Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Expression, Purification, and Identification of rIGF-I & Cloning and Expression of MGF

Author LiangYaLi
Tutor LiuXiaoHui
School Shanxi University
Course Biochemistry and Molecular Biology
Keywords Insulin -like growth factor-I Inclusion bodies Refolding Force growth factor stress
Type Master's thesis
Year 2006
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IGF - Ⅰ (insulin-like growth factor-Ⅰ, IGF-Ⅰ) is a function of a wide range of regulatory factors. Since 1976 Rinderknecth isolated from human blood after, IGF-Ⅰ scholars at home and abroad has been a research focus. Many reports show its various cells such as vascular endothelial cells, nerve cells, vascular smooth muscle cells proliferation, the differentiation promoting effect, IGF-Ⅰ also has the effect of improving insulin sensitivity and glucose of diabetic microvascular complications, peripheral neuritis and other diseases also have a therapeutic effect. As a natural source of IGF-Ⅰ very limited blood IGF-Ⅰ content of less than 1mg/100L, and mainly in the form of non-active compounds, so relying on the natural purification of blood IGF-Ⅰ unable to meet the basic research and clinical applications. In genetic engineering, cell engineering, fermentation engineering and enzyme engineering as the mainstay of the rapid development of modern biotechnology research and development and mass production of genetically engineered drugs provides a possible way, so the use of biotechnology to explore expression of recombinant IGF-Ⅰ much attention. The experiment was constructed successfully engineered strain BL21 (DE3)-pExSecI-IGF-Ⅰ on the basis of further rIGF-Ⅰ expression conditions and refolding conditions were optimized. Experimental results show that in 37 ℃, IPTG at a final concentration 0.8mol / L 3 hours under the conditions of induction was highly expressed, the product form of inclusion body, inclusion bodies after denaturation and refolding use Sephcryl S100 gel purification systems, isolated purity greater than 90% rIGF-Ⅰ, Western-Blot analysis having immunological activity, MTT assay showed that it has to promote the in vitro cultured mouse NIH3T3 fibroblast proliferation of biological activity. It was also reported that in recent years, the discovery of a skeletal muscle IGF-Ⅰ heterogeneous physical stress growth factor (mechano growth factor MGF), it is in force induced stimulation of insulin-like growth factor - Ⅰ gene exon generated by alternative splicing of occurrence in normal physiological conditions are less common, but skeletal muscle or myocardial mechanical load increase in the state increased expression through autocrine or paracrine function, is considered to have involved in muscle the process of repair after injury. MGF muscle atrophy therapy, exercise rehabilitation training, muscle, nerve repair, treatment of motor neuron diseases, improve the prognosis of myocardial infarction and stroke, etc. have broad application prospects. MGF in structure than IGF-Ⅰ increase in the C-terminal 40 amino acids, so this experiment on the basis of the IGF-extending through the three-step PCR cloned MGF gene of E. coli-based MGF constructed prokaryotic expression vector, and complete Expression and Identification of the preliminary work. Keywords: Insulin-like growth factor - Ⅰ: inclusion: Repeatability: force growth factor stress

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