Expression Of Fusion Protein Of Newcasstle Disease Virus In Transgenic Rice

In the past decade, due to the rapid progress of pharmaceutical biotechnology and plant genetic engineering, it has became attractive in the biomedical research to use transgenic plants as bioreactors to produce high-value medical peptides and proteins. It is well-known that Newcastle Disease Virus (NDV) could cause Newcastle Disease, one of the hastiest and highest destructive diseases for chicken, and its fusion protein serves as the key factor both for virulence and immunity. In present study, we attempted to construct transgenic rice as a novel bioreactor to express the fusion protein of NDV, and to evaluate the immunogenicity of the expressed product in rice.Three expression cassettes which containing the fusion protein gene of Newcasstlc Disease Virus (NDV F) under the control of maize ubiquitin (Ubi) promoter or rice Gtl promoter, named pGNDV, pGNDV-1 and pUNDV, respectively. In which, the hygromycin phosphotranferase (HPT) and beta-glucuronidase (GUS) genes were used as the selectable marker and report gene, respectively. Morevoer, in order to enhance the expression of NDV F gene in the seed of transgenic rice plants, the coding sequence of rice glutelin signal peptide was fused between Gtl promoter and NDV F coding region, resulted in two constructs pGSNDV and pGSNDV-1. These binary vectors were transformed into Agrobacteriun tumeficiens EHA105 by freeze-melt method, which was confirmed by both PCR analysis and restriction enzyme digestion of plasmid from recombinant Agrobacterium,The primary callus derived from mature embryos of rice (Oryzy saliva L.) cultivar Guanglinxiangjin was used for optimization of Agrobacterium-mediated transformation of rice, and finally three different expression cassettes containing the NDV F gene were introduced into rice, and 24 independent transgenic rice lines were regenerated. The results from hygromycin resistance test and PCR analysis indicated that the T-DNAregion containing the NDV F chimeric gene had been integrated into the genome of transgenic rice plants. Moreover, our results also showed that there was a significant influence of the induction period of primary callus on the frequency of Agrobaclerium-media6ed transformation of rice, the frequency of transformation was higher when using calli 12 days after induction than others.During the stage of 15 days after pollination, leaf and immature seeds were collected from transgenic plants and their parent lines and the total RNAs were exacted for RT-PCR analysis. The results showed that the transcription of NDV F gene could be detected in both leaves and immature seeds of two transgenic rice plants in which the NDV F gene was under the control of Ubi promoter, while in the case of Gtl promoter, NDV F gene transcription could only be detected in immature seeds of five transgenic plants. ELISA analysis was carried out by using murine-specific NDV polyclonal antibodies to detect NDV F product in total salt soluble proteins from both leaves and mature seeds of all transgenic rice plants. The results showed that there was an obviously positive reaction in the case of leaf from transgenic plants Y22 and Y23 and of seeds from transgenic plantsY2, Y5, Y12 andY22. The expression of NDV F protein in these transgenic plants was further confirmed by Western blot.The total salt soluble proteins were extacted from leaf of transgenic plant Y22 and mature seeds of transgenic plant Y5, respectively, and used for immunization testing in mice. Six weeks old BALB/c mice were immunized intraperitoneally with 400|ig Y22 leaf proteins or Y5 seed proteins in complete Freund's adjuvant for the first inoculation and then in incomplete adjuvant for the second time. The mice were bled 15 days after the second immunization, and the titration of NDV-specific antibodies was detected by indirect ELISA in sera from mice group injected transgenic events. The results showed that titration of antibody could reach 1: 139. 3+35. 78 in mice immunized with Y22 leaf protein and 1: 105.6+43.82 in those with Y5 seed protein, while it was only 1-. 26. 39...