Constraction Of A Full-length CDNA Clone Of Porcine Deltacoronavirus And Preparation Of Antibody Against Its S Protein

Porcine deltacoronavirus(PDCoV)is a type of single-stranded,positive-sense RNA virus with envelope,belonging to,Deltacoronavirus,Coronaviridae,Nidovirals.PDCoV could induce pig diarrhea in all age pigs and death in piglets,the clinical syptoms and mortality induced by PDCoV are similar,but milder to those of porcine epidemic diarrhea virus(PEDV).PDCoV was first discovered in Hong Kong,China in 2012,since then,it has been reported in the United States,Canada,China,South Korea,Thailand,Myanmar,Japan and other countries.Since the virus was reported,it has been detected in pig farms in many areas of China,indicating PDCoV is prevlent in China,has caused economy loss to the pig industry.In this study,719 diarrheal disease samples collected from 18 provinces in China from 2016 to 2018 were used for PDCoV and PEDV detection.The results showed that among 719 samples,94 were PDCoV positive,with a positive rate 13.07%;264 were PEDV positive,with a positive rate 36.72%;29 were both PDCoV and PEDV positive,the co-infection rate was 4.73%.On the other hand,we cloned full-length cDNA of PDCoV into a plasmid based on bacterial artificial chromosomes(BACs).Sequences of full-length cDNA of PDCoV-CH-HA3-2017 strain preserved in our laboratory,CMV promoter,HDV and BGH were analysed by online software NEBcutter.After analysis of the possible 16028 restriction sites,five cleavage sites BamHI,BsiWI,BspDI,Avr? and Hind?were selected to construct PDCoV full cDNA clone.Firstly,XbaI,SalI,PstI and SphI in the MCS region of pBeloBAC11 vector were removed by homologous recombination,and then three enzyme sites BsiWI,BspDI and Avr? were inserted between BamHI and Hind?,achieving transformation of the pBeloBAC11 vector.After this,CMV-PDCoV-poly(A)-HDV-BGH was mainly divided into five parts:A,B,C,D,and E.These five parts were sequentially inserted into the pBeloBAC11 vector in the order of E,D,A,B,and C by means of homologous recombination.In the process of inserting fragment E,the 23976th base,thymidine was converted into cytosine nucleoside using primers in a manner of silent mutation to achieve the introduction of rescue markers.The construction of the full-length cDNA clone of PDCoV was successfully completed.The bioinformatics software DNASTAR was used to analyze the S gene sequence of PDCoV-CH-HA3-2017 strain,and the possible epitopes were predicted.Finally,the antigenic epitope encoded by base between 766-1173 in S was selected and named PDCoV-S-ep.The PDCoV-S-ep gene was amplified by PCR,constucting prokaryotic expression vector pET-28a-PDCoV-S-ep.His-PDCoV-S-ep was successfully expressed under the induction of 1 mM IPTG at 16?.After purifying the protein of interest using Ni+ affinity chromatography,the protein was used to immunize BALB/c mice.The antibody of his-PDCoV-S-ep was obtained.This study investigated the prevelence condition of PDCoV and PEDV in China,providing data support for the prevention and control of PDCoV and PEDV in China.A full-length cDNA clone of PDCoV was constructed,laying a foundation for the construction of the reverse genetic system of the virus.The antibody of S protein was manufactured,providing a tool for mechanisam research and virus detection of PDCoV.