Prokarotic Expression Of Duck Enteritis Virus Glycoprotein H And Development Of An Indirect ELISA For Detection Of The Viral Antibody

Duck plague is a major infectious disease of duck,muscovey and geese, which is caused by a Alphaherpesvirus,Duck Enteritis Virus(DEV). This virus can cause infection in disparity age and sex duck. Both morbidity and death rate are high and present the growth, and affect the fast development of the duck industry.The gH gene of Duck Enteritis Virus (DEV) vaccine strain was amplified by PCR method using three pairs of specific primers. PCR products were successively cloned into pET28 to generate three prokaryotic expression vectors, pET28- gH, pET-gH1347 and pET-gH1707.The results showed that the gH gene in pET-gH1347 or pET-gH1707 without signal peptide and transmembrane domains were successfully expressed in the E.coli BL21 (DE3) host cells, but complete gH gene in pET-gH was not. Western-blot analysis proved that two expressed proteins had good immunoreactivity against DEV antibody. Therefore, recombinant gH proteins can be expressed in vitro in E. coli, which helped for further research on gH function and DEV detecting method.The DEV recombinant envelope glycoprotein (gH) was expressed by E.coli and purified in Ni2+-Agarose column through native conditions purification method. Using purified production, an indirect ELISA for detection of anti-DEV antibodies was developed and its optimal reaction conditions were determined: coating antigen for 4℃overnight at a concentration of 3μg/mL, serum sample (1∶8 0) and Goat anti-Duck IgG being incubated at 37℃for 60min or 30 min, the substrate for ELISA being incubated at 37℃for 10 min. The ELISA assay was confirmed to have a good reiteratively, specificity and sensitivity by reiteratively test, and cross test . The present study confirmed that the indirect -ELISA was a rapid, sensitive, economic and specific method for serologically detection of DEV infection ..