Construction Of Recombinant Duck Plague Virus Expressing HA And NA Gene Of H5N1 Subtype Avian Influenza Virus

Highly Pathogenic Avian Influenza caused by avian influenza virus (AIV), is a highly contiguous disease. AIV can infect almost all wild and domestic poultries and cause severe economic losses for the poultry industry. Efforts to developing a safe and effective vaccine against AIV are cotinuing.Being able to induce humoral and cellular immune responses in animals,live virusvector vaccines can provide effective protection. Duck plague virus(DPV),one of herpesviruses,has a large genome that contains many non-essential regions for inserting and expressing foreign genes,thus it is frequently used as live vaccine vector.In this study,Using extracted total DNA from duck plague virus (DPV) vaccine strain infected Chicken (Gallus domesticus) embryo fibroblasts(CEF) cells as template, the 1.0 kb TK gene nonessential for viral replication was amplified by polymerise chain reaction(PCR) and cloned into T-easy vector to obtain pGTK. According green fluorescent protein vector pEGFP-Cl sequence, one pairs of the primers used to amplify CMV,EGFP and its integrality gene expression case which lie in the plasmid pEGFP-C1 were inserted into TK site which lies in pGTK obtain the transfer vector pGTK-EGFP. Two pairs of primers were synthesized according HA and NA sequence of H5N1 Subtype avian influenza virus published on genbank, one pairs of the primers used to amplify the HA gene, the other to amplity the NA gene,the both of them were then inserted into the resulting transfer vector pGTK-EGFP in the EGFP multiclonal sites to obtain transfer vector pGTK-EGFP-HA-NA. The recombinant virus, designated rDPV-HA-NA was generated by cotransfection of CEF with pGTK-EGFP-HA-NA and the DPV34F2 vaccine virus.The recombinant viruses offer a base for the study of the immunological function of AIV HAandNA ,and they represent potential vaccines against AIV.In this research we construct the recombinate pGTK-EGFP transfer vector which offer a base for the study of the DPV bivalent gene engineering vaccine. Thereby rDPV-HA-NA was able to provide protection against challenge with H5N1 subtype of HPAIV. Recombinant DPV vaccine will be a promising substitution of conventional whole virus inactivated vaccine to prevent and control AIV. This is a great breakthrough in thedevelopment of AIV vaccine.