Cloning Of F Gene Of NDV Isolated In Hebei Province, Construction Of Expression Vector In Plants And Studies On Transformation Of Potato

Newcastle disease (ND) , also called Asian fowl plague or pseud-fowl plague, is an avian acute, highly contacted, contagious disease caused by Newcastle disease virus(NDV).The disease currently has a worldwide distribution and causes tremendous loss to poultry industry. It was defined as a list A disease by the Office International des Epizooties (OIE) .To date, vaccation against NDV is a common practice worldwide excluded some developed countries. The development of the traditional conventional vaccine is based on training a large number of pathogenic microorganisms. It not only brings the vaccine manufacturing limitations, but also the insecurity of potential factors. Such as inactivation of pathogenic microorganisms is not complete, leading to the spread of highly virulent; virulence might return stronger and so on. However, using plant bioreactors to produce vaccine shows several important advantages as a safe, cost-effective and large-scale production system in comparison with traditional ones. So transgenic plant vaccine has the important significance and great practical value.Referred to the reported sequence, one pair of nucleotide primers were designed and synthesized. F gene of NDV HBW-1 strain was amplified by RT-PCR. 1700bp DNA fragment was amplified and cloned into pMD19-T simple vector respectively. And then transformed into E.coli TOP 10. The specific recombinant plasmid was identified by molecular weight, PCR and restriction endonuclease analysis. The results indicated that the resultant constructs contained the gene of interest F at right orientation of the insert. The nucleotide sequence of the F gene of HBW-1 was sequenced and amino acid sequence was inferred and compared. The result showed the homology of nucleotide sequence and amino acid sequence of F genes with those (eg. B1, BP01, LaSota, JS-2, CH2000 et al.) of reported strains were 83.3%-99.1% and 87.9%-99.1%.In this paper, one plant efficient expression vector was constructed with complete gene F of NDV, so as to express main antigen of NDV. The recombinant expressing plasmid (PBI121-F) was identified by PCR and restriction enzymes analysis. The results indicated that the fragment were correctly inserted into the PBI121 expressing vector. The plasmid vector pBI121 was transformed into Agrobacterium tumefaciens LBA4404 via improved freeze-thaw method. These could afford the basic research for transgenic maize and tomato as bioreactor to produce plant gene engineering vaccines.The F gene was transformed by the Agrobacterium tumefaciens LBA4404 transformants into potato leave discs and stem segments callus. The explants thirty two plantlets were regenerated on selective media with Kanamysin. The presence of the F gene was confirmed preliminarily by PCR in transgenic plants surviving sections. One transformant was obtained in the experiment. It made a good foundation for producing new gene engineering vaccines against NDV using transgenic plants as bioreactors.