The Establishment Of A High-throughput Screening Model For HCV NS3 Serine Protease Inhibitors

Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma, which has been a worldwide health crisis. To date, there has been no vaccine against HCV and specific therapy available. The lack of an in vitro viral replication system and the small animal model of HCV infection hampers anti-HCV studies and drug screening. Recently,the assay based the virus-encoded enzyme is often as the subrogate. It is conceivable in celluar context to evaluate the candidate drug. So it is significant to develop a cell based assay for measurement of HCV NS3 serine protease activity in an effort to screen potential inhibitors.The HCV NS3 serine protease (NS3/4A serine protease) plays a pivotal role in the viral maturation and replication. So it is generally considered to be one of the most attractive targets for design of new anti-HCV drugs. We established a novel cell model targeted at the HCV NS3 protease. The recombinant reporter gene EGFP5AB, which could still represent green fluorescence, was engineered by inserting the NS5A/5B peptide into EGFP. Cleavage of NS5A/5B sites by NS3 serine protease results in disappearance of fluorescent light that is easily detected by fluorescence microscope. Therefore, we can evaluate NS3 protease activity in the cellular context by the performance of green fluorescence. The results were described from the two parts as follows.Part 1. Prokaryotic expression systemFirst, we constructed the recombinant gene EGFP5AB, in which the NS5A/5B peptide was inserted into the 172-173 amino acid of EGFP. Construct the recombinant plasmid pET-28a-EGFP5AB and transform it into E.coli BL21(DE3). Clone the N-terminal fragment(1-172aa) of EGFP into pET-28a(+) vector and clone the C-terminal fragment (173-238aa) into pET-22b(+) vector. They are named as pET-28a-E172N and pET-22b-E172C, respectively. Cotransform them into BL21(DE3) and confirm the positive colony by double antibiotics screening. BL21(DE3) with plasmids was induced by 1mM IPTG at 30℃for 4h. The results showed that: the group expressing EGFP5AB protein represented green fluorescence, while the group coexpressing the N-terminal frangment and the C-terminal frangment was negative. So we could determine the EGFP5AB to be the reporter gene in prokaryotic system.Then, we detected the activity of HCV NS3 serine protease. The sequence MAPI-NS4A ( 21～34aa)-GG-NS3( 2～181aa) which was named as NS3/4A for short was amplified with overlap PCR. Construct the recombinant plasmid pET-22b-NS3/4A. We constructed the recombinant plasmids of pET-28a-EGFP and pET-28a-EGFP5AB, then transformed them into BL21(DE3) as control. Cotransform pET-22b-NS3/4A with pET-28a-EGFP and pET-28a-EGFP5AB into BL21(DE3) respectively. Confirm the positive colony by double antibiotics screening. BL21(DE3) with plamids was induced by 1mM IPTG at 30℃for 4h. The results showed that: the phenomenon of the group coexpressing NS3/4A and EGFP was similar with the group expressing EGFP separately, they both represented green fluorescence. However, the group coexpressing NS3/4A and EGFP5AB was negative. It indicated the NS3 serine protease had destroyed the spatial structure of EGFP5AB but EGFP. We concluded that the NS3 serine protease could recognize the cleavage site of NS5A/5B inserted into EGFP.Part 2. Eukaryotic expression systemSimilarly, we chose the reporter gene firstly. pcDNA3.1(+) was used as the expression vector. Construct the eukaryotic plasmid pcDNA3.1-EGFP5AB and transfect it into the HeLa cell lines via lipofectamine2000. Plasmid pcDNA3.1-E172N-2A-E172C was constructed by inserting the viral F2A peptide into the 172-173 amino acid of EGFP. Transfect it into HeLa cell lines to achieve equal expression level of two proteins. After 48h, the results showed that: the group expressing EGFP5AB protein represented green fluorescence, while the group coexpressing the N-terminal frangment and the C-terminal fragment was negative. Therefore, the EGFP5AB also could be used as the reporter gene in eukaryotic system.Then, we detected the activity of HCV NS3 serine protease. We constructed the recombinant plasmid pcDNA3.1-EGFP5AB-2A-NS3/4A, in which the EGFP5AB gene and NS3/4A gene were linked via F2A peptide. Construct the recombinant plasmid pcDNA3.1-EGFP-2A-NS3/4A in the same way. Transfect them into HeLa cell lines to achieve equal expression level of two proteins, respectively. After 48h, The results showed that: the group coexpressing NS3/4A and EGFP represented green fluorescence. However, the group coexpressing NS3/4A and EGFP5AB was negative. It indicated that the NS3 serine protease had destroyed the spatial structure of EGFP5AB but EGFP. We concluded that the NS3 serine protease could recognize the cleavage site of NS5A/5B inserted into EGFP in HeLa cell lines.In conclusion, in both prokaryotic an eukaryotic systems, we developed an assay for measurement of HCV NS3 serine protease activity. The activity could be indirectly indicated by the performance of green fluorescence. It provide the prerequisite for the HTS screening of potential NS3 serine protease inhibitors.