Prokaryotical Expression Of The Major Antigen Regions Of Glycoprotein B Gene Of Duck Enteritis Virus In E.Coli And Development Of An Indirect ELISA Assay For Detecting Antibody Against DEV

Duck virus enteritis (DVE), also called duck plague(DP) is an acute contagious viral disease affecting birds of the order Anseriformes(ducks, geese and swans) , it caused by duck enteritis virus. Virus genome encodes several glycoproteins. Among them glycoprotein B is one of major proteins presented on the envelope of virus. It is the major immunity protein as well. In this study, Duck enteritis virus are propagated on the CEF monolayers and then the viral DNA are prepared from the purified virions which purified by ultra centrifuge. The DNA sequence of DEV was got from the GenBank (access number: EF203709). Based on the hydrophilicity, antigenity and surface probality analysis of DEV's gB using biosoftware DNAstar,two major protective antigen encoding regions of gB gene were chosen. The target genes were cloned into pMD18-T Vector and then constructed using prokaryotic expression vector pET-32a(+) after being confirmed by PCR and restriction endonuclease analysis. The recombinant plasmids were transformed into expression Escherichia. coli BL21(DE3)pLysS and the recombinant fusion proteins could be highly experessed in inclusion body form in the presence of IPTG. The Western-blotting analysis and ELISA tests unfolded the excellent immunogenicity of the recombinant proteins which might be used as coating antigens to develop the ELISA method for DEV specific antibody diagnosis as well as a constituent of subunit vaccine for DEV prevention.The indirect ELISA for detection of antibodies of DEV was established using recombinant fusion protein pET-32a-gBa as coated antigen with the optimal working parameters, including 2.5μg/mL of the pET-32a-gBa protein antigen for coating, the best sealing buffer is 0.15% BSA and the sealing time is 24h (4℃), testing sera dilution at 1:800 and the sera reaction time is 90min (37℃), second antibody dilution at 1:1000 and the reaction time is 30min, the standard of determining as positive sample is S/P≥0.446. Several positive sera stored in our lab were tested by the indirect ELISA and this assay was confirmed to be specific and sensitive by the results ,suggesting its application prospection. In our study, two major antigen regions of DEV gB gene were successfully expressed in inclusion body form and purified. They lay the foundation for the futher studies of the protein structure and function of DEV gB. The simple and fast diagnosis method using pET-32a-gBa expressed as the diagnosis antigen can be a technique support for futher developing the commercial kit of diagnosis, prevalence survey and control & eradication program of DEV in China.