CN102344930A - Simple and convenient chemical industrial technology for acidic fibroblast growth factor (aFGF) - Google Patents
Simple and convenient chemical industrial technology for acidic fibroblast growth factor (aFGF) Download PDFInfo
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- CN102344930A CN102344930A CN2011102743965A CN201110274396A CN102344930A CN 102344930 A CN102344930 A CN 102344930A CN 2011102743965 A CN2011102743965 A CN 2011102743965A CN 201110274396 A CN201110274396 A CN 201110274396A CN 102344930 A CN102344930 A CN 102344930A
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Abstract
The invention relates to a simple and convenient chemical industrial technology for prokaryotic expression and purification of humanized acidic fibroblast growth factor (aFGF), and aims to construct engineering bacteria of recombinant haFGF (Human Acidic Fibroblast Growth Factor) gene by using a recombinant DNA (Deoxyribonucleic Acid) technology, realize expression of a large amount of humanized FGF-1 in prokaryotic cells and obtain a target gene with higher purity through simple purification steps. haFGF is produced by using the biological engineering method, so that the technology has the advantages of easiness in forming scale production capacity, high yield and low cost.
Description
Technical field
The present invention relates to the easy method of the acid fibroblast growth factor (aFGF) in prokaryotic expression and purifying people source.
Background technology
Fibroblast growth factor (Fibroblast Growth Factor is called for short FGF) is a protein families, has had now found that 9 members.Their common characteristic are to combine with heparin, therefore also it are referred to as heparin-bounding somatomedin.Acid fibroblast growth factor (aFGF; Also be called AFGF) be the important member in this family; It is one type to deriving from the polypeptide that mesoderm and neuroectodermal various kinds of cell have nutrition and short division Differentiation, at first by Thomas in 1984 from the ox brain separation and purification obtain.1986, Michael Jaye cloned human acid fibroblast growth factor (haFGF) gene first from human brain, and has measured nucleotide sequence, and the research of aFGF has got into the DNA reorganization stage thus.
Research shows that haFGF has biologic activity widely, is bringing into play important effect at aspects such as injury repairing, vascularization and neural nourishings.Because these biological functions, aFGF is expected to be developed to the polypeptide property medicine of extensive clinical value.Because haFGF is the endogenic trace activity substance of human body, be difficult in body fluid or tissue, extract, thereby yield is not high, economic implications also greatly reduces.
Summary of the invention
The objective of the invention is to adopt recombinant DNA technology to make up the engineering bacteria of reorganization haFGF gene, realize the great expression of fibroblast growth factor-1 in prokaryotic cell prokaryocyte in people source, and the purification step through simplifying, the higher target protein of purity obtained.Produce haFGF with this biological engineering method, easy row becomes scale throughput, and output is big and cost is low.
The present invention is through the method for external pcr amplification, and amplification obtains the aFGF gene from the eDNA of the hepG2 in people source, and is connected with expression vector pET-26b with the EcoRI restriction enzyme site through NdeI.Subsequently plasmid is transformed into e. coli bl21 (DE3) and obtains efficient soluble-expression, obtain the acid fibroblast growth factor albumen of natural structure.
Description of drawings:
Fig. 1 be the AFGF abduction delivering per hour take a sample the SDS-PAGE electrophoretic analysis as a result figure (M is marker; 1 for inducing preceding sampling; 2 for inducing the back sampling).
Fig. 2 is AFGF ultrasonic cell disintegration SDS-PAGE electrophoretic analysis figure (M is marker, and 1 is supernatant, and 2 are deposition) as a result.
Fig. 3 be SDS-PAGE electrophoresis detection AFGF through each step effluent liquid of Q sepharose purifying as a result figure (M is marker; FT is that stream is worn liquid; W is a buffer A wash-out, and 1～25 is that 0～1M NaCl gradient elution each several part is collected liquid).
Fig. 4 be SDS-PAGE electrophoresis detection AFGF through each step effluent liquid of sp sepharose purifying as a result figure (M is marker; FT is that stream is worn liquid; W is the bufferA wash-out, and 1～7 is that 0～1M NaCl gradient elution each several part is collected liquid).
