Under The Application Of Fibroblast Growth Factor 9(FGF9) Using Plant Oil Body System

The research and development of medical science and molecular biology are mutually dependent and rich. With the mutual penetration of molecular biology and pharmacy in various fields, medical science has reached the molecular level. At present, the use of transgenic animals or transgenic plants to produce pharmaceutical proteins is a major application of molecular biology in medicine. Plant oil body system for expressing heterologous proteins has been a hotspot in the bioreactor research, it not only has the advantages of low production cost,convenient transportation and storage stability, but also simplifies the process of the protein separation and purification, and does not contain pathogenic organisms that damage human health, campared to other expression systems such as animals’ and micro-organisms’. These advantages provide favorable conditions for the large-scale production of heterologous proteins.Human fibroblast growth factor 9(FGF9) is a member of fibroblast growth factors superfamily,which contains 22 members in human. And it encodes a protein of 207 amino acids weighing approximately 23 k Da. FGF9 is present in various human body tissues where it effectively promotes mitosis and cell growth. FGF9 on the diagnosis of the clinical research of ovarian cancer, cartilage disorders, promoting hair growth and gonadal differentiation has been more and more concerned by people. In view of the wide and important application value of FGF9 in clinic and the low production cost, the use of plants(Arabidopsis, safflower) to specific expression of recombinant FGF9 protein should meet the high demand market of FGF9.The main research results are as follows:1. Construction of rh FGF9 expression vectors: we constructed an rh FGF9 gene expression cassette for insertion on p OTB. The new recombinant plasmid, named p OTB-rh FGF9, was transformed into Agrobacterium EHA105.2. The p OTB-rh FGF9 plasmid was transformed into A. thaliana via the floral-dip method.The Flora-Dip method infected the Arabidopsis inflorescence and used the basta for screening in order to obtain transgenic Arabidopsis strains T1, T2 and T3 generation. And then using RT-PCR for further validating and screening the T3 generation lines to obtain the Arabidopsis positive plant seeds that were used for detection at the molecular and protein levels:(1) As a result SDS-PAGE and Western blot indicated that expression of target protein oleosin-rh FGF9 in Arabidopsis seeds.(2) MTT activity test results demonstrated that the oil bodies expressed oleosin-rh FGF9 from the transgenic Arabidopsis thaliana had a remarkable proliferation effect on NIH/3T3 cells.3. The transgene safflowers were obtained by Agrobacterium-mediated transformation.Transgenic seedlings in screening medium containing Basta have obtained. The survival of seedlings through the grafting technique. PCR detection for the transgenic safflower at the molecular level: PCR amplification results indicated that the target gene rh FGF9 had been successfully integrated into the safflower genome.It was the first time that transgenic Arabidopsis and safflower were used in an oleosin-fusion expression system for specific expression of the recombinant human fibroblast growth factor 9(rh FGF9). And the recombinant protein exhibited biological activity through the cell experiments. This result laid the foundation for the expression of exogenous protein in the plant oil body expression system and the feasibility of a large scale production of rh FGF9.