| Global outbreak of bird flu not only leads to economic loss, but also threat human health seriously in many countries. Expression of the avian influenza virus antigen protein by plant bioreactor, to develop an edible, cheap and safe avian influenza subunit vaccine, as a new idea, will be of great inportance in fighting against the pandemic spread of bird flu. This study constructed two high effective plant expression vector, then the hemagglutinin gene of avian flu virus was tranfered into the legume plant Lotus corniculatus"Leo"by Agrobacterium tumefaciens, obtained 30 positive transgenic Lotus corniculatus plants, after RT-PCR and Western Blot assay, it is proved that the avian influenza hemagglutinin gene was successfully expressed in Lotus corniculatus, then screened, enlarged, transplanted the transgenic Lotus corniculatus which contain higher level of the hemagglutinin protein. Major results obtained are as follows:1. By genetic recombination technology, the artificially modified gene of avian influenza hemagglutinin MHA and nuclear matrix attachment region MAR RB7 were amplified by PCR, based on the vector pBI121, we constructed two high effective plant expression vectors: pMHA (plant expression vector pBI121, CaMV 35S promoter, MHA gene, NOS terminator); pMARs-MHA (plant expression vector pBI121, nuclear matrix attachment region MAR RB7, CaMV 35S promoter, MHA gene , NOS terminator).2. The two vectors pMHA and pMARs-MHA were transformed into Agrobacterium tumefaciens LBA4404, then infected Lotus corniculatus "Leo" cotyledon and hypocotyls. By Kanamycin resistance screening and PCR identification, we obtained 10 strains of positive transgenic Lotus corniculatus which were infected by vector pMHA, and 20 strains of positive transgenic Lotus corniculatus which were infected by vector pMARs-MHA. Further testing Southern-blot were carried out, DNA samples were fixed onto the solid phase carrier, a homologous sequence show a strong hybridization signal by the combination with the specific labeled probe, proving that the avian influenza hemagglutinin gene has been successfully imported into the genome of Lotus corniculatus, and exsisting in single copy and double copy.3. The RNA of transgenic Lotus corniculatus were extracted by Trizol, obvious bands were shown on the 1700 bp site after RT-PCR amplification, proving that the avian influenza hemagglutinin gene in Lotus corniculatus could be transcripted into RNA. Then extracted transgenic Lotus corniculatus total protein, the proteins in the SDS-PAGE gel were transferred onto film by vacuum transfer, with a diluted mouse anti-H5N1-HA polyclonal antibody and secondary antibodies diluted goat anti-mouse-HRP 1:10000, 66 KD area showed a strong hybridization signal on the film as we expected, confirming that the avian influenza hemagglutinin protein were successfully expressed in Lotus corniculatus. By Elisa detection, five transgenic Lotus corniculatus which contain higher amout of avian influenza hemagglutinin protein were screened out. 4. The transgenic Lotus corniculatus stems were cut into one centimeter bits, then transfered to the cutting medium, 25℃light training until the seeding. After harden, the small plants were transplanted to the nutrient soil, in good control of the substrate, humidity, temperature and light conditions, the transgenic Lotus corniculatus plants were robust, providing sufficient germplasm materials for new strain breeding.5. By genetic recombination technology, based on the vector pET-30a, we constructed the prokaryotic expression vector pET-MHA. The vector was transformed into E. coli BL21 and induced with IPTG for 4 h at 37℃. After ultrasonic vibration, centrifugation, SDS-PAGE electrophoresis, 66 KD area showed a stick band on the gel as we expected, while the empty vector and strain without induction did not show obvious band, confirming that the avian influenza hemagglutinin protein were successfully expressed in E. coli, layed the foundation for the standard antigen and antibodies in the future.
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