Expression Of Keratinocyte Growth Factor-1 (KGF-1) In Transgenic Safflower

Plant could produce different biologiclal agents in the tuppenny costing as a new bioreactor, for the past few years, the application of transgenic plant in the production of vaccines, antibodies, opypeptide, enzymes and other pharmaceutical proteins has become possible both domestic and international gene engineering. The advantage of plant bioreactor in pharmaceutical protein production is that low cost, safe, and easy mass production, etc. Plant bioreactor system possess more development potential than microorganisms or animals bioreactor systems, furthermore, there is no animal ethics problem.Keratinocyte growth factor-1(KGF-1), a member of fibroblast growth factor (FGF), namely FGF-7, was discovered from the culture supernatant of human embryonic lung fibroblast cell by Rubin in 1989. It was found that KGF-1 could promote cell proliferation, migration and differentiation effectively via binding to a specific FGF receptor, and it is closely related to embryonic development, tumor formation and development and immune reconstruction. Currently, KGF-1 protein in commercial is low-yield and expensive. So we need to try different ways to express KGF-1 protein for meeting market demand.Safflower(Carthamus tinctorius L.) is classified the Asteraceae (Compositae) annual herb, also known as Sichuan safflower, thorn safflower, carthami, flos. Safflower is the oil and medical use of crop, Its dry tubular flower is commonly used of acrid, warm in traditional Chinese medicine, and it could promote blood circulation to remove meridian obstruction, removing blood stasis and analgetic. Safflower contain rich safflower yellow and a small amount of red pigment, which are a natural food colorant. Linoleic acid of safflower seed oil is high grade cooking oil and widely used in pharmaceutical and chemical industries.The recombinant plasmid vector p1390R(35S)-KGF-1 was transformed into receptor safflower by Agrobacterium tumefaciens to express KGF-1. By this way, not only safflower regeneration system was optimized but also the exogenous protein expressed. This established the basis for the production of KGF-1, and made the necessary preparation of techniques and theoretics for the other valuable pharmaceutical proteins produced in plant expression system. The results were summarized as follows:l.Gene earn and vector construction:Basis on the plant prefer codon of KGF-1 gene and the pUC19-KGF-1 plasmid for template, KGF-1 gene had been amplified through PCR and cloned in the p1390R vector, we successfully obtained the plant expression vector p1390R-KGF-1 contain KGF-1 gene;2.Preparation of engineering strain LBA4404:The vector p1390R-KGF-1 was introduced into Agrobacterium tumefaciens strain LBA4404 by freezing-melting method, and we successfully obtained engineering strain LBA44404, and positive clones were confirmed using PCR technique.3.Optimization of hormone conditions, hygromycin resistance and antibiotics for transgenic safflower, the best density as follows:hormone 6-BA 1.5mg·L-1and NAA 0.5mg·L-1, hygromycin 6mg·L-1, callus induction differentiation medium contained Cef 100mg·L-1 and Carb 200mg·L-1, regeneration bud lengthen and root medium contained Cef 200mg·L-1 and Carb 100mg·L-4.Genetic transformation of Safflower:The resulting vector was introduced into safflower leaf via A.tumefaciens-mediated transformation. Resistance plants were obtained in selecting media which contained hygromycin 6 mg·L-1.5.Molecular biology assays:KGF-1 has been transformed into safflower cell and chromosome successfully by the PCR, Southern blot, RT-PCR, SDS-PAGE protein electrophoresis and Western blot assay.