Transient Expression Human Keratinocyte Growth Factor-1 In Tobacco

Human keratinocyte growth factor-1(rhKGF1) is a important member of the FGF's family,it could promote proliferation of epithelial cell in various tissue. So the KGF1 possess the effection of wound repair of epithelial cell in many position include skin, cornea, bladder, kidney, lung and bowel, and the KGF-1 also has the important significance in ontogenesis and tumor healing. The traditional method of obtain the rhKGF-1 is that extract the rhKGF-1 from animal tissue and prokaryotic express it in Ecoli. But the former method is too expensive and lower production, the latter method is not stable express in Ecoli. and it largely express in inclusion body which the inclusion body denaturation and renaturation could increase the procedure of protein expression and after renaturation the activity of protein became lower. This research use the reconstructive potato virus X (pGR107) as the vector to transient express the rhKGF-1 in tobacco (Nicotiana benthamiana), to seek aconvenient way of low-cost and mass production of rhKGF1.The pGR107 is a binary expression potato virus X vector, it could use the T-DNA sequence in Ti plasmid of Agrobacterium tumefaciens to put the virus gene in injured plant cells, then the virus gene systemic proliferate in the whole plant. This research is to synthesize rhKGF1 gene sequence using pET3c-hKGF1 as a template in PCR method, inserted it into the binary expression potato virus X (pGR107) vector. through the way of freezing and thawing transformed it into the competent Agrobacterium transformed GV3101, then the obtained GV3101-pGR107-rhKGF1 infected tobacco. Green fluorescent protein (smGFP) as a reporter gene is used to establish the potato virus X (PVX) transient expression system. Through UV lamp (UV Products, model B 100AP,365nm) observe Tobacco infected by GV3101-pGR107-smGFP for 20 days to determine the time of reaching the maximum of exogenous protein accumulation. Law, then use the way of SDS-PAGE Western blotting to determine whether rhKGF1 is successfully expressed in tobacco.The results show that We have successfully constructed pGR107-rhKGF1 binary vector and transformed it into competent Agrobacterium tumefaciens GV3101, the obtained GV3101-pGR107-rhKGF1 infected tobacco, by ultraviolet light after observing it for 20 days and determine 7-9 days for maximum protein expression. SDS-PAGE electrophoresis and westernblot assay indicated that the target protein of rhKGF1 have successfully expressed in tobacco. Finally we successfully found a convenient way of low-cost and mass production of rhKGF1.