie
/
Séquence d'acides aminés
/
Protéines virales non structurales
/
Structure secondaire des protéines
langue:
Chinois
Texte intégral:
Journal of Southern Medical University
Année:
2009
Type:
Article
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