Construction Of Porcine Integrin αvβ3 Eukaryotic Expression Vector And Preliminary Research On PEDV Infection

Porcine epidemic diarrhea( PED) is an intestinal infectious disease which incidence and mortality rate are very high so that it caused enormous economic losses. It had demonstrated that porcine aminopeptidase N( pAPN) is a cell receptor of PEDV, but only rely on the pAPN protein still can not explain to relevant theoretical problems in PEDV infection. As a result, the research of PEDV receptor has important scientific significance and provides the basis for the research of antiviral drugs. In recent years, a growing number of research found that integrin can be used as many viruses receptor, which has trends of become a co-receptor.In this study, the porcine integrin αv and porcine integrin β3 amino acid sequences were analyzed by hydrophobic interaction which published in GenBank. Interception with hydrophilic amino acid sequences of the extracellular domain. Then we artificial synthesis the two gene fragments which had optimized of Escherichia coli codon. The two gene fragments were cloned into prokaryotic expression vector pGEX-6p-1, and then induced by IPTG. The recombinant proteins were purified and immunized 50 μg to 8 weeks BALB/c female mice respectively.Three days after the third booster immunization, drawing the blood from eyeball. Serum antibody titers were tested by indirect ELISA method. Then used Western blot method to identify whether the preparation of two kinds of polyclonal antibodies were able to identify ST cells, IEC cells, PK15 cells and small intestinal tissues of piglets. Artificial synthesis porcine integrin αv and porcine integrin β3 gene sequence were connected to the pCAGGS-HA eukaryotic expression vector, then the recombinant plasmid pCAGGS-pIαv and pCAGGS-pIβ3 were transfected into CHO cells. CHO cells were infected of PEDV after recombinant protein expressed. Statistical analysis of cell infection rate at different time points after the infected of the virus.The results showed that the synthetic genes were successfully obtained. Recombinant protein pGEX-6p-1-αv and pGEX-6p-1-β3 were obtained in the prokaryotic expression system. Western blot showed that the truncated porcine integrin αv had a good antigenicity from No.37 to No.557 amino acid, and the truncated porcine integrin β3 had a good antigenicity from No.30 to No.714 amino acid. The preparation of porcine integrin αv and porcine integrin β3 polyclonal antibody titers were 1: 6 400 and 1: 12 800, respectively, which can identify ST cells, IEC cells, PK15 cells and small intestinal tissues of piglets. The eukaryotic expression vector of pCAGGS-pIαv and pCAGGS-pIβ3 was constructed, and co-transfection group was higher than single-transfected group of the integrin expression, and the integrin αvβ3 proteins expression level were the highest in CHO cells after transfection 24 hours to 36 hours. After inoculation with PEDV in this period of time, PEDV infection rate of CHO cells which over expressed integrin αvβ3, was significantly higher than that of control group.This study we obtained the polyclonal antibodies of porcine integrin αv and porcine integrinβ3 which can identify natural integrin protein binding in various cells or tissue. The CHO cells which over expression of integrin αvβ3 enable increased of PEDV infection rate. Thus lays the foundation for the research on the mechanism of PEDV infection and the interaction between virus and receptor.