Cloning, Expression, Purification And Identification Activity Of Human KGF-2Gene

Skin is one of the most important immunity organs of human body. The repair ofthe damage of skin is a complicated process and it’s introduced by several growthfactors and cell factors. Human fibroblast growth factor family (FGFs) are the majorfactor of those ones who participate in growing of the connective tissues and thehealing of skin. Human Keratinocyte growth factor-2(KGF-2) is one member of theFGFs.KGF-2plays a very important role in the healing process of tissue organ damagecaused by the acute and chronic inflammation, ulcer and physical and chemical burn,and KGF-2is irreplaceable in the embryonic development process among the highervertebrates. It participates in and regulates the forming and differentiation of multipletissues and organs. KGF-2has great prospect and it can cure many diseases relevantwith epidermis harm in clinical ways.The main content of this article is successfully building a highly expressedrecombinant human KGF-2vector, and through preliminary optimization offermentation conditions, we get a stable and high expression E.coli strain. With thedevelopment and optimization of the purification process, we establish a stable andsimplified process which can get a high purified target protein and with greatapplication value. The purified protein has a certain biologic activity after activitydetection and thus it lays a fundamental base for future research use. The following isthe main result of this study:1. With the optimization of the codon, successfully build a pET-30a(+)-hKGF2recombinant expression vector, and get a high-performance and solubly expressed KGF-2recombinant protein with the optimization of expression conditions.2. Optimization of the major process parameter of the primary fermentationconditions with confirming the medium gradients and induction temperature andinduction time and induction pH, and confirm the high-density fermentation conditionin this base.3. Establish the purification process suitable for scaling up, and confirm a simpleand easy purification process for future scaling up. Get high purity target protein withhigh pressure homogeneous, Heparin affinity chromatography, ultrafiltration desaltingand DEAE anion chromatography method.4. rhKGF-2has a very significant role in promoting growth on culturedkeratinocytes, and it may also promote the growth and differentiation oftissue-engineered skin’s epidermal keratinocytes in3D gas-liquid culture system.