Selection And Identification Of Specific Single Domain Antibody(VHH) Against Porcine Epidemic Diarrhea Virus(PEDV)

Porcine epidemic diarrhea(PED)caused by PEDV virus appears as sporadic outbreak resulting dramatic economical loss to swine industry.As a member of the genera Alphacoronavirus in the family Coronaviridae of the order Nidovirales,PEDV induces characteristic manifestations such as watery diarrhea,dehydration,emesis and high mortality of suckling pigs.PEDV is an enveloped virus containing 28 kb positive-sense,single-stranded RNA genome with a poly(A)tail.A total of 7 open reading frames(ORFs)encoding 4 major structural proteins(S protein,E protein,M protein,N protein)and 3 non-structural proteins are comprised of virus genome.Single domain antibody fragment(sd Ab),referred to as VHH,is the antigen-binding fragment of naturally occurring functional heavy-chain antibody(HCAbs)devoid of light chain found in camelids or sharks.VHH possesses 4 framework regions(FR 1-4)and 3 variable complement determining regions in structure.The FRs work as scaffold to support CDRs that mediate antigen-binding interaction.Comparing with conventional immunoglobulin,VHH have smaller molecular weight of 16 k Da and more flexible CDR domain,which facilitate binding inaccessible epitopes in deeper cavities of antigen surface.Moreover,VHH shows other merits over intact immunoglobulin that is characterized with high stability,high yield and ease of production or purification.Take all merits into consideration,VHH is deemed to an alternative to conventional immunoglobulin in diagnosis and therapeutic applications.The current study aimed to develop a novel strategy to construct a specific immunized repertoire using T7 phage display system and select specific sd Abs with potential for further therapeutic or diagnostic application.The research content is as follows:(1)The PEDV vaccaine was subjected to immunize the camel.The peripheral blood lymphocytes(PBLs)were isolated from blood sample using lymphocytes separation medium and over 107 cells counted by hematocytometer were prepared.Total RNA was extracted using Axyprep Toatal RNA extraction kit from PBLs and first-strand c DNA was synthesizedusing reverse transcriptase M-MLV with oligo-d T primers.The gene encoding VHH was amplified using nested PCR.The repertoire was obtained by gene ligation to T7 Select 10-3b Eco R I/Hind III arms and in vitro packaging with packaging extracts.The library was subjected to 4 rounds of biopanning and a total of 3 subclones were selected by phage-ELISA.(2)The subtype VHH fragments were then digested and subcloned into p ET-30 a vector.The recombinant plasmids were transformed into BL21(DE3)strain.The recombinant VHH was expressed as soluble protein in E.coli.The protein was identified with SDS-PAGE and Western Blot.The ELISA results indicated the high affinity and low cross activity of recombinant antibody.The determinations of intracellular interaction were further carried using indirect fluorescence assay(IFA)and immunocytochemistry assay(ICC).In the present study,the high quality phage library was constructed using novel T7 system.Subclones were expressed as soluble and functional protein.Extracellular and intracellular affinities were both determined and proved that their potential for therapeutic and diagnostic purpose.