表达不同氨基端二级结构HCVNS3/4A点突变质粒的构建及在Huh7细胞中的表达

Construction of point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal and their expressions in Huh 7 cells

目的构建表达不同氨基端二级结构HCVNS3/4A的点突变质粒,并在Huh7细胞中进行表达。方法以pSG5/M-H05-5/4A为模板(A1-1),根据分型标准设计点突变引物。首先构建4个单点突变质粒: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),pSG5/M-H05-5(B2-2)/4A(B2-2)(S61A)。然后,分别以A1-2,B2-1,B2-2为模板,再将第80位的赖氨酸点突变为谷氨酸(L80Q),构建另外3个双点突变质粒:pSG5/M-H05-5(B1-2)/4A(B1-2),pSG5/M-H05-5(A2-1)/4A(A2-1)和pSG5/M-H05-5(A2-2)/4A(A2-2)。每个质粒均进行序列测定验证点突变成功。用FuGene6转染试剂将构建物转染入Huh7细胞,并应用间接免疫荧光试验和Western blotting检测构建物的表达。结果免疫荧光实验检测到NS3的4种亚细胞定位方式:点状,弥漫样,面包圈样,及短棒状。Western Blotting亦显示构建物均成功表达,同时发现A2-1和B2-1亚型NS3/4A存在不完全切割现象,表明与其他亚型相比,A2-1和B2-1NS3 in cis丝氨酸蛋白酶活性较弱。结论成功构建表达不同氨基端二级结构HCV NS3/4A点突变质粒,为抗不同亚型HCV的深入研究提供基础。

Objective To construct point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal, and express the constructs in Huh 7 cells. Methods Using pSG5/M-H05-5/4A as the template （AI-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（BI-1）/4A（BI-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 （LSOQ） 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. Results 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 contructs and weak in cis and in trans NS3 serine protease activities of subtypes A2-1 and B2-1 in comparison with other subtypes. Conclusion 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.