Construction Of Porcine PDCoV Phage Single-chain Antibody Library And Selection Of Antibody To PDCoV N Protein

Porcine deltacoronavirus(PDCoV)is a newly discovered coronavirus in recent years which can cause intestinal disease in pigs.The major clinical manifestations of PDCoV are dehydration,diarrhea and vomiting.PDCoV can infect pigs of different ages,and causes high infection and fatality rate in piglets.It has been detected in clinical samples from diarrheal pigs in many countries and regions and caused great losses to the swine industry since its outbreak.Nowadays,vaccines and antibody drugs target to PDCoV are urgently to be developed.Single-chain variable fragment antibody(scFv),also called single-chain antibody,is a small molecule genetic engineering antibody.It has strong penetration,short half-life,low immunogenic and is easy to operation by genetic engineering,so it has been widely used in many fields such as immune diagnosis,antivirus,targeted therapy and so on in recent years.At present,scFv is obtained mainly by phage display technology.Instead of cell fusion,the method only need use PCR to amplify the antibody gene.Then the antibody gene fused with phage coat protein gene to expression and displayed on phage surface,which can simulate antibody expression in vitro.Since it greatly reduces the operation difficulty,the invention of antibody library technology promoted the rapid development of genetic engineering antibody.In this study,a procine single chain antibody display library was constructed by using phage display technology,and phage antibodies specifically bind to PDCoV-N were obtained by repetitious biopanning.The screened scFvs were expressed in eukaryotic cells and then antibody reactivity was verified by several assays.Hope this study can lay a foundation for the detection and prevention of PDCoV.The main research contents are as follows:1.Construction of porcine PDCoV single-chain antibody libraryRNA was extracted from spleens of PDCoV infected pigs and then reverse transcribed into cDNA.Primers were designed to amplify the heavy(VH)and light(VL)chain variable regions of antibody,and scFv gene was spliced by overlap PCR.The scFvs and the plasmid pSEX81 were digested,linked,and then electro-transformed into XL 1-Blue competent cells to construct a single-chain antibody gene library.The cumulative storage capacity of VH-VLκ was about 6.5×107cfu/mL,while that of VH-VLλ was about 1.7×108 cfu/mL.PCR result showed that the correct rate of the inserted gene fragment was about 80%.Infect the primary antibody gene library by M13KO7 helper phage to construct the phage single-chain antibody primary library,and the titer was detected as 4.5×109pfn/mL.2.Enrichment and screening of anti-PDCoV N protein antibodyUse PDCoV N protein as the target protein to enrich and screen the antibody library for 4 rounds.The final enrichment ratio was about 310 times,indicating that the specific phage antibody was successfully enriched.Pick up 96 colonies randomly from the 4th round output plate for PCR identification and the positive number was 11.After sequencing and sequence analysis,only five strains are correct and have no terminator codon.Finally,phage-ELISA showed that there are three scFvs with high affinity to N protein:scFv-25,scFv-27 and scFv-53.3.Expression and reactivity detection of specific scFvs.Selected scFvs were digested and linked to the eukaryotic expression vector pFUSE-hIgG-Fc2,then transfected 293T cells to obtain recombinant protein.Western blot analysis showed that two antibodies were successfully expressed,and the antibodies in the supernatant could be purified by Protein A.ELISA results showed that both antibodies react with PDCoV but not PEDV,TGEV and PSV,indicating that antibodies have good specificity.Western Blot results showed that the expressed antibody could bind to N protein but not other proteins of PDCoV.Indirect immunofluorescence assay showed that both antibodies could specifically recognize PDCoV.In summary,a porcine PDCoV single-chain antibody library was constructed in this study,and two anti-PDCoV N protein scFv strains were screened and successfully expressed,which provided some references for the treatment and prevention of PDCoV.