Fig. 5 is that AFGF is through superdex 75 purifying peak figure.
Fig. 6 is that the SDS-PAGE of AFGF analyzes purifying purity figure as a result, and what the SDS-PAGE checking obtained is highly purified albumen, purity of protein＞95%.
Embodiment
1, the prokaryotic expression of acid fibroblast growth factor
The aFGF gene in coding human source is connected with expression vector pET-26b with the EcoRI restriction enzyme site through NdeI.Subsequently plasmid is transformed into e. coli bl21 (DE3).
The picking mono-clonal also inserts and to contain 10mL LB and final concentration is in the substratum of 100 μ g/ml kantlex, and 37 ℃ of shaking tables, 250rpm were cultivated about 16 hours.
Above-mentioned bacterium liquid inserted in 1: 100 ratio contain 1000mL LB and final concentration is in the substratum of 100u g/ml kantlex, 37 ℃ of shaking tables, 250rpm is cultured to OD600 and is about 0.8, and bacterium liquid is placed cooled on ice.Add IPTG to final concentration be 0.8mM, induce under 30 ℃ of conditions and receive bacterium after 4 hours, centrifugal, 5000rpm, 20min abandons supernatant, thalline-80 ℃ preservation.AFGF abduction delivering SDS-PAGE electrophoretic analysis as a result result sees that (M is marker to accompanying drawing 1; 1 for inducing preceding sampling; 2 take a sample after inducing 4h).
Conclusion: the aFGF theoretical molecular is 17.4kDa, and band meets the purpose stripe size among the figure.And from sampling, find out, induce AFGF acquisition great expression after 4 hours
2, cytoclasis
The thalline of collecting places on ice and melts, and is suspended among the Lysis buffer (50mM TrispH8.0,5mM EDTA, 1mM PMSF, 1mM DTT) in 1: 10 (m/v) ratio.Cell is through Ultrasonic Cell Disruptor cracking (450w, broken 9.9s stops 9.9s, altogether 6min).Centrifugal 13000rpm, 20min is to remove impurity such as cell debris.Cleer and peaceful deposition in the collection, the proteic solubility of SDS-PAGE electrophoresis detection (electrophoresis result shows that this albumen is soluble protein).
AFGF ultrasonic cell disintegration SDS-PAGE electrophoretic analysis result sees accompanying drawing 2
Conclusion: show in the electrophorogram that aFGF is a soluble protein.
3, ion exchange chromatography purifying target protein
The albumen of above-mentioned collection is diluted one times with lysis buffer equal-volume.Get 5mLQ sepharose, after Lysis buffer balance, with the slow upper prop of the supernatant liquor of above-mentioned collection, about 0.5mL/min collects flow through.After identical buffer washing, with 0～1M NaCl wash-out.Every pipe is collected 2ml, and SDS-PAGE detects.Collect the higher albumen of purity.
Get 5mL sp sepharose, after lysis buffer balance,, collected flowthrough the slow upper prop of albumen that a last step collects.After identical buffer washing, with 0～1M NaCl wash-out.Every pipe is collected 2ml, and SDS-PAGE detects.Collect the higher albumen of purity and be used for next step experiment.
The SDS-PAGE electrophoretic analysis result of aFGF behind q sepharose and sp sepharose purifying sees accompanying drawing 3,4.
Conclusion: through Q sepharose purifying, the albumen major part is present in stream wears in the liquid, and foreign protein is attached on the resin, thereby has obtained partial purification.Pass through the purifying of sp sepharose again, protein binding is to resin, and the part foreign protein is present in stream to be worn in the liquid, and albumen is further purified.Purifying through the two-wheeled ion exchange chromatography obtain purer target protein, but purity must improve further also.
