Phage Display And Identification Of Active Peptides Targeting Porcine Epidemic Diarrhea Virus S Protein

Porcine epidemic diarrhea(PED)is a highly contagious intestinal infectious disease caused by the porcine epidemic diarrhea virus(PEDV),which is characterized by acute diarrhea,vomiting,dehydration and even death of piglets.The mortality of piglets after infection is as high as 90%.The clinical symptoms,pathological changes and epidemiology of PEDV infection are similar to those of transmissible gastroenteritis of swine(TGE),and mixed infection often occurs.It is difficult to make a definite diagnosis through clinical symptoms,and laboratory testing methods must be used to make a differential diagnosis.Since its appearance in the UK in the 1970s,PED has gradually spread to the world and erupted on a large scale in China in 2010,which causing serious economic losses.Therefore,researches on vaccines and inhibitors of PEDV is particularly important.PEDV S protein is a spike protein on the virus surface,and also the largest structural protein of PEDV.It plays a key role in the process of virus particles invading host cells through membrane fusion after binding with cell surface receptors.In addition,S protein contains multiple neutralizing epitopes,which can induce the host to produce neutralizing antibodies.Because S protein has many important functions,therefore most of the current studies on the structural proteins of PEDV focus on S protein.Because PEDV S protein plays an important role in the process of virus invasion into host cells,our laboratory carried out semi-flexible molecular docking with the S1-CTD region of PEDV S protein as the target,and designed a series of active peptides with antiviral effects targeting the S1-CTD region of PEDV S protein by using computer software.Firstly,this study constructed a PE-sumo-PEDV-S1 prokaryotic expression vector for the expression of PEDV S1 recombinant protein,and then purified PEDV S1 recombinant protein as the basic material for subsequent experimental researches.Secondly,this study successfully displayed two active peptides 116306 and 118337 by phage display technology,and provided a new method to express peptides.Finally,the antiviral functions of the two displayed peptides were identified and the achieved good results.The surface plasmon resonance test showed that peptides 116306 and 118337 had good affinity with PEDV S1 recombinant protein,with K_Dvalues of 736 p M and 17.94μM respectively.Peptide 116306 has a lower K_Dvalue,indicating it has higher affinity and higher antiviral potential.The effect of two active peptides on the viral transcription level was detected by fluorescence quantitative PCR.The results showed that compared with the viral control group,the peptide treatment group could significantly reduce the viral copy number,and both peptides had good antiviral effect.The indirect immunofluorescence method identified that peptide 116306 and 118337 had the effect of inhibiting virus proliferation,and it was dose-dependent.Even if the concentration of peptide is very low,it still has a certain inhibitory effect,when the concentration of peptide is more than 50μM PEDV can hardly be detected.To sum up,this study successfully displayed active peptides and identified that they both have good anti-PEDV activity,which can be used as small molecular inhibitors of anti-PEDV.This study provided certain test bases for the prevention and treatment of PEDV and the development of peptide vaccines.