Expression Of Keratinocyte Growth Factor-1 In Transgenic Tobacco And Safflower

Keratinocyte growth factor (KGFl) is a member of the fibroblast growth factor family, also described as FGF7. KGF1 is produced by cells of mesenchymal origin and secreted via the way of paracrine secretion. It can bind to epithelial cell-specific receptors and plays important roles in wound repair, embryonic development, tumor generation and immunologic reconstitution. KGF1 promotes cell proliferation and highly expressed during wound repair. KGF1 also can promote epidermal cell proliferation, migration, differentiation. Particularly, it facilitates the proliferation and differentiation of skin hair follicle and sebaceous gland precursor cells. Previously, we expressed KGF1 in E. coli and found that the expression level was low and mainly formatted into inclusion body, which results in difficulties in KGF1 purification, and limits the usefulness of KGF1 in basic and applied pharmacology and pathology researches. In this study we used a tobacco viral expression system to improve the expression efficiency and activity. It has the favorable characteristic of a short cycle, high relative content of protein, and protein post-translational modification and so on.In this study, we used tobacco (Nicotiana benthamiana) and safflower (Carthamus tinctorius L.) plants to express human recombinant KGF1 by the potato virus X (PVX)-based vector (pGR107) and oil body expression vector pCAMBIA1390Do (p1390Do) via agrobacterium-mediated transformation. In order to increase the expression of KGF1,the KGF1 gene sequence optimizated according to insects preferred codon usage patterns, taking into account of the structure of mRNA and enhancement the stability. Respectively, it was a low-cost, convenient, safe, and exact way.The plant expression vectors pgR.107-KGF1-smGFP and p1390Do-KGF1 was obtained by cloning KGF1 gene into the pgR107-smGFP and p1390Do vector, respectively. The pgR107-KGF1-smGFP and pgR107-smGFP plasmids were transferred into agrobacterium tumefaciens strain GV3101 using a freeze-thaw method. Then, the agrobacterium strains were infiltrated onto the underside of tobacco leaves. After p1390Do-KGF1 was transferred into A. tumefaciens strain EHA105, then KGF1 gene was introduced into safflower plants via agrobacterium-mediated leaf disc transformation. The transgenic tobacco and safflower plants were determind by PCR, RT-PCR and Western blot. Using the heparin affinity to purify the KGF1 protein expressed by tobacoo through which we can do the cell viability and animal wound healing experiment.The main reason is as follows:1. This research has established successfully in transient expression vector of tobacoo pgR107-KGF1-smGFP and oil body expression vector of Carthamus tinctorius p1390Do-KGF1. KGF1 protein is highly expressed in tobacoo and its expression quantity reaches 530ug fusion protein in each fresh tobacoo’s grass with the expression product having cell biological activity.2. KGF1 protein expressed in tobacoo has wound healing activity whose healing rate is apparently higher than the negative, while no significanct difference compared with the positive. Ensured by Masson staining results, KGF1 protein expressed in tobacoo is consistent with the positive in the effect of healing wound tissue.3. Transfer the KGF1 gene into Safflower genome using agrobacterium-mediated method, and then acquire 57 transgenic safflowers by the selection of hygromycin B. After PCR amplification test, there are 11 safflowers having the target gene (589bp band),19.29% conversations. We can make sure that KGF1 gene has already been integrated into the genome of safflowers.The research expressed KGF1 protein successfully by using the tobacoo transient expression system, carrying out the cell and animal experiment and ensure the biological activity of KGF1 protein. Also, the research insert the KGF1 gene into transgenic safflowers successfully which lay the foundation of producting KGF1 by using the plant expression system.