| Duck viral enteritis (DVE) is an acute, heat, haemorrhagic and lethal disease caused by duck enteritisvirus (DEV). The disease was epidemic in many countries and regions which cause high morbility,mortality and large econmic losses. It is one of the important diseases impairing poultry production.Waterfowl and migratory birds are the media of DEV transmission and carried the virus for several years,which takes huge difficulty the prevention and control.Avian influenza (AI) is a deadly infectious disease of poultry caused by type A influenza virus.Waterfowl are not only the susceptibility of influenza virus in poultry and has become a huge reservoirfor influenza virus. Waterfowl played an important role in the spread and ecological evolution of theH5N1subtype of HPAI. The HA gene is one of the most important virulence genes in AIV. HA caninduce neutralizing antibody to generate protective immunity. Hence, HA gene is the preferred targetgenes of the genetically engineered vaccine.In this study, DEV Clone-03strain was preferred as the parental virus and the nonessential geneThymidine kinase (TK) gene was chosen as target for the insertion of foreign genes to construct TK genedelection recombinant virus. The plasmid pTK-EGFP constructed by our lab and DEV Clone-03genomicDNA were co-transfected into CEF cells. After screened and purified, we obtained a pure recombinantvirus which expressed enhance green fluorescent protein, named as TK-rDEV-EGFP. The sequencingresult of recombinant region showed that EGFP expression cassette was inserted into the TK gene ofDEV genome. The replication kinetics of the recombinant virus was determined and compared with thatof DEV Clone-03. The TCID50of TK-rDEV-EGFP was lower than DEV Clone-03. TK-rDEV-EGFP waspassaged to25th in CEF cells. We detected the EGFP gene every five passage by PCR method, andobserved the green fluorescent protein under the fluorescence microscope. The results showed that TKgene was non-esserntial gene for the replication of DEV, which can carry the foreign gene expressionalong with DEV genome. Recombinant birus TK-rDEV-EGFP keep well genetic stability.On the basis of the construction of the recombinant virus TK-rDEV-EGFP, we chose the H5N1avianinfluenza virus (AIV) HA gene as the target gene to construct the transfer vector pTK-HA. PlasmidpTK-HA and DEV Clone-03genomic DNA were co-transfected into CEF cells. We obtained a purerecombinant virus which expressed H5N1AIV HA protein, and named as TK-rDEV-HA. Thesequencing result of recombinant region showed that HA expression cassette was inserted into the TKgene of DEV. Indirect immunofluorescence assay (IFA) can detect HA protein after TK-rDEV-HAinfected CEF cells6h, HA expression incresed as the infection duration. While, the western blot detectedthe HA expression at48h post infected TK-rDEV-HA. May be the IFA is more sensitive than westernblot emthod. Repliacation kinetics results showed that the TCID50of TK-rDEV-HA was similar to TK-rDEV-EGFP but loser than DEV Clone-03. Compared with TK-rDEV-EGFP, TK-rDEV-HA showed noobvious difference with respect to virus replication and shape of virus plaque. TK-rDEV-HA waspassaged to25generations in CEF cells, the recombinant virus was detected by PCR and western blot every five passage. The results showed that HA gene was genetically stable and the the protein expressedstablely. The recombinant virus TK-rDEV-HA kept the character of the parental virus. The recombinantvirus TK-rDEV-HA has the potential feature to be a candidate for the construction of DVE-AI covalentlive vectored vaccine. |
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