Fundamental Crystallography Study And Activity Test On Canine Respiratory Coronavirus Main Protease With An Inhibitor

Cornavirus infects human and a large variety of animals,causing different kinds of respiratory and intestinal diseases.SARS-Co V and MERS-Co V typically have triggered a huge panic in human society,still being a magnificent threat to the health of human lives.Canine respiratory coronavirus,CRCo V,is classified in the Bete coronavirus genus,which also include SARS-Co V and MERS-Co V.CRCo V is one of the main pathogens causing Canine infectious respiratory disease,CIRD,causing considerable economic loss.After coronavirus adhere and invade into host cells,viruses hijacked the hosts ribosomes to translate their genome into polyprotein 1a and also polyprotein 1ab.As main protease is responsible for cutting the most conserved cleavage sites in the process of polyprotein dissection,main protease plays a key role in the whole coronavirus replication process.Functional main protease could be a necessary condition for the generation of virus replication transcription complexes.As a consequence,coronavirus main protease is considered as a vital important tartget for related drug design.Targeting main protease,Prof.Rao ‘s team based on the three-dimensional structure of SARS-Co V main protease at atomic resolution,successfully designed and synthesized a compound N3 with high inhibiting efficiency basing on Michael addition reaction.As the main work of this thesis is encompassing CRCo V main protease cloning,expression and purification,crystallization screening and optimization,X-ray diffraction,data collection,data processing and preliminary strcture determination,and also enzymology experiments focusing on CRCo V main protease and its inhibitor,ect,it is clearly interpreted that we ? utilize an excellent strategy to produce the authentic CRCo V main protease;bring up with a set of feasible protein purification condition and procedure;figure out the significant role of the reduce reagent(DTT)in the buffur system;? bring forward related practical crystallization technique;analysis the process and the outcome of diffraction data processing and structure determination;? successfully verify outstanding inhibition effect of the inhibitor N3 through quantitative demonstration,ect.This research provides new information for understanding the structure and function of main protease of the coronavirus family,and provides an important basis for further drug development based on the structural insight.