丙型肝炎病毒NS3/4A丝氨酸蛋白酶瞬时表达小鼠模型的建立

摘要

目的:建立丙型肝炎病毒NS3/4A丝氨酸蛋白酶体内活性评价模型.方法:利用NS4A/B是NS3/4A丝氨酸蛋白酶作用底物的特性,构建融合基因NS3/NS4A /B-SEA P,底物片段NS4A/B插在NS3/4A和人分泌性碱性磷酸酶(SEAP)之间,融合基因表达后SEAP的分泌依赖于有活性的NS3/4A在NS4A/B位点的切割.将含融合基因的质粒NS3/4A(△4AB)SEAP通过水动力转染技术转染到小鼠体内,检测小鼠血清中SEAP的活性,高活性的SEAP是该评价体系成立的证据.结果与结论:在瞬时表达NS3/4A的小鼠血清中检测到了高活性的SEAP,建立了可用于评价抗NS3/4A的小鼠体内瞬时模型.%Objective: To develop a system for monitoring specific hepatitis C virus(HCV) NS3/4A protease activity in mouse. Methods: According to the characteristic of NS3/4A, a fusion protein NS3/4A/4B-SEAP was constructed, in which HCV NS3/4A protease was fused to secreted alkaline phosphatase(SEAP) through the NS3/4A protease decapeptide recognition sequence, NS4A/B, which spans the NS4A and NS4B junction region. Theoretically, the secretion of SEAP into plasma depends on the cleavage of NS4A/B by HCV NS3/4A protease. This theory was judged by the activity of SEAP in the plasma of mouse transient expressing NS3/4A protease. Results and Conclusion: High activity of SEAP was detected in the plasma of the mouse transient expressing NS3/4A protease,the transient mouse model for expressing HCV NS3/4A protease was successfully constructed.