Indirect ELISA Based On Protein E Of Tembusu Virus

Duck Tembusu virus(DTMUV)is an infectious disease characterized by decreased egg production,decreased feed intake and duck associated with neurological symptoms and growth retardation in egg ducks.Since the outbreak of Tembusu disease in April 2010,this virus has been endemic in large scale in China,resulting in enormous economic losses to the breeding industry,especially the duck industry.Until now,the disease is still prevalent in some regions.In order to better prevent and control the disease,we conducted a study on the establishment of laboratory test methods for the disease.This study identifies laboratory-stored DTMUV for whole genome determination.By RT-PCR identification and whole genome sequencing,it was confirmed that the DTMUV stored in the laboratory was one of the branches of the FX 2010 strain;the full length of the genome was 10991 bp,containing a large open reading frame,encoding the polyprotein of3425 amino acids.Genome similarity to the FX 2010 strain(accession number MH454168.1)published by NCBI is up to 99.52%.In addition,a strain isolated from animal diseases was identified by LY.The full length genome of LY strain was 10990 bp,which was similar to that of Shandong strain(accession number:JX965381.1)up to 98.92%.The strain was in the same branch with Shandong strain,and its affinity was very close.In this study,the E protein of DTMUV was expressed and identified in prokaryotic cells.The E protein was the main antigen region of DTMUV.A 1530 bp fragment of E was designed and cloned into p ET-28a(+)expression vector based on the structural E protein gene sequence of LY strain.After PCR identification and sequencing,it was confirmed that the p ET28a-E vector contained the target protein E.PET28a-E was transformed into E.coli BL21(DE3).After induction with 1.0 m M IPTG for 4.0 h,a lot of target proteins were obtained.The target proteins were purified by nickel ion affinity chromatography and identified by SDS-PAGE electrophoresis and Western blot.SDS-PAGE results showed that the target protein was about 55 k Da,as expected,indicating that the target protein was expressed,and Western blot results showed that the E protein had good immunogenicity.This study established an indirect ELISA method for the detection of expressed DTMUV E protein.The positive serum of DTMUV was used as the primary antibody to establish an indirect ELISA method for detecting DTMUV antibody.After optimizing the reaction conditions,the optimal reaction conditions were as follows:antigen.The dilution of the primary antibody was 1:400,blocked with 1%BSA for 1 h,incubated with one antiserum for 1.5 h,incubated with the secondary antibody for 1.0 h,visualized with TMB chromogen for 10 min,and read the values on a microplate reader by using OD₆₂₀.The critical value of negative and positive was determined to be 0.791.When the OD₆₂₀ value of the sample to be tested was greater than or equal to 0.791,it was judged as positive.When the OD₆₂₀ value of the sample was greater than or equal to 0.791,it was judged as negative,and the sensitivity reached 1:1600.The sensitivity was higher.The specific experiments were carried out for vimentin pestivirus,avian influenza virus and reovirus.And the he results showed that the indirect ELISA method established in this study had good specificity and did not react with other viruses.In conclusion,we successfully expressed and purified the main antigen region E protein of DTMUV,and obtained a lot of target proteins with good antigenicity.Then we established an indirect ELISA method for detecting DMTUV by using purified E protein as inclusion antigen.