Porcine Epidemic Diarrhea Virus S Protein Expression And The Preparation Of Its Specific Single Domain Antibodies

Pig epidemic diarrhea(PED)is a common disease of pigs,and which causes serious economic loss to the pig industry.Currently,the vaccination is the major measure to prevention and control of the PED.Many factors cause unfavorable influence on the prevention and control of the disease,such as immune blank period,high density cultivation with the deteriorated environment,vaccination failure,antibiotics abuse,and co-infection of the immune suppressive disease,like porcine reproductive and respiratory syndrome(PRRS)and Porcine circovirus(PiCV),as well as the existing of the risk of disease transmission from the traditional live virus vaccine(Attenuated vaccine).Phage display technology is a technique can display the antibody molecules on the phage surface,and can identify the antigen specific antibody by screening.The antigen epitope of pig epidemic diarrhea virus(PEDV)was mainly distributed in the spike protein(S protein)of the virus.The spike protein specific single domain antibodies can be used in the diagnosis,treatment and prevention of PED by applying phage display technology to construct PEDV S protein specific phage antibody library,and selection and preparation of PEDV S protein specific single domain antibodies from the antibody library.In this study,we immunized Bactrian camel with PEDV,and extracted total RNA after the separation of peripheral blood lymphocytes,and synthesized the cDNA by RT-PCR,and amplified the VHH gene fragments by three round of PCR with single domain antibodies specific primers.We successfully constructed a primary single antibody library with the capacity of 5.49 × 106?by ligating the VHH gene fragments to pCANTAB5E vector,and electrotransformation to competent Ecoli TG1.We also expressed and purified the spike protein of PEDV by cloning the S gene fragment to the prokaryotic expression vector pET-28a,and transformed into competent Transetta-DE3.We enriched and screened the antibody library three rounds by using the S protein as the antigen,and identified the S protein of PEDV specific single domain antibody by phage-ELISA.