4, gel permeation chromatography is further purified target protein
The albumen of above-mentioned collection is concentrated into about 5mL, and last superdex 7510/300 is further purified.Earlier with S75gelfiltration column buffer (PBS pH7.4,5mM EDTA) balance pillar, last appearance back is with identical damping fluid drip washing albumen before the upper prop, and the component collector is collected elutriant 1mL/ pipe.Albumen behind the wash-out is after the checking of SDS-PAGE electrophoresis, and being concentrated into concentration is 1mg/mL, and-80 ℃ of refrigerators are preserved.The final aFGF protein content that obtains is 4.5mg.
AFGF sees that through superdex 75 purifying peak figure accompanying drawing 5 and SDS-PAGE analyze purifying purity result and see accompanying drawing 6,
Conclusion: be further purified through superdex 75, what the SDS-page checking obtained is highly purified albumen, purity of protein＞95%.
Claims (5)
1. easy method through the acid fibroblast growth factor (aFGF) in procaryotic cell expression and purifying people source; It is characterized in that the acid fibroblast growth factor (aFGF) that this method is originated through procaryotic cell expression and purifying people through following steps: the prokaryotic expression of acid fibroblast growth factor; Cytoclasis; Ion exchange chromatography purifying target protein, ion exchange chromatography purifying target protein
2. the easy method of passing through the acid fibroblast growth factor (aFGF) in procaryotic cell expression and purifying people source as claimed in claim 1 is characterised in that the acid fibroblast growth factor (aFGF) in said people source is from the supernatant liquor of escherichia coli high-level expression, to prepare purifying.
3. the easy method of passing through the acid fibroblast growth factor (aFGF) in procaryotic cell expression and purifying people source as claimed in claim 1 is characterised in that the expression temperature of the acid fibroblast growth factor (aFGF) in said people source is 37 ℃
4. the easy method of the acid fibroblast growth factor (aFGF) through procaryotic cell expression and purifying people source as claimed in claim 1, the cleansing temp that is characterised in that the acid fibroblast growth factor (aFGF) in said people source are room temperature or 4 ℃.
5. the easy method of the acid fibroblast growth factor (aFGF) through procaryotic cell expression and purifying people source as claimed in claim 1 is characterised in that after acid fibroblast growth factor (aFGF) the liquid nitrogen cold shock in said people source in-80 ℃ of preservations.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110078815A (en) * | 2019-05-24 | 2019-08-02 | 温州医科大学 | A kind of recombination human acidic mechanocyte growth factor large-scale producing method |
| CN110151977A (en) * | 2019-05-29 | 2019-08-23 | 新乡医学院 | A method for protecting the stability and activity of aFGF protein drugs |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1400305A (en) * | 2001-07-26 | 2003-03-05 | 广州暨南大学医药生物技术研究开发中心 | Recombinant acid fibroblast growth factor (aFGF) gene engineering bacterium and method for preparing recombinant aFGF by using said bacterium |
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2011
- 2011-09-16 CN CN2011102743965A patent/CN102344930A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1400305A (en) * | 2001-07-26 | 2003-03-05 | 广州暨南大学医药生物技术研究开发中心 | Recombinant acid fibroblast growth factor (aFGF) gene engineering bacterium and method for preparing recombinant aFGF by using said bacterium |
Non-Patent Citations (3)
| Title |
|---|
| 余瑛: "人酸性成纤维细胞生长因子在大肠杆菌和酵母中的表达、纯化及其特性分析", 《中国博士学位论文全文数据库（医药卫生科技辑）》 * |
| 刘青 等: "人酸性成纤维细胞生长因子在大肠杆菌的高效表达", 《吉林大学学报（医学版）》 * |
| 李校堃 等: "人酸性成纤维细胞生长因子的高效表达", 《华南理工大学学报（ 自然科学版）》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110078815A (en) * | 2019-05-24 | 2019-08-02 | 温州医科大学 | A kind of recombination human acidic mechanocyte growth factor large-scale producing method |
| CN110151977A (en) * | 2019-05-29 | 2019-08-23 | 新乡医学院 | A method for protecting the stability and activity of aFGF protein drugs |
| CN110151977B (en) * | 2019-05-29 | 2022-11-29 | 新乡医学院 | Method for protecting stability and activity of aFGF protein medicament |
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