| Infectious bronchitis (IB) caused by infectious bronchitis virus (IBV) is an acute and highly contagious viral disease of chickens. The commercial modified live and killed vaccines against the virulent IBV were available. But these conventional vaccines have many defects, such as lacking cross immunity between different serotyes of IBVs, the possibility of contamination of foreign and vertical transmissible pathogens, and the virulent recovery of vaccine virus. Therefore, it is necessary to develop safe, efficient, economic IB vaccine.The expression of antigens in transgenic plants has been increasingly used as an alternative to the classical methodologies for antigen expression in the development of vaccines. Several viral and bacterial antigens have been expressed and produced in transgenic plants. Antigens produced in transgenic plants are capable of invoking protective immune responses against important pathogens as it has already been demonstrated. Based on the analysis of genetic variantion of SI gene of IBV, the plant expression vector was constructed by fusing S gene of IBV-H52 with pBI121. Potato plants were genetically transformed with cDNA construct encoding the full-length glycoprotein S of IBV under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter by the Agrobacterium system. Transgenic potatoes screened on (he media containing kanamycin and carbenicillin were analyzed by PCR and Southern, Northern and Western blot.The main results presented in this thesis are as follows:1. The specific primers were designed and synthesized according to the published sequence of SI gene of IBV-Beaudette strain. cDNA of SI gene were amplified by reverse transcription polymerase chain reaction (RT-PCR) from viral RNAs of IBV-SC021202 isolate, which was extracted with Trizol reagent. The PCR products about 1.9kb in length were cloned into pGEM-Teasy vector for sequencing. Sequencing results showed that SI gene of the nephropathogenic isolates IBV-SC021202 was 1855bp in length and encode a protein of 618 amino acids which consist a signal peptide (1-18aa) that were to be cut of in eukaryotic cell and SI subunitof600aa(19-618aa).The nucleotide and deduced amino acid sequences of the SI gene of IBV Sc021202 isolate were compared with those of the other 31 IBV strains from GenBank with such softwares as DNAstar, OMIGA.The result showed that there are HVR and relatively conserved region in it. Insertion and deletion were both found in it and the mutation at 281,285 and 465 aa were unique. The SI gene of IBV SC021202 isolate showed high homology with that of nephropathogenic IBV X and J strains (94.4% and 86.2% in nucletide sequence and 94.5% and 87.7% in amino acid sequence), low homology with that of nephropathogenic IBV Holte strain and Gray strain (75.1% and 72.5% in nucletide sequence and 73.8% and 73.1% in amino acid sequence), and relatively constant value of homology with the other IBV strains (72.6% and80.8% in nucletide sequence and 73.1% and 81.8% in amino acid sequence).2. The ORF of S gene of IBV H52 strain, encoding a peptide composed of 1162 amino acid residues, was cloned into the plant expression vector BI121 under the control of 35S promoter. PCR and restriction analyses confirmed that the recombinanl plasmids containing the target gene were obtained. Recombinant plasmids were transferred into Agrobacterium tumefaciens EHA105 by tri-parenlal mating method. PCR and restriction detection also confirmed that the target gene was transferred into Agrobaclerium tumefaciens.3. Different potato plants were genetically transformed with cDNA construct encoding the full-length glycoprotein S of IBV, responsible for the neutralizing antibody induction against the virus, under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter by the Agrobacterium tumefaciens mediated system. The plantlets grown on media containing kanamycin and carbenicillin were analyzed by PCR and found that IBV-S gene could be amplified from 48 of 51 (94%)...
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