Newcastle Disease F Antigen Protein Gene Plant Expression Vector And Transformed Into Potato

Newcastle disease (ND) caused by Newcastle Disease Virus (NDV) is an acute deadly infectious disease in poultry industry , and the most effective measure to control it is to vaccinate .But the use of traditional vaccines is restricted by the disadvantages they inherit such as higher cost , inconvenience to operate , short protection period , the danger that 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.According to the conservative sequences of NDV-F and the cholera toxin B subunit(one of the effective adjuvants in immunity),we designed two group primers(p1, p2) and (p3, p5) , and cloned the CTB and the F gene of NDV (NDVF )gene separately , then spliced two different gene fragments of CTB and NDVF by recombinant PCR into the fusion gene CTBF2 in vitro firstly. Redesigned the other 3' race (p4) containing the leading sequence to ER (endoplasmic reticulum) , and another fusion gene CTBF2 was synthesized with p1 and p4 by recombinant PCR. After sequencing, CTBF1&CTBF2 fusion gene were inserted into eukaryotic expressing plasmids and four efficient plant expression vectors were successfully constructed , which are named pBI121CTBF1, pBI121CTBF2(including CaMV35S promoter), pBI12pCTBFl , pBI12pCTBF2 ( including patatin class I promoter ) ,in which pBI121CTBFl and pBI12pCTBFl have the leading sequence .The two fusion genes CTBF1 and CTBF2 were transformed by the Agrobacterium tumefaciens LBA4404 transformants into potato leave discs and stem segments . The explants 12 plantlets were regenerated on selective media with Kanamysin .The presence of the CTBF1 and CTBF2 gene were confirmed by PCR and PCR-Southern blot in transgenic plants surviving sections . RT-PCR results showed that the fusion genes CTBF1 and CTBF2 have been integrated into the genome of potatoes.