Screening The Dominant Epitope Regions Of Glycoprotein C And Glycoprotein E Of Duck Enteritis Virus

Duck viral enteritis (DVE), also known as duck plague (DP), is acute, heat and haemorrhagic and lethal disease which infects birds of the order Anseriformes (ducks, geese and swans). The diease is caused by duck enteritis viral (DEV), which has the characteristics of wide prevalence, rapidly spreading, high incidence and mortality. DVE was epidemic in many countries and regions and caused considerable economic losses to the commercial duck industry. Glycoprotein C and Glycoprotein E are the important envelope proteins of DEV; Glycoprotein C has good immunogenicity and antigenicity, also could stimulate the T-Lymphocytes proliferation; Glycoprotein E has neutralization activity, has good antigenicity within extracellular region and intracellular region. It was involved in virus attachment and played a role in cell-to-cell spread. In this study, by the means of overlapping peptides expressed in E.coli combination with Western blot to identify the dominant epitopes region of Glycoprotein C and Glycoprotein E of DEV. At the same time, the dominant epitope regions lay a foundation for the research of diagnostic reagents, epitope vaccin and so on.In order to map the dominant epitope regions on gC and gE of DEV, a set of partially overlapping fragments of 58-85aa which overlay the extracellular region of Glycoprotein C and the extracellular and intracellular region of Glycoprotein E were designed. Each protein was synthesized 7 pairs of primers. PCR products were cloned into the expression vector pET-32a and then these fragments confirmed by sequencing. Each fragment was expressed in Escherichia coli Rosetta(DE3)pLysS. The gC-1(27-96aa), gC-2(74-149aa), gE-5(241-325aa) and gE-7(433- 490aa) were then detected by Western blot with positive serum against DEV. With the purpose of identification the dominant epitope regions on gC and gE of DEV, we screend the epitopes secondly. The results of Western blot showed that gC-2.1(67-104aa), gE-5.3(290-325aa), gE-7.3(449-475aa) and gE-7.4(463-490aa) have good antigenicity. At the basic, five pairs of oligonucleotides overlaying gC-2.1, five pairs of oligonucleotides overlaying gE-5.3 and six pairs of oligonucleotides overlaying gE-7.3 and gE-7.4 were designed respectively. Each pairs of oligonucleotides was cloned into the expression vector pET-32a. The expression proteins were purified to analyze the antigenicity by Western blot. gC-2.1-B(73-88aa), gC-2.1-C(79-94aa), gE-5.3-B, gE-5.3-C(296-315aa) and gE-7.3-B~7.4-B(455-485aa) were incubated with the positive serum of DEV. For the purpose of decreasing the numbers of amino acid, we screened the epitopes sequentially. The result of the fourth identification of the epitopes showed that the region of 73-88aa is the domain epitope region of Glycoprotein C of DEV; the regions of 303-313aa, 317-326aa and 461-481aa are the the domain epitope regions of Glycoprotein E of DEV.According to the result of this study, the antigenic epitopes on gC and gE of DEV were contrasted with others sequence of DEV on amino acid by DNAMAN. The results showed the dominant epitope regions were conservative. Meanwhile, the study may be useful for understanding the construction of DEV.