HCV NS3/4A蛋白酶胞内荧光检测方法的建立

目的:建立丙型肝炎病毒NS3/4A丝氨酸蛋白酶胞内荧光检测方法。方法:利用EGFP分子内合适位点可以插入一定长度外源片段而不影响荧光性能的特性,构建EGFP分子内插入NS3/4A蛋白酶识别序列NS5AB的EGFP-5AB重组分子。将EGFP-5AB与NS3/4A蛋白酶共表达,若短肽链被切断,则EGFP的两个部分解离,荧光消失,从而可以监测HCV NS3/4A蛋白酶的存在。通过将NS5AB插入三种不同位点,寻找最合适的插入位点;将EGFP-5AB转染进入不同宿主细胞,验证其在不同细胞的表达情况并选择最佳宿主细胞。结果:确定EGFP 173-174氨基酸位点是合适的插入位点;确定CHO-K1为理想的荧光检测系统宿主细胞;在构建的细胞模型中,能够检测到EGFP被切割后的条带,但检测不到荧光信号,说明EGFP-5AB蛋白被有效切割,该方法可以检测到NS3/4A丝氨酸蛋白酶的存在。结论:成功构建了一种在哺乳动物细胞中检测NS3/4A蛋白酶切割活性的荧光检测方法。

Construction of a Fluorescence Detection Method for HCV NS3/4A Protease Activity

Objective: To develop a visualized cell method for detecing the existence of HCV NS3/4A protease.Methods: Based on the character that the insertion of NS5AB sequence into certain site of EGFP molecule will keep its fluorescence,constructed EGFP-5AB recombinant molecule.When the NS3/4A was coexpressed in the same cell,the EGFP will be digested into two parts and lost its fluorescence.So the model can be used to detect HCV NS3/4A protease.Three insertion sites were compared to find the best site for insertion;three host cells were transfected to find the best for fluorescence detection.Results: After optimization,the EGFP 173~174 aa is chosen as the best site for NS5AB insertion,CHO-K1 cell is chosen as the best host cell.The digested EGFP1-173 fragment can be detected in the cell without fluorescence,which proved that the method can detect HCV NS3/4A existence sensitively.Conclusion: A fluorescence detection method for HCV NS3/4A protease activity is successfully constructed.