Effects Of Pig Intestinal Mucosa Proteins M6P/IGF2R And SI On PEDV Replication

Porcine epidemic diarrhea virus(PEDV)is the pathogen of Porcine epidemic diarrhea(PED).The disease mainly causes highly infectious intestinal diseases,intestinal epithelial villi atrophy,diarrhea,dehydration and loss of appetite of infected pigs,and suckling piglets have extremely high mortality rate.It has been affecting and perplexing the development of the pig industry all over the world and causing huge economic losses to the pig industry.Therefore,it is necessary to study the infection process of PEDV such as invasion and replication in cells.Receptor interaction of coronavirus(Co V)is mediated by trimeric spike glycoprotein(S),which forms a unique large spike on the envelope of coronavirus.Co V uses a variety of cellular proteins as functional receptors for cell entry.The interaction of virus surface glycoprotein S protein and host cell receptor is the first step of infection and is also the main determinant of virus tropism.At present,the research on PEDV receptor is quite controversial and cannot explain some theoretical problems in the process of PEDV infection,pig aminopeptidase N has been gradually rejected as the receptor of PEDV.Therefore,the research on PEDV infection process is of great significance.In order to study the mechanism of PEDV infecting cells,the virus overlay protein-binding assay and mass spectrometry were used to screen proteins interacting with PEDV in pig intestinal mucosal tissue proteins and to study their effects on PEDV-infected cells.First of all,in order to obtain purified PEDV,we carried out two rounds of plaque purification on the PEDV GDgh strain isolated from the laboratory.Monoclonals were selected for mass culture and virus supernatant was collected.Supernatant of the virus was concentrated by ultracentrifugation with 20% sucrose as the base,and the obtained virus particles were observed by electron microscope negative staining.The typical crown structure of coronavirus could be clearly seen.By measuring and comparing TCID50 of virus before and after concentration,it can be seen that ultracentrifugation can effectively improve virus titer.In this study,small intestinal epithelial mucosal tissue protein of piglets was used for SDS-PAGE,and purified PEDV was incubated after membrane transfer,and then the purified PEDV was incubated with PEDV S1 monoclonal antibody for color development.Compared with the virus group without incubation,it can be seen that there is an obvious band between 250 k Da and 300 k Da in the test group,and the corresponding protein strips are cut off for mass spectrometry analysis.M6P/IGF2 R and SI were preliminarily screened as candidate proteins.Their eukaryotic expression vectors were constructed by segments and their corresponding small interfering RNA were designed to study their roles in PEDV infection.The results show that both M6P/IGF2 R and SI interact with PEDV S2 protein.Laser confocal results show that both M6P/IGF2 R and SI are co-located with PEDV S2 in Vero E6 cells.Overexpression of M6P/IGF2 R can promote the replication of PEDV,while interference of M6P/IGF2 R can inhibit the replication of PEDV.On the contrary,overexpression of SI can inhibit the replication of PEDV and interference of SI can promote the replication of PEDV.M6P/IGF2 R protein can promote PEDV invasion into cells,while SI protein can inhibit PEDV invasion into cells.The research of PEDV receptor is still at the exploratory stage,and the related research on the receptor has always been a research hotspot for researchers.Up to now,there is no clear definition.This study can provide some reference basis for further research on the interaction mechanism between PEDV and host cell proteins and the screening of drug targets in the future,and provide some theoretical support for the prevention and control of porcine epidemic diarrhea in China.