Hcv Ns3/4 A Protease Inhibitor Cell Screening For The Establishment Of The Model Building Method

HCV that has a worldwide’s high infection rate is a small, enveloped, single-positive-stranded RNA virus, with a nonstructural (NS) proteins NS3/4A serine protease which placeplay an important key roles in cutting in the nonstructural region. NS3/4A serine protease is located in at the third end of the N-terminal of NS3protein along with the NS4A cofactor, whose combination Strengthened enhanced the role of the NS3serine protease’s. NS3/4A serine protease cutting cuts’s four non-structural proteins in its downstream:NS3/NS4A/NS4B/NS5A/NS5B, and process transforms HCV protein precursor into a number of important viral enzymes. In addition, NS3also has other biological founctions:RNA helicase and NTP enzyme. NS3/4A is also involved in HCV to evadeing the host innate immune system response in HCV, Providing providing the conditions for HCV’s continued chronic infection. As a result, It can be seen that NS3/4A is a multifunctional molecule in HCV. Inhibition of its activity can not only blocksed HCV replication, translation and post-translational protein precursor’s processing, but also letbrings the antiviral effect of Interferon interferon into fully play its antiviral activity.In view of those the above NS3/4A features, also meanwhile considering that NS3/4A Serine serine protease is a target relatively easy to be controlled, Learning learning from the successful experience of protease inhibitor therapy for HIV infection, Many many research institutions institutes and pharmaceutical companies have begun to research specifically targeted antiviral therapy for HCV, especially HCV NS3/4A serine protease selective inhibitors. NowAt present, many kindsa variety of candidate drug have entered been brought into clinical trials, like such as VX-950, SCH-503034, ACH-806and MK-7009, etc. Those drugs associated with PEG-IFNa, increased SVR (up to55-69%), but they have serious side effects, Often frequently cause causing skin rashes, itching, anemia or heart, kidney dysfunction. Therefore, continue to find the safe and effective NS3-4A protease inhibitors is necessary.This subject project uses EGFP as reporter protein, to established a cell model screening the HCV NS3/4A protease inhibitor, and subsequently to then optimized it. The fundamental Main principle is described as below.:At first, choosing choose appropriate sites in EGFP internal to insert NS5A/5B sequence, form reporter molecules-EGFP5AB, which still have green fluorescent. When NS5A/5B sites in EGFP internal is cutted by NS3/4A protease, reporter Protein protein is broken into two parts and Fluorescence fluorescence disappeareddisappears. So, this model can visually evaluate the activity of the NS3/4A serine protease by by detecting the fluorescent phenomenon, finally screening the NS3/4A serine protease inhibitor. Based on this monitoring system, we can Screen and evaluate the effect of the HCV NS3/4A serine protease inhibitor, providing basic information for the screening and evaluation of new anti-HCV drug.Content and results of this study are as follows:The first part is optimizing the reporter molecules, host cell and transfection conditions.The appropriate insertion sites in EGFP for NS5AB was choosedchoosen, to build reporter molecules.According to the literature, we choose sites in EGFP after155Aa (between the155th Aa and the156th Aa),158Aa (between the158th Aa and the159th Aa), and173Aa (between the173th Aa and the174th Aa) in which NS5AB sequence (NS3/4A’s recognition sequence) is inserted respectively. Then build plasmid: zE1555AB, zE1585AB and zE1735AB in PcDNA3.1vector.Similarly, the control group plasmid is build built in PcDNA3.1vector:(F2A sequence is inserted between NS5A and NS5B sequence) zE1555AB-2A, zE1585AB-2A and zE1735AB-2A;(reporter molecules sequence is connected with streamlined scNS3/4A sequence by F2A sequence) zE1555AB-2A-NS3/4A, zE1585AB-2A-NS3/4A, zE1735AB-2A-NS3/4A and zEGFP-2A-NS3/4A. Also pcDNA3.1(+)-EGFP is buildbuilt.Then, the appropriate host cell is Selectedselected, and the transfection conditions is optimized:above plasmids were all respectively transiently transfected into mammalian cell:HeLa, CHO-Kland HEK293A, optimizing the transfection conditions. By observing the green fluorescence, choose CHO-K1as the appropriate host cells, and173Aa sites as the appropriate sites for NS5AB to insert.The second part step is the detection of prokaryotic expression’s detection, optimization of the screening molecules and finally establishing establishment of the cell screening model.Reporter molecules was constructed in prokaryotic plasmid to and was transformed into E. Coli, whose expression of EGFP protein is observed. Build EGFP and E1735AB plasmid in pET-28a vector and NS3/4A plasmid in pET-22b vector. Transfected those prokaryotic plasmid into E. Coli respectively and Induced induced E. Coli to express protein. By detecting, the expression of the fluorescence was conformed consistent with our expectation. Besides, we find found that after the induced expression of the E. coli containing NS3/4A gene, morphological changes occurred in E. Coli’s morphological changed..Optimization the screening molecular model and finally obtain the cell model for screening which is well worked.Build molecules in Eukaryotic eukaryotic expression vector:zE1735AB-2A-NS3/4A and zE1735AB-IRES-NS3/4A and choose the well worked plasmid: zE1735AB-IRES-NS3/4A, which is finally transfected into CHO-K1cells, to establish the cell model for screening. By Western Blot detection, the size of the bands conformed with the expected molecular weight of EGFP fragment that is cutted by NS3/4A. The cell model for screening NS3/4A protease inhibitor finally established.Above results suggest that, we established a method which can be used visually to screen NS3/4A protease inhibitor in mammalian cell. NS3/4A protease inhibitor can be preliminary filtered just by observing reporter protein’s green fluorescent state. This laid the foundation for the preliminary screening and validation of the NS3/4A protease inhibitors. Just by changing the recognition sequence, this model can also be used to detect a similar protease’s activity and to filter corresponding inhibitor.