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| Construction of a Fluorescence Detection Method for HCV NS3/4A Protease Activity |
| YAN Xing1,2, SHI Ming-lei2, CHEN Na2, WANG Yang2, ZHANG Yan2, WANG Zheng2, LI Xiao-chen1, ZHAO Zhi-hu2 |
1. College of Life Science, Shaanxi Normal University, Xi’an 710062, China;
2. Institution of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China |
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Abstract 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.
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Received: 15 February 2012
Published: 25 July 2012
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