Molecular Cloning And Sequencing Of NA Gene Of Avian Influenza Virus

Avian influenza A viruses are widespread among domestic poultry and are recently isolated from humanswith respiratory illness in China.Although this avian is not highly pathogenic for avian species,these viruses have recently been transmitted to mannalian species,including humans.Neuraminidasen is one of two kinds of surface glycoprotein.The purpose of this research is to amplify the NA gene.It will provide the foundation for the study of molecular and gene chip of AlV.We obtained allantoic fluid from SPF chicken embryo inoculated with AlV.Then we extracted the vival RNA using guandidihium isotheeyanate.In the present study,one pair of specific primers to NA gene of Nl subtypes were designed to amplied cDNA of Nl by RT-PCR,purified the production of PCR used reagents made by ourself and cloned the production added A-tail into pGEM-T easy vector by T-A ligation.Then the JM109 competent cells were transformed with the recombinant plasmid.Consequently ,the rocombiant plasimid was amplified to test the inserting of DNA fragment.Sequencing the NA fragment of the positive clone showed that we cloned the whole length of NA gene,the Nl subtype was composed of 1345 nucleotides,and encoding 434 amino acid.The highest homology is up to 98% compared with the published gene sequence in genebank,there is minor variation in some nucleotide sequences.The product cloned will provide reliable evidence for the establishment of quick and accurate molecular biological diagnostic method and lay a profoundf foundation for the study of gene engineering vaccine of AI.