T7Phage Display For Neutralisating Epitopes Of Porcine O Type Foot And Mouth Disease Virus

Foot-and-mouth disease (FMD) is a highly contagious, acute vesicular disease of cloven-hoofed animals, caused by Foot-and-mouth disease virus(FMDV). This disease can quickly spread in avariety of species between animals and has been ranked as type A by the Office International Des Epizooties, World Organization Animal Health. The capsid of FMDV is constituted of60copies each of the four proteinsVP1, VP2, VP3and VP4, in which VP1, VP2and VP3form the capsomeres, VP4is inside of virus particle. The VP1capsid protein is the dominating antigen associated with immunogenicity of the viruses,which has been identified as the major immunogenic site for neutralizing antibodies. This has formed the basis for the peptide approach to vaccination against FMD.The T7Phage display is a newly developed system for displaying of exogenous peptides or protein domains by fusion expression on the T7surface. In this system the displayed proteins maintain the irrelatively independent conformation and biological activities. This process is initiated by fusing a foreign gene to the T7surface capsid accessory protein gene p10B. Phage T7is a desirbale vector because it is not only a small type phage with the features of convenient rapid growth and stbale system, but also antigen can be displayed on its capsid as fusion portein.The study was aimed at VP1120-160of Pocrine Foot and mouth vrius (FMDV) type O, which was displayed on T7phages using T7Select415-1b vector.1. The construction of recombinant T7phageThe neutralizing epitopes gene from VP1residues120-160of FMDV type O reported in GeneBank was designed and synthesized. The gene sequence optimized according to codon preference was ligated into bacteriophage T7vecter, and recombinant bacteriphage T7was Packaged with capside proteim in vito. Recombinant bacteriophage was named T7-VP1120-160and identified by PCR and sequencing.Then the phages were amplified and purified. A large amount of the phages were harvested, detected with SDS-PAGE and Western-blotting.The recombinant T7phage expressing VP1120-160is constructed successfully. The expression of VP1fusion protein and its antigenicity were confirmed in the recombinant phages by SDS-PAGE and western-blot assay. The target DNA was cloned into T7vecter correctly. The molecular weight of the fused proteins of the recombinant bacteriophages was about41kD and possess immunogenicity.2. The biological properties of the recombinant phageThe recombinant phage biological properties were researched after the particles were amplified and purified. Phage thermostability, pH stability, optimal multiplicity of infection(MOI) and one-step growth curve of phage were determined.The plaque of the phases is circular, transparent and clear boundary. The titers of the phage was5.0×1010pfu/mL, and it was still above1010pfu/mL after incubation at60℃for20min. The phage was stable between pH4to11. The optimal MOI was10-4～10-5. Comparing with most of the other phages, one-step-growth test showed that the phag had a higher adsorption rate, shorter incubation period and higher lytic efficacy. The latent period and the rise period of the phage were about30min and150min, respectively, and the average burst sizewas was about126pfu/cell. The phage with glycerol as stabilizer can be stored for6months at low temperatures. The freeze-dried phage can be stored for a short time at37℃and for a longer time at4℃.3.The immunogenicity of the recombinant PhageThe young piglets were devided into three groupes. The piglets in the first group were immunized with the recombinnant phages. The2th group was the synthetic peptide vaccine of swine foot-and-mouth disease(Type O) control, and the3th grous was blank control respectively. Animals were boosted two weeks post the primary immunization. Blood samples were collected before immunization,2, and4weeks after first immunization. VP1120-160specific sera antibodies were detected with indirect ELISA and LPB-ELISA respectively.The anti-VPl serum antibody titer of the the phage group was much higher than that of the synthetic peptide vaccine group. And the result of LPB-ELISA shows that4/5of the phage vaccinated pigs got protection from virus of FMD challenge theoretically, while no pigs immunized with the synthetic peptide vaccine were protected.In short, that the recombinant phage has great application prospects. The work establishes the foundation for the development of new vaccines for the prevention of foot-and-mouth disease.