Yeast Two-hybrid System Screening Host Protein Of N Protein Interaction Of Porcine Epidemic Diarrhea Virus And Identification Of N Protein Epitopes

Porcine epidemic diarrhea(PED)is an acute contact intestinal infectious disease caused by porcine epidemic diarrhea virus(PEDV)infection.The disease is characterized by diarrhea,vomiting,dehydration,especially the lethality of lactation piglets within 7 days can be as high as 100% in clinical practice.Since 2010,PED with PEDV as the main pathogen broke out in China,swept the whole country and the surrounding Asian countries quickly and brought huge economic losses to the pig industry in China and Asian countries.PEDV is a small enveloped positive strand RNA coronavirus,with four structural proteins respectively including spike protein(Spike,S),membrane protein(Membrane,M),small membrane protein(Envelope,E)and Nucleocapsid(Nucleocapsid,N).The N protein is an important multifunctional structural protein and is highly conserved,thus screening for PEDV N protein interacting with host cells,as well as screening for N protein epitopes offered help for exploring the pathogenesis of PEDV mechanisms and specificity new diagnostic research.The specific research contents and results are as follows:(1)In order to obtain the host protein interacting protein of porcine epidemic diarrhea virus N protein.In this study,pGBKT7-N which is a recombinant vector of the porcine epidemic diarrhea virus N protein was constructed.and pGBKT7-N was used as bait plasmid to screen ten positive clones interacting with N protein from the cDNA Library of porcine alveolar macrophage by yeast two hybrid method.The results showed that there were six proteins corresponding to FTH1,LGALS3,CORO1 C,SNRPG,KRTAP5-3 and ZNF598,two proteins among them of LGALS3 and SNRPG may be related to the proliferation and apoptosis of the virus,which has the value of further research,Therefore,the two proteins were tested for return,selfactivation and Co-IP experiments.It was confirmed that LGALS3 and SNRPG protein have specific interactions with N protein and excluded the possibility of false positives.This study laid a foundation for further study on the function of N protein and the pathogenic mechanism of porcine epidemic diarrhea virus.(2)In order to screen and identify the antigenic epitope core region of N protein,the truncated N gene was 4 DNA fragments cloned into plasmid pCold I to construct 4 recombinant plasmid pCold I-N1,pCold I-N2,pCold I-N3,pCold I-N4 induced by IPTG and the expressed fragment was expressed by Western blot.The results showed that only N1(1-126aa)and N2(107-230aa)reacted with N protein monoclonal antibody,indicating that the epitope region of N protein was in the region of N1-N2(1-230aa).By the strategy of the same method,the region of N1-N2(1-230aa)was truncated to 10 overlapping DNA fragments A1?A2?A3?A4?A5?A6?B1?B2?B3?B4,which were cloned into plasmid pCold TF respectively,constructed 10 recombinant plasmids and IPTG induced segment fusion protein prokaryotic expression protein were successfully expressed;The expression of A1(1-190aa),A2(1-150aa)and B1(1-150aa)were detected by Western blot.The results show that A1(1-190aa),A2(1-150aa)and B1(1-150aa)and B1(1-150aa)were used to construct the recombinant plasmid p Cold TF.(1-140aa)reacts with the N protein monoclonal antibody,while the other fragmented proteins do not react,confirming that the antigenic epitope of the N protein is in the 107-140 aa region.The epitope of N protein in this study laid a foundation for the establishment of specific virus diagnostic methods.