Cloning And Expression Of Duck Enteritis Virus GB Gene And Preliminary Application Of Its Product

Duck viral enteritis (DVE), or duck plague (DP), was caused by duck enteritis virus (DEV).DVE was an acute, heat and septicaemic contagious viral disease that naturally affecting birds of the order Anseriformes (ducks, geese and swans). DEV is currently grouped in the alphaherpesvirinae subfamily of the herpesvirus family. But the molecular study of DEV has fallen behind those of other herpesviruses. Until now, The DEV genomic features and physical map have not been elucidated. Glycoprotein B (gB) is a major envelope protein of herpesvirus and plays an important role in various aspects of virus infection, including virus attachment, penetration and cell fusion. Therefore, the research on gB have significance not only the function and characterization in the molecular biology of the virus but the clinical diagnosis, prevention, genetically engineered vaccine.In the present study, using genomic DNA of duck enteritis virus as a template, a degenerate PCR, three TAIL-PCRs and a Long-PCR method were performed, the DEV UL25, UL26, UL26.5 ,UL27 sequences was obtained. DNA sequence analysis revealed a 1797bp open reading frame (ORF) encoding a 599 amino acid (aa) polypeptide homologous to herpesvirus UL25 proteins, a 2124bp ORF encoding a 708aa polypeptide homologous to herpesvirus UL26 proteins, a 1074bp ORF encoding a 358aa polypeptide homologous to herpesvirus UL26.5 proteins, a 2796bp ORF encoding a 931aa polypeptide homologous to herpesvirus UL27 proteins, that is gB. These sequences have been submitted to GenBank with Accession number EF554399, EF554400, EF554400, EF554401, respectively.According to the antigenic analysis of DEV gB protein , two pair of primers were designed , with which the gene fragment coding the high antigenic domain of DEV N-terminal and the middle gB protein were amplified from the DPV genome . Two segments were cloned into pET32a vector to obtain the recombinant plasmids pET-gB1and pET-gB2. Two recombinant plasmids were transformed into E. coli BL21, and expressed in very high level as inclusion body after induced with IPTG. The expressived product analyzed by 12% SDS- PAGE,Its molecular weight were 42.4KDa and 54.6KDa as expected. The expression were optimized with proper inducing conditions of 0.4 mmol/L IPTG and 3 hours induction,0.2 mmol/L IPTG and 5 hours induction, respectively.Western-blot analysis proves that the fusion proteins(gB1, gB2) have specific antigenicity.The inclusion body were solubilized in urea sulution (8mol/L) .The purified protein were obtained by His·Bind affinity chromatography. Then an indirect ELISA was established to detect antibody against DEV with the purified gB1 protein as the coating antigen. The optimal operation methods were: A protein concentration of 6.5μg/mL was coated overnight at 4℃on 96-well microtiter plates and the plates were subsequently blocked 3h with 1% BSA, then the optimal dilution of serum was 1∶80 and incubated for 45 min, the anti-duck immunoglobulin conjuate was added and incubated for 30 min at 37℃, and then the indicator OPD was added and incubated for 15 min at 37℃, 2mol/L H2SO4 was added before OD was measured at 490nm. The positive criterion of this ELISA assay is OD the tested serum﹥0.4 and OD the tested serum/OD the negative serum﹥2.0.The ELISA was performed on 700 sera from which were preserved from Shandong ,Jiangsu provinces ,they were detected by igB1-ELISA and iDEV-ELISA by duck plague virus as the coating antigen respectively. The agreement ratio between the two methods is 95.6 %.