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Studies On Preparation And Bioactivity Analysis Of Recombinant HFGF-2 In Pichia Pastoris

Posted on:2011-11-11Degree:MasterType:Thesis
Country:ChinaCandidate:Y LiFull Text:PDF
GTID:2120360305954534Subject:Biomedical engineering
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Basic fibroblast growth factor (basic fibroblast growth factor FGF-2) was isolated from bovine pituitary and brain tissues by the Gospodarwiz in 1974. which is a representative member of FGF family without glycosylation sites . pI9.6. It has a strong affinity with the heparin for a single-chain cationic peptides. It is widespread in various organizations, a bioactive peptide, sensitive for acid and heat, as it can stimulate 3T3 fibroblast multiplication. There are four forms of FGF-2 with AUG as the start codon of the relative molecular mass of 18000 and with CUG as the start codon of the relative molecular mass of 22000,22500 and 24000.Relative molecular mass of 18000 is its main activity form.In this study we will investigate FGF-2's main activity form. Including large-scale fermentation,purification and biochemical analysis for industrialization.It is a broad-spectrum mitogens, also the morphogenesis and differentiation-inducing factor, showing significant chemotactic effect on mesoblast and neuroectodermal cells . Its target cells are fibroblasts, vascular endothelial cells, cartilage cells and bone cells, and so on. Now it proved vascular endothelial cell growth factor, can promote wound healing and tissue repair, certain cell regeneration. It plays an important role in embryonic growth, development and physiological processes.This study made use of laboratory synthesis of FGF-2 gene cloned into the expression vector pPICZαC gained the pPICZαC-FGF-2 expression plasmid. The pPICZαC-FGF-2 expression plasmid was transformed into Pichia pastoris X-33, establishing both the advantage of prokaryotic and eukaryotic systems of rhFGF-2 Pichia yeast,identifying high expression rhFGF-2 yeast strains by PCR and other technique. After study on optimal conditions for fermentation, ferment in 80 L fermentor, a large-scale expression, discuss downstream purification. The results showed that we got rhFGF-2 in Pichia yeast, secretory expression rhFGF-2 can be induced by methanol, the expression had a cumulative effect, the output increased with the prolonged time, reached the fastigium after 48 h. This study provides experimental data for further using Pichia pastoris as a large-scale production bioreactor for rhFGF-2 as well as preliminary study on the biological activity of FGF-2. This study carried out in the following aspects:1 The expression of rhFGF-2 in Pichia pastorisThe results showed that the rhFGF-2 in Pichia pastoris expression system can secrete rhFGF-2 by methanol induced, expression on 1st, peaked on 3rd, the 4th begin to decline. After SDS-PAGE., can see the stained bands in the corresponding molecular weight (18kD). 2 Optimization the Expression of rhFGF-2 in Pichia yeastPichia yeast can be well upgrowth under the conditions of pH3.0~ pH 7.0, it is facility to optimize fermentation conditions. High expression of the selected strains were inoculated into pH3.0, pH3.5, pH4.0, pH4.5, pH5.0, pH5.5, pH6.0, pH6.5, pH7.0 medium to ferment, expression product was detected by SDS-PAGE.The results showed that, pH6.0 is the optimization condition.3 Preparation rhFGF-2 in Pichia yeastPichia pastoris is a single-cell eukaryotes, its advantage is quite suitable for large-scale fermentation to product heterologous proteins. this study explored the expression rhFGF-2 in 80 L fermentor for large-scale fermentation technology. Focus on the greater impact of fermentation pH, fermentation medium composition, dissolved oxygen degrees (DO value), methanol added acceleration, fermentation time and other factors. The results showed that: containing 0.5% peptone FM21 medium, pH 6.0 or so, the cell density reached about 180g·L-1 begins to induce the expression; the more suitable conditions for fermentation is that the final methanol flow acceleration is 0.08 ml·s-1, DO value is between 20% to30%. After the fermentation supernatant were isolated by cation-exchange purification,the high purity rhFGF-2 was obtained.4 analysis the biological activity of rhFGF-2After SDS-PAGE, ELISA, western blot, MTT test analysis and other biological activity for purified rhFGF-2. The results showed that the expression of a large-scale fermentation of recombinant protein electrophoresis rhFGF-2 can see the stained bands in the corresponding molecular weight (18 kD), ELISA tests, has the color reaction contenting 2.23μg·mL-1, western blot detection with the standard protein bands in the same location.To sum up, this experiment design a high-secreting expression of rhFGF-2 in Pichia yeast, establish the large-scale fermentation and purification methods for rhFGF-2,and provide the data of biological activity of rhFGF-2.
Keywords/Search Tags:FGF-2, large-scale fermentation of Pichia pastoris, biological activity
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