Abstract
The neuraminidase gene is responsible mainly for virus release from the infected cells, thus increase the incidence of virus shedding from infected birds. For production of mammalian expression DNA vaccine encoding for the NA1 gene of the HPAI H5N1 subtype, we used gateway technology for cloning and its Benefits include uses a one hour reversible recombination reaction, without using restriction enzymes, ligase, sub cloning steps, also it provide a very useful method or the directional cloning thus eliminate the need or analysis of the recombinant plasmid for antisense cloning.. a well-defined Egyptian field isolate was used to amplify the full length open reading frame of NA1 gene. The isolate was first propagated on SPF-ECE and gave 9 log10 heamagglutination test ( HA) titer and 7 log 10 heamagglutination inhibition test (HI) titer when using anti-H5 antisera. Sequencing analysis of the cleavage site revealed the presence of the polybasic amino acid sequence PQG↓ERRRKKR↓GLF. Sequencing of the full length NA1 gene showed that the open reading frame is about 1302 bp with many glycosylated and protein C phosphorylation motifs. For the production of the mammalian expression DNA plasmid, the full length NA1 gene was first cloned into the entry vector (pENTR D/SD topo) and transformed into E. coli Topo 19 host strain. plasmid purification analysis revealed that the recombinant entry vector carry the NA1 insert has about 7.6Kbp when analyzed with the traditional agarose electrophoresis, while using RealtimePCR assay, all the tested clones gave a positive cycle threshold ranges from 32.66 – 34.49. This positive plasmid preparation was used in homologues recombination with the mammalian expression vector (pDEST 40). Real time PCR analysis of the plasmid preparation from the transformed E. coli with this destination vector revealed that the all the tested clones gave a positive cycle threshold ranges from 14.19 to 15.91. In conclusion, Cloning of the neuraminidase gene of the highly pathogenic avian influenza virus H5N1 subtype have been done using the gateway cloning strategy as step for production Of DNA vaccine coding for N1 against H5N1

Key words: avian influenza; DNA vaccine; neuraminidase gene; highly pathogenic;cloning ;gateway technology