Research Of Effectiveness Of Hepatitis A Virus3C Protease Recognition Sequence

Hepatitis A virus (HAV), a small RNA virus, which can mainly cause liverdamage, was first discovered by Feinslone in1973. HAV3C proteinase is its ownunique proteolytic enzymes, with about20kD molecular weight, having S (serine)/C(cysteine) protease properties, and the active center is Cys-His-Tyr. Although HAV3Cproteinase doesn’t directly involved in viral RNA replication, it can correctlyhydrolyse precursor polyprotein to forcefully guarantee for the formation of capsidand replication of viral. As a proteolytic enzyme,3C protease has a high degree ofspecificity, which plays a very important role in the viral life cycle, inhibiting itscleavage activity, blocking viral replication, and therefore the HAV3C proteinase isalso one of targets for treatment of drug.Currently, the method for identifing the P3C enzyme recognition sequence istranslation of multimeric protein in vitro (rabbit reticulocyte lysate), and incubateswith cell lysates of prokaryotic expression P3C at30oC for7h. Byimmunoprecipitation and SDS-PAGE to determine the cutting strips, and the P3Ccleavage recognition sequence was determined by protein N-terminal sequencing.Inthis study, the ProIL1B-Gluc protease detection system was used, which has beenreported in the literature. According to the methods of the gene synthesis andmolecular cloning, fusion protein genes were successfully constructed, which containsix different length restriction recognition sequences (P2B-P2C) of HAV3C protease(P3C).293T cells were co-transfected by P3C and these fusion protein genesrespectively to analyze changes in fluorescence measured values, and itscorresponding Western blot, which can be quickly and accurately identified its P3Ccleavage recognition sequence. In this study, a P2B-P2C minimum recognition sequence LRTQSF was found.Meanwhile, we explored the relationship between the fluorescence ofProIL1B-Gluc and the transfection concentration of P3C plasmid, and optimized thedetection time. Experimental results show that the fluorescence of ProIL1B-Glucincreases with the increase of transfection concentration of P3C plasmid, and theoptimal testing time should be about12h after transfection, which suggest thatProIL1B-Gluc is a good reporter gene for HAV3C protease.In vitro tests, we verified that as the ProIL1B-Gluc and P3C incubation timeextension, the fluorescence value significantly rised, and there is a clear Western blotcleavage fragment. Which demonstrated that the P3C still has a recognition sequencefunction in vitro.Our results contribute to Pro1L1B-Gluc reporter gene to other smallRNA viral3C protease extend, in order to detect and identify the appropriaterestriction enzyme recognition sequence. Also hope that with the aid of this luciferasereporter gene to3C protease, we can further study the3C protease at the molecularlevel.