Expression And Characteristic Analysis Of Synthetic Human Epidermal Growth Factor And Fungal Immunomodulate Protein In Transgenic Tobacco Plant

The genetic engineer will be necessary in exploitation of new products to retain objective (especially in the real curatorial proteins) proteins in the future. Tobacco is a model plant in transgenic research, and there were so many genes expressed successfully in transgenic tobacco plants including genes from plant, microorganism and animal. In this paper, tobacco was used as host plant, and the foreign gene was transformed into tobacco using agrobacterium-mediated procedure to get high expressed foreign protein.In the research of hEGF, to improve the expression level of foreign protein, the gene was modified as following: all the gene was synthesized using the plant basis base;and the KDEL sequence was synthesized in the C terminal of the protein;the expression frame was composed of duplicated 35S promoter and Ω enhancer element;beside the expression frame, the MARs sequence was used to get steady transform and expression of this gene. EL1SA assays showed that the accumulation level of recombinant hEGF in plants was elevated at great magnitude and the hEGF content in transgenic tobacco was up to 0.29% of the total soluble protein. To confirm whether this recombinant hEGF has the biological function, Vero E6 cell elongation assay and cell proliferation by MTT experiment were conducted and the results showed the plant produced hEGF has the same biological activity as the commercial products. All of these data conclude that after modification the accumulation level of recombinant hEGF in transgenic plant can be highly improved. On the other hand, the expression vectors were constructed, which was composed of special promoter in tomoto fruit and the hEGF gene. This is the basic study for the research of odible vaccine.Fungal immunomodulate protein was found in the 80s of the last centry, which was from the edible fungi Ling-Zhi, and played an impotant role in immunomodulation of Ling-Zhi. In this paper, a new FIP gene was cloned from Garnodum japanicum, which was 335bp, and composed with 111 amino. The gene shared 86% homologous with others, which was named as FIP-g/c and submitted to NCBI, the accession number was AY987805. The gene was expressed in E. coli BL21, and the foreign protein accumulation reached 36.46mg/L. After purified with Ni-NAT, the protein was used to immunity rabbit.At the same time, LZ-8 was cloned from Garnodum lucidum, which was the same in base composition with the existent sequences. The FlP-g/a and LZ-8 was constructed into plant expression vectors respectively to be transformed into tobacco plant using agrobacterium-medlaled procedure. EL1SA assays showed that the accumulation level of recombinant FIPs (LZ-8 and FlP-gja) in plants was up to 0.18% and 0.31% of the total soluble protein. The biological activity in vitro was performed using Vero E6 cell line. And a 600bp TNF-ot gene was used as probe to blot with the RNA from Vero E6 cell supplemented with recombinant protein from transgenic tobacco plants. The result showed obviously improvment of mRNA level of TNF-a gene, which illustrated that the FIPs from transgenic plant shared the same biological activity with the crude one. In this study, the FIPs were successfully expressed in transgenic tobacco plant firstly, which was a new way to use this gene.