Construction Of IEC Yeast Two-hybrid CDNA Library And Sreening Of The Host Proteins Interact With The PEDV Structural Proteins

When viruses entry into animal body, the body first to start inflammation, interferon antiviral response, apoptosis and so on a series of innate immune response, initially by these mechanisms to resist the invasion of the virus. However, many viruses can break through the innate immune barrier, get into the animal body and lead to disease. Therefore, exploring virus invasion and pathogenic mechanism is very important for the virus prevention.Moreover,the study on protein interaction between virus and host is unusual for understanding the virus pathogenic mechanism.Porcine epidemic diarrhea(PED) is one of the intestinal diseases which is caused by porcine epidemic diarrhea virus(PEDV). The main features of the disease is highly transmissible,acute enteritis, watery diarrhea, vomiting and dehydration, the age of the pigs are susceptible, piglet morbidity rate was 100%, the mortality rate is as high as 80%-100%, brought huge losses to the pig industry in china.In recent years, researchers engaged in the receptor and signaling pathways of porcine epidemic diarrhea virus, there are aspects of interaction between PEDV and host cells. But, PEDV and host cell interaction research is limited. Therefore,this study is intended to explore the interaction of PEDV structural proteins with the host proteins, find out the effect on PEDV replication and mechanisms.This research is based on the PEDV structural proteins as bait, using yeast two hybrid technique, to explore the host proteins have interaction with PEDV structural proteins from the porcine intestinal epithelial cells(IEC) or susceptible cell Vero E6. First of all, construction and identification of IEC yeast two hybrid c DNA library successfully, library titer is about 8.4 × 108 CFU/m L, the insert ds c DNA fragments size is 0.5-2 kb, the average is about 1.1 kb. Then constructs the bait plasmids and detect the self activation and toxicity, constructed 10 bait plasmids successfully, among them, 8 bait plasmids have no toxicity and self activation. Using yeast two hybrid technique for protein in Vero E6、IEC cells that interaction with PEDV structural proteins. Now, 25 cell proteins have been found, including 22 kinds of Vero E6 cell proteins, 3 kinds of IEC cell proteins. By BLAST analysis, select part of the protein to be verified and have further study. The yeast two hybrid system of rotary verification and immunoprecipitation experiments, have confirmed that there is an interaction between the PEDV N protein and NDUFB3(NADH dehydrogenase[ubiquinone]1 beta subcomplex, 3).