The Role Of Porcine Integrin αvβ1 In The Course Of PEDV Infection Preliminary Research

Porcine epidemic diarrhea(PED)is an acute and highly contact intestinal infectious disease caused by porcine epidemic diarrhea virus(PEDV),which is extremely harmful to newborn piglets.To the world pig industry caused huge economic losses.The mechanism of PEDV invasion into host cells is still unclear,and the receptor research is still controversial.Integrins can act as infection receptors or infection-related proteins for a variety of viruses.Previous laboratory studies have shown that porcine integrin αvβ3 is associated with PEDV infection.Is integrin αvβ1 in the same family as integrin αvβ3 associated with PEDV infection? Therefore,in this study,the effect of porcine integrin αvβ1 on PEDV infection was studied,providing a new theoretical basis for the understanding of PEDV infection mechanism and the research and development of antiviral drugs.To verify the role of porcine integrin αvβ1 in PEDV infection,polyclonal antibodies against porcine integrin β1 were prepared.The porcine integrin β1 gene was cloned into the prokaryotic expression vector p GEX-6p-1 to express the porcine integrin β1 gene by artificial synthesis of the extracellular domain gene.Polyclonal antibody against porcine integrin β1 was prepared by immunizing New Zealand white rabbits with the purified recombinant protein by gel cutting method.The results showed that the prokaryotic expression vector of porcine integrin β1 was successfully constructed and expressed in BL21,mainly in the form of inclusion body.The molecular weight of the recombinant protein was about 102 k Da.Polyclonal antibody was obtained by immunizing New Zealand white rabbits with purified recombinant protein,and the titer of antibody was determined by indirect ELISA to reach 1:12 800.Western Blot analysis showed that the porcine integrin β1 polyclonal antibody could specifically recognize the protein of integrin β1 in small intestine of piglets.In order to verify the role of porcine integrin αvβ1 in PEDV infection of Vero E6 cells,eukaryotic expression vector of porcine integrin β1 was constructed and successfully expressed in Vero E6 cells by liposome transfection.Vero E6 cells were treated with overexpression and si RNA silencing technology,and then PEDV was inoculated.The changes of m RNA and protein levels of PEDV were detected by q PCR and Western blot,respectively.The results of integrinαvβ1 overexpression showed that compared with the control group,the m RNA level of PEDV ORF3 gene and the protein level of PEDV N in experimental group were extremely significantly increased 24 h after inoculation(p<0.01).After 48 h of inoculation,the m RNA levels of PEDV ORF3 gene and the protein levels of PEDV N in experimental groups were also significantly increased(p<0.05).Compared with the control group,the m RNA level of PEDV ORF3 gene and the protein level of PEDV N in experimental group were significantly decreased(p<0.001).These results indicate that porcine integrin αvβ1 promotes PEDV infection of Vero E6 cells.In order to verify the role of porcine integrin αvβ1 in the adhesion and internalization stage of the process of PEDV infection of Vero E6 cells,the porcine integrin αvβ1 was overexpressed and silenced in Vero E6 cells and then inoculated with PEDV,and the m RNA level of PEDV was detected by q PCR.The results of overexpression of porcine integrin αvβ1 showed that compared with the control group,the PEDV ORF3 gene m RNA level in the experimental group was significantly increased during the adhesion and internalization stage of infection(p<0.05).The results of silencing integrin αvβ1 showed that compared with the control group,the PEDV ORF3 gene m RNA level in the experimental group was extremely significantly reduced during the adhesion and internalization stage of infection(p<0.01).The above results indicate that porcine integrin αvβ1 can promote the adhesion and internalization stage of Vero E6 cells infected by PEDV.In conclusion,porcine integrin β1 polyclonal antibody was successfully obtained in this experiment.It was confirmed that porcine integrin αvβ1 promoted PEDV infection of Vero E6 cells,and played a role in PEDV adhesion and internalization stages.It provides a material basis for subsequent researches on the mechanism of PEDV infection and broadens the understanding of the mechanism of PEDV infection.