Efficient Expression Of Broad Spectrum H5N1 Antigen Protein In Lotus Corniculatus L.

Lotus corniculatus L., also named five-leaf grass and desert alfalfa, is a legume trefoil herbaceous perennial widely distributed in the world. Because of its rich nutrition and good quality, strong adaptability and other characteristics, it’s widely cultivated in pasture and garden ornamentals. It’s an excellent model plant for gene transformation to legumes. It is also an ideal material for the production of transgenic plants Vaccine. In this research, we use L. corniculatus L. as the host plant to produce plant vaccines. An efficient Agrobacterium-mediated genetic transformation system had been established and optimized in our laboratory. In this paper, two plants fusion expression vectors harboring HA genes with broad-spectrum resistance against all the highly pathogenic H5N1 avian influenza subtypes, were constructed and used for L. Corniculatus transformation. Major results were as follows:1. Plant expression vector p3HA-LTB was constructed to harbor the 3 full-length HA genes from HK97, HK07 and SX06 strains of H5N1 highly pathogenic avian influenza, respectively. Plant expression vector pHA-2HA1-LTB was constructed to harbor the full-length HA gene from strain HK97,2 HA1 genes from strains HK07 and SX06, respectively. In both of the above vectors, LTB and PMI were fused in as the adjuvant and selective genes, respectively.2. The two plant fusion expression vectors p3HA-LTB and pHA-2HA1-LTB were transfered into agrobacterium strain LBA4404. The agrobactrium thus obtained was used to infect the petiole cotyledon explants of L. corniculatus according to the protocol established in our laboratory. A total of 17 and 64 D-mannose resistant transgenic plants were obtained from of p3HA-LTB and pHA-2HA1-LTB, respectively. Positive transgenic plants were further selected out by of PCR and RT-PCR analysis of the transgenic plants.3. Five positive genetically modified plants were selected randomly from the positive lines selected out above, and used for further detection in protein level using SDS-PAGE electrophoresis and ELISA methods. The results showed that the target genes were expressed properly4. The transgenic Lotus plants with high levels of the target genes expression were further transferred to wood chips for rooting. The well-grown plants and transplanted to the nutritive soil with nursery vermiculite and matrix by 1:1 ratio, and then transplanted to the field. Seeds of the transgenic plants were collected and used for the further analysis.