Expression Of S1 Gene Of Infectious Bronchitis Virus In Transgenic Potatoes

Infectious bronchitis (IB) caused by infectious bronchitis virus (IBV) is an acute and highly contagious disease in chickens. The control of IB depended on completely the vaccination with the attenuated IB vaccine. But IB attenuated vaccine has many defects,such as the possibility of contamination of foreign and vertical transmissible pathogens,and the virulent recovery of vaccine virus. Therefore,exploring the safe and efficient new vaccines has become the hotspot in the research of IB control. In 1992,Mason presented the idea of producing vaccine with transgenic plant in Science,which brought about the new hope in IB control. To explore the feasibility of IB edible vaccine,we have transformed SI gene of IBV into potato and researched the immunogenicity of it's expression product. The findings are as follows:1. A pair of primers for IBV SI gene were designed and synthesized according to the published nucleotide sequence of IBV SI gene and multiclone sites of expression vector pBI121. IBV SI gene was amplified with recombinant pBS plasmid containing SI gene of IBV as template by PCR. Then,the SI gene was inserted into pBI121 under the control of 35S promoter. The recombinant vector pBI121 containing SI gene of IBV was confirmed by PCR and double digestion of restriction endonuclease. Agrobacterium fumefaciens EHA105 with the recombinant vector pBI121 was obtained by tri-parental mating method.2. The potato transformation system that was mediated by Agrobacterium fumefaciens EHAlOS.was established,The optimal callus-forming mediums and callus-differentiating medium were selected and found for potato transformation system. The rates of calli and shoot differentiation were respectively 100% and more than 90% for transgenic potato with SI gene of infectious bronchitis virus.3. Potato stems and Agrobacterium fumefaciens containing recombinant vector were co-cultured at 28Cfor 48 hours and transplanted onto callus-inducing medium at 24C for 7 days. And then,the explants were transplanted onto differentiation medium and cultured at 24C for 21 days. Resistant buds rooted and grew into plants in medium with kanamycin for 20 days,and 83 plants were obtained. The regenerated resistant plants were identified by molecular biological methods. 90% of them were positive by PCR. Southern blot analysis showed that IBV SI gene had been integrated into genomic DNA of potato and most transgenic plants have two copies of IBV SI gene. Northern blot analysis indicated that most transgenic plants could transcribe normally SI gene of IBV though the level of transcription was different in the different tansgenic plants. But gene silencing was also found in onetransgenic plant.4. Transgenic plants produced microtubers in inducing liquid medium in dark. After a week,the microtubers can be observed. And a month later,a lot of microtubers were obtained with weight ranging from 0.2g to 1 .Og. Meanwhile,some transgenic plantlets and microtubers were transplanted into fields for reproduction and they survived. Immunity assay with BALB/C mice showed that expression product of transgenic potatoes with SI gene of IBV had immunogenicity,and ELISA antibody liter reached respectively 1:20 to 1:40 and 1:80 to 1:160 with doses of 0.5g and Ig.