Construction Of Recombiant Marek's Disease Virus Expressing HA And NA Gene Of Avian Influenza Virus

HA and NA gene were the major protective antigens for AIV. To elucidate the relationships among AIV isolates, HA and NA gene was amplified by RT-PCR, then cloned into pMD18-T and sequenced. The results of sequencing showed that 1707bp of HA cDNA covered the complete open readingframe, encoding 568 amino acid residues and that NA cDNA also covered the complete open readingframe, encoding 469 amino acid residues. The first 16 amino acid residues were composed of signal peptide of HA and HA1 contains 330 amino acid residues, HA2 was 222. When compared with other AIV(H5N1) HA gene published on Genbank, there were identity of 91.0%-98.7% at nucleotide acid level . Results indicated that HA gene was greatly variant. While, NA gene was not directly derived from other avian influenza virus and nucleotide acid identity was above 95.2%.Marek's Disease Virus have been used as safe vector to construct vector vaccine to control avian virus disease. In the construction of recombinant virus , transfer vector were considered important step. Based on the sequence analysis of the genome of MDV, the US2 gene was chosen as the insertion site for the construction of transfer vector. The DNA of MDV genome was extracted , and the 2.6kb homologous flank was amplified , then the segments was ligated in vitro and subcloned, the transfer vector was named as M2. Based on the transfer vector M2, transfer vector containing the SV40 controlled EGFP was constructed , after transfection in chicken fibroblast cells infected with MDV, the reporter gene was expressed 72 hours later. This results lay foundation for recombinant vaccine development .For ideal vaccine, it was difficult to provide protection against all subtypes of AIV because HA and NA gene of AIV were highly frequent antigenic variation.In this study, to produce a new kind of vaccine inducing cross-protection in chickens, recombinant viruses co-expressing HA-NA genes of AIV were constructed. To construct transfer plasmids, HA gene of H5N1 and GFP gene were cloned into the vector of MDV. And the NA gene was cloned into transfer plasmid containing HA gene, which was constructed previously. Then these recombinant vectors were transferred on CEF cells infected with parent MDV. Based on the expression of reporter gene, recombinants MDV was selected by fluorescent light plaque, purified by several cycles cloning. rMDV-HA-NA plaque, and identified by PCR. rMDV-HA-NA was stable genetic properties and could express foreign genes of AIV.In a word, recombinant transfer plasmid was constructed in this stdy and this provided great help for new vaccine. rMDV-HA-NA was able to provide protection against challenge with H5N1 subtype of HPAIV. This is a great breakthrough in the development of AIV vaccine. Recombinant MDV vaccine will be a promising substitution of conventional whole virus inactivated vaccine to prevent and control AIV.