Preparation And Identification Of Single Domain Antibody Specific For Spike Protein Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus(PEDV)is the major pathogen causing pigs' acute and contact intestinal infectious diseases.PEDV can persist in pigs.The main symptoms are vomiting,diarrhea and dehydration.Both breeds and pigs are susceptible.The high mortality rate of piglets caused by the onset of infection has caused huge economic losses to the pig industry.At present,China adopts vaccine immunization methods to prevent and control swine epidemic diarrhea(PED).However,there is a blank period of immunization for immunization.The immune function of piglets is imperfect and PED is prone to occur.Single-domain antibodies prepared by genetic engineering can make up for this deficiency.The PEDV Spike protein(S protein)is located on the surface of the virus and contains multiple neutralizing epitopes and receptor binding domains of the virus,which is closely related to the antigenicity and adsorption invasion of the virus.S protein is the main target for the development of novel genetic engineering vaccines and passive immunological preparations.Single-domain antibodies have high water-solubility,low immunogenicity,high antigen-binding activity,and high stability.As passive immunization,they have rapid effects and do not require incubation period.Once they are imported,they can immediately acquire immunity and can effectively prevent and control diseases..In this experiment,S protein was used as a screening antigen to screen from the constructed phage antibody library.A positive clone with relatively strong binding ability to PEDV in Phage ELISA was selected,and a single domain antibody gene fragment was obtained by plasmid recombination.After being ligated with the expression vector pET-28 a and chemically transformed into the prokaryotic expression cell Transetta-DE3 to induce expression and purification,a single domain antibody with high binding activity to the PEDV S protein was obtained and subjected to ELISA and immunofluorescence assays.The identification of its immunological activity resulted in the following conclusions:(1)The specific phage antibody library was well-enriched and 20 positive clones with certain binding activity were obtained after screening by Phage ELISA.(2)Six different single-domain antibody sequences were obtained by gene sequencing,and a single domain antibody(S7)plasmid was extracted and cloned into the prokaryotic expression vector pET-28 a to obtain the recombinant expression plasmid pET-28a-VHH.,IPTG-induced expression and purification,to obtain a single domain antibody without contaminating protein;(3)Western-Blot test confirmed that single-domain antibody has immunological reactivity;single-domain antibody can be neutralized with PEDV to produce antigen and antibody,and has high antigen-binding activity.Single-domain antibody was confirmed by cellular immunofluorescence test.S7 can produce antigen-antibody binding reactions with antigens in cells.