Prokaryotic Expression Of The Epitope Domain (S1-CTD) Of Porcine Deltacoronavirus And The Developement Of An Indirect ELISA For Antibody Detection

Porcine deltacoronavirus is a new type of porcine intestinal coronavirus,it can cause diarrhea,vomiting and dehydration in suckling piglets.It is one of the important causes lead to the death of newborn piglets.Since the first discovery of Hong Kong in2011,PDCov have appeared in the United States,South Korea and China,and have become a new potential threat to to the breeding industry in various countries.In the present study,the S gene cloning and bioinformatics analysis of PDCo V strains SCNC201705 and SCNC20107 strains in sichuan were carried out in this study,and the prokaryotic expression vector pet28a-S1-CTD was constructed,and the indirect ELISA detecting PDCOV antibody was developed based on the expressed protein.1.Cloning and bioinformatics analysis of PDCo V S gene in SichuanTwo pairs of specific primers were designed with reference to the PDCo V S gene sequence(Genbank: KY436218).The complete S gene of two PDCo V S was amplified and cloned into p BM16A-TOPO vector to obtain recombinant cloning plasmids p BM16A-NC05S1 and p BM16A-NC07S1,p BM16A-NC05S2,p BM16A-NC07S2.The gene sequence obtained by sequencing the recombinant cloning plasmid is analyzed by bioinformatics software;The homology analysis of the gene showed that the cloned S gene in this study had a homology of 99.3%-99.6%with the previously cloned S gene(CHN-SC2015 strain)in our laboratory and the homology of the domestic isolate was significantly higher than that of the foreign isolate;The phylogenetic analysis showed that the cloned strains in this study were closely related to the strains circulating in mainland China,and the evolution distance from the strains(Genbank:KT266822.1)in Sichuan was the closest,but it was far away from the epidemic strains in the Korea,United States and Southeast Asia;The secondary structure analysis showed the S protein is mainly composed of ?-helix and?-fold,followed by random curl,and at least ?-turn,and four structures alternately appear;The S protein is hydrophilic by pro-hydrophobicity analysis and transmembrane region prediction.Protein,a signal peptide exists in the 19aa-20aa position,the N-terminal S1 region is located on the inner side of the membrane,and the C-terminus S2 is located on the outer side of the membrane;The B cell antigenic site prediction results show that the S1 region contains the major B cell epitope of the S protein.2.Prokaryotic expression of PDCo V S1-CTD gene and preparation of polyclonal antibodyAccording to the results of S gene bioinformatics analysis,this study designed a pair of specific primers to amplify the S1-CTD gene(S1 gene 832bp-1848bp)and construct the recombinant expression plasmid p ET28a-S1-CTD.The recombinant plasmid was identified by double enzyme digestion and sequencing,and then transformed into the host bacterium BL21(DE3).The positive recombinant bacterium was induced by IPTG,broken by ultrasound and purified by Ni+ column,and the target protein with a size of 41 k Da was obtained,mainly in the form of inclusion body.Polyclonal antibodies were prepared by immunizing rabbits with inclusion body proteins after hemodialysis renaturation.Polyclonal antibody titers were detected by indirect ELISA and its specificity was identified by western-blot and indirect immunofluorescence.The results showed that the target protein was correctly expressed and renatured,and the prepared polyclonal antibody hadgood biological activity.It laid a solid foundation for the establishment of ELISA diagnostic method.3.Establishment and preliminary application of indirect ELISA based on S1-CTD proteinBased on the purified and renatured S1-CTD recombinant protein,an ELISA method for detecting PDCo V antibody was established.The optimal coating concentration of antigen was 1?g/well,the optimal dilution of serum was 1:50,the optimal dilution of HRP-conjugated secondary antibody was 1:500,and the optimal closure time,reaction time and color development time were optimized.A total of 40 PDCOV negative serum were analyzed by the indirect ELISA the positive criteria of this ELISA were as follows: when OD450?0.377,it was judged as positive,when OD450<0.309,it was negative,and between the two values was suspicious;In each repeatability test,the coefficient of variation is less than 10%;Moreover,there is no cross-reactivity with reference positive serum such as CSFV,PRRSV,PCV,PRV,JEV,Po RV,PEDV,TGEV,etc;Compared with the virus neutralization test method,the total coincidence rate of the ELISA method is 83.3%.Finally,a total of 231 sera collected from Sichuan in 2018,which showed the PDCOV-antibody positive rate was 2.59%.The indirect ELISA constructed in this study provides a new method for antibody detection of PDCo V,which can be used for serological diagnosis and epidemiological investigation of the disease.