| Duck viral enteritis(DVE) is an acute, heat and hemorrhagic contagious viral disease, whose pathogen is Duck enteritis virus(DEV), causing huge economic losses to to the waterfowl breeding. Currently, due to the complexity of neutralization test, high cost of I-ELISA based on DEV which is difficult to purify, and I-ELISA based on certain dominant antigen region of DEV glycoprotein only to detect single antibody, there exists some problems, so a simple, low cost and applied for antibody comprehensive detection method is needed.In this study, we screened B-cell linear antigenic domain region of DEV g B firstly, then compared with the epitopes of DEV g C, g D, g E, which had been identified in previous study of our labtorary, prepared tandem repeated multi-epitope, and established I-ELISA for serum detection of DEV. This study afforded significant data for the study on function of DEV g B. At the same time,the work could be founder to monitoring for the rapid diagnosis of disease, epidemiological investigation and immune antibody titer determination of DEV, and be diagnosed in early infection, which might replace the existing the commercial DEV ELISA kits with market prospects as well.To screen and identify B-cell linear antigenic domain region of DEV g B, a set of overlapping peptides covering the whole region were expressed, purified and screened by multistep of Western-blot by expression system p ET-32 a of Escherichia coli. The results showed that the antigenic domain region were identified successfully by the DEV infected duck serum, which were located in N-terminal111-150aa(ATDRPHGLMNDQDTHLDGERLIRGVQSTRE I)and506-530aa(QELTRDNRTELALDLLGAMRGDKTR) region, which is now known to be the smallest of the dominant antigenic region identified.The dominant epitope peptides of g B, g C, g D and g E of DEV were confirmed by indirect enzyme linked immunosorbent assay(I-ELISA) for evaluation of their antigenicity, which were in accordance with the the identification results of Western-blot further. The results showed that the results of I-ELISA test were consistent with Western-blot, at the same time,that the antigenicity of g B was the strongest, g C followed, g D weaker, and g E weakest.Experimental immune serum antibodise of DEV have been tested by I-ELISA, as results showed that the antibodies against gB occurred at the first week after vaccination, then reached its peak at the third week, and the antibodies levels decreased slightly later, which was in the same pace with DEV. Antibodies against g C, gD and g E also occurred at the first week, then maintained at a relatively low level until the sixth week, however, reached a peak at the eighth week, and the antibodies against g C, g D and g E kept pace with each other almostly.the antibodies against g B reached its peak earlier than the antibodies against g C, g D and g E, with higher reactivity, compared with the antibodies against g C, g D and g E.By comparing the g B, g C, g D, g E antigenicity, the best dominant antigenic region of glycoprotein were chosen, and two tandem epitope peptides DEV-1-GP and DEV-2-GP were designed and synthesized, using a gene splicing by overlap extension protocol with preferred codons for Escherichia coli. The resultant two tandem repeat multimer between 1 to 7 were expressed and as soluble fusion proteins in E. coli. The results showed DEV-2-GP2 was identified with the stongest antigenicity and the expression of the supernatant by Western-blot and I-ELISA, which can be used as diagnostic antigen.An indirect ELISA based on the recombinant DEV-2-GP2 protein was developed and theirs optimal reactive conditions were determined. Using the cut-off value of 0.35, The established method could distinguish anti-serum against DEV from anti-serum of some common pathogens of duck such as duck enteritis virus, gosling plague virus and Ana I aviadenovirus which shows that the established I-ELISA was with great specificity. The limit of detection was 1:320, Intro-batch was 1.50%~8.12% as well as the Inter-batch was 2.56%~8.89%, which shows that the established I-ELISA was with great sensitivity and repetitiveness. Compared with neutralization test(recommended by OIE) and the commercial DEV ELISA kits, the total agreement of DEV-2-GP2 was 82.8% and 76.7%, respectively. I-ELISA established could well monitor the fluctuation of antibody after DEV attenuated vaccine. In duck farms serum samples with DEV attenuated vaccine and no DEV attenuated vaccine were detected against antibody of DEV, the positive rate is 50% and 4.2% by using this ELISA method respectively. The results showed this method may be applied to the detection of antibodies of DVE for the detection and serological surveys of duck enteritis virus, which provides an effective technical support. |
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