Establishment Of A Stable Eukaryotic Cell Line Expressing Truncated E2 Protein Of Classical Swine Fever Virus And Its Application In Serodiagnosis

Classical swine fever(CSF)is an OIE-listed highly contagious disease of pigs and has caused huge economic losses every year to the pig industry worldwide.The disease is caused by classical swine fever virus(CSFV),a member of the Pestivirus genus within the Flaviviridae family.CSFV is an enveloped virus with a positive-stranded RNA of about 12.3 kb and highly homologous to other pestiviruses,including bovine viral diarrhea virus(BVDV)and border disease virus(BDV),which causes serological cross-reaction and misdiagnosis between CSFV and other pestiviruses.The glycoprotein E2 of CSFV is anchored at the outer surface of the virion and is important to induce the production of neutralizing antibodies during infection.At present,the crystal structure of BVDV E2 has been revealed and two Ig-like domains formed a unique protein architecture which has an elongated ?-stranded domain with a new fold at C-terminus.This unique protein architecture may also exist in CSFV and lead to cross-reactivity between CSFV and BVDV.To reduce the cross-reactivity between CSFV and BVDV,while keeping a good antigenicity,we referred to the crystal structure of the BVDV E2 protein to predicate structure of the CSFV E2 protein.Based on the putative structure of CSFV,we expressed the truncated antigenic domain of the CSFV E2 protein(E2B/c/D/A)in eukaryotic expression systems.The truncated E2 protein was identified by SDS-PAGE and Western blotting analysis,indicating a band approximately 26 kDa.Meanwhile,we constructed the CHO stable cell line to express the E2B/C/D/A.Subsequently,the cell supernatant containing E2B/C/D/A were collected and purified with Strep,agarose affinity chromatography.In order to establish an efficient indirect ELISA(iELISA),with high sensitivity and specificity,the reaction conditions were optimized.The optimal concentration of coating antigen in the iELISA was 2.5 ?g/ml,the dilutions of sera and secondary antibodies conjugated to horseradish peroxidase(HRP)were determined to be 50 times and 20 000 times,respectively,by checkerboard titration method.The cutoff value has been calculate by a series of sera based on E2B/C/D/A ELISA.The antisera against BVDV,BDV,porcine reproductive and respiratory syndrome virus(PRRSV),porcine circovirus type 2(PCV2),pseudorabies virus(PRV)and CSFV were used to confirm the cross-reactivities of the test.The result shows that only CSFV E2 antibody positive sera were detectable,indicating that the current E2B/C/D/A-based iELISA is specific.Then,a sensitivity test was performed by 8 sera(4 positive sera and 4 negative sera)using a serial dilutions range from 50 to 6 400 times.Subsequently,the iELISA was evaluated with 288 clinical swine sera and compared with IDEXX CSFV antibody test kit.The coincidence rate between E2B/c/D/A-based iELISA and IDEXX CSFV antibody test kit was 90.8%.Taken together,the E2B/c/D/A-based iELISA is specific and sensitive,and can be used for clinical diagnosis.