3C Protease Expression, Purification And The Research Of Activity Inhibition

3C protease,which cleaves the virus non-structure poly-peptide plays very important role in the process of Picornavirus replication.3C protease is one of important targets of Picornavirus' drug treatment investigation.If inhibit its catalytic action,we can active suppression the dissection of precursor protein,and intercept Picornavirus replication at last.Picornaviridae is the minimal RNA virus of animal as known.There are seven generas in the Picornaviridae.All of them are the pathogenys that cause serious human disease.Therefore,the investigation of 3C protease will provide scientific information and academic knowledge to the therapy of these serious disease which pose threat to the public health.The cleavage activity of 3C protease is highly specific.The protease identify the site Leu Glu Vul Leu Phe Gln↓Gly Pro,and the cut is between Gln—Gly,we called the site of Q-G.Regard of its specific cleavage activity,we conceive that the protease could be gained through the method of genetically engineering,and we can authenticate the enzymatic active of recombinate 3C protease in vitro,it is also can establish the foundation of developing a new type of genetic engineering protease in the future.We get the antibody of 3C protease through animal experiment,and inhibit the enzymatic active of 3C protease by antibody in order to restrain the Picornavirus replication.First of all,The encoding region of human rhinovirus(serotype 14)3C protease have been cloned and inserted into the vector of the pBV220and screen out the recombinant successfully,by transforming the vector to the BL-21.We gained a large mount of 3C protease by induced expression high performance and stabilize in Bacillus coli.We purify the protein by CM Sepharose Fast Flow and we got the 3C protease whose purification above 90%by emendation.In addition,we express and purify the fusion protein of Interleukins-11 which has the tangential point of 3C protease as a target protein of 3C protease.We refold the 3C protease and detect its specific cleavage activity in vitro.The result that the proteases we expressed can cleave the fusion protein of interleukins—11 in vitro,which could prove that our product can be manipulated as a tool protease in the research undoubtedly.Furthermore,we cultivate Veto cell and use 3C protease' antibody which is gotten by immunize cony pig to inhibit the active of 3C protease in order to restrain the Picornavirus replication.The rudiment experiment didn't have some evident positive result.We want to provid available method of therapy to the clinic through this experiment.Otherwise,the proteolytic ferment like 3C protease of Coronavirus genus has some autoploidy in sequence and space conformation with 3C protease.Our research could help us to comprehend and study the proteolytic ferment like 3C protease of Coronavirus genus and also provide scientific information and academic knowledge to the therapy of disease like SARS which originated by Coronavirus.