CN104293821A - Gene cloning, expression and application of recombinant human fibroblast growth factor-20 - Google Patents

Abstract

The invention discloses a method for the expression and production of FGF (fibroblast growth factor)-20. By the method of bioinformatics for optimization design, full length FGF-20 nucleotide sequence is synthesized, and is connected with pET series carriers, the expression carrier connection is introduced into proper escherichia coli host cells, a high expression quantity and stable inheritance engineering strain is obtained through screening, a high density fermentation method, an inclusion body expression FGF-20 purification method and a corresponding quality detection method are established, and mass production of rhFGF-20 is possible. On the basis, clinical application of the FGF-20 in tissue repair and degenerative diseases of the nervous system and effect of the FGF-20 in occurrence, development and migration of tumors can be further explored.

Description

The gene clone of recombinant human fibroblast growth factor-20, expression and application

Technical field

The invention belongs to biomedicine field, relate to a kind of method being produced into FGF-20 (FGF-20) of optimization, and for the expression vector of the method, host, express, purification process and possible clinical application.

Background technology

Fibroblast growth factor (Fibroblast Growth Factor, FGF) be the cell polypeptide growth factor that a class is extensively present in Various Tissues, 23 members are found at present, molecular weight is 16kD ~ 34kD, about 120 aminoacid sequences of high homology (30% ~ 70%) are contained in its central zone, to deriving from mesoderm and neuroectodermal broad variety cell has biologic activity widely.FGFs in vivo content is very micro-but widely distributed, and the generation of wherein most of FGFs member and fetal development, tissue injury reparation, neuroprotective and nutrition, tumour, development and transfer have substantial connection, are with a wide range of applications.

FGF-20 is a newcomer of FGF family; belong to FGF9 subfamily (FGF9, FGF16, FGF20); produced by endoplasmic reticulum-Golgi Secretory Pathway; play important in tissue maintenance and wound healing and to act on widely; in recent years, its focus becoming research and development in neural protection and treatment and hereditary hearing impairment disease is studied.2000, Japanese Scientists Shigeki Ohmachi found that FGF-20mRNA is the highest at cerebral tissue especially cerebellar tissue substantia nigra compacta expression amount first from rat brain, indicated that it is a kind of neurotrophic factor neurone to provide protection.HFGF-2 0 assignment of genes gene mapping, in human chromosomal 8p21.3-p22 region, is made up of 3 exons and 2 introns, and the polypeptide of 211 amino-acid residue compositions of encoding, molecular mass is about 25kDa, is a kind of basic protein.HFGF-2 0 has the high homology of 95% with the aminoacid sequence of mouse.Structurally, FGF20 and FGF9, FGF16 are similar, and its homology difference 70% and 62%, all lacks typical N terminal signal sequence, can be secreted into extracellular.The multinomial research of in vitro and in vivo shows, FGF-20 is formed in differentiation and development, vasculogenesis, tumour and also plays an important role in other cell development processes.

FGF-20 has good application prospect in tissue repair.Identical with most of FGFs member, FGF-20 can with a series of FGFR specific binding, there is short epithelium and the mitotic function of mesenchymal cell.With FGF-1, FGF-2 functional similarity gone on the market at present, can be developed into the externally applied medicine into treatment burn and scald and chronic ulcer.Meanwhile, send out FGF-20 after deliberation, to tissue adherence injury repairing, there is good effect.Jeffers etc. study the effect of FGF-20 to ulcerative colitis and inflammatory bowel animal model, find that the FGF-20 of preventive dose effectively can reduce the scope of mucosa injury and alleviate the severity of mucosa lesions; Alvarez etc. study discovery, and the oral mucositis of FGF-20 to Chemo-radiotherapy Induced has therapeutic action equally; Maclachlan etc. study discovery, and FGF-20 obviously can increase the survival rate of mouse after lethal dose ionizing rays.It is expected to, FGF-20 has huge application prospect in clinical directions such as ulcerative colitis, inflammatory bowel, oral mucositis and radiotherapy radiation preventions.At present, the novel growth factor FGF-2-20 of CuraGen company of U.S. research and development, is used for the treatment of the Orphan Drugs of the oral mucositis (OM) because radiotherapy causes, and has completed I phase clinical study by FDA approval.(Jeffers?M?et?al.Gastroenterology，2002，123(4)：1151-1162；Alvarez?E?et?al.Clin?Cancer?Res，2003，9(9)：3454-3461；Maclachlar?T?et?al.Int?J?Radiat?Biol，2005，81(8)：567-579；Beenken?A?et?al.Nat?Rev?Drug?Discov.2009Mar；8(3)：235-53.)

FGF-20 has good application prospect in degenerative neural disease treatments such as parkinsonisms.Parkinsonism (PD) is the nervous system degenerative disease caused by a kind of substantia nigra dopaminergic neuron sex change disappearance, morbidity is between 10-405/10 ten thousand, be current a kind of serious and common middle-aged and old nervous system degeneration diseases, in its pathogenesis, the effect of inherited genetic factors is more and more subject to people's attention.Clinical for PD patient gene organize examination find, FGF-20 is just in time positioned at No. 8 the short arm of a chromosome desmic regions the closest with PD relation, and its coding albumen there is obvious neurotrophic properties in normal cerebral tissue; FGF-20 albumen especially has expression in normal cerebral tissue in cerebellum, may have regulating effect to the growth of central nervous system; Add the survival ability that FGF-20 can significantly improve dopaminergic cell in vitro in cell cultures, promotion cytodifferentiation is dopaminergic neuron.These phenomenons all point out FGF-20 may play a significant role in nerve retrograde affection process.Restructuring FGF-20 albumen is acted on mouse by Ohmachi etc., find its midbrain Dopamine neuron survival significantly improve and other neuronic survival rates without considerable change, show that FGF-20 a kind of has optionally neurotrophic factor to dopamine neuron; The research such as Takagi shows, add FGF-20 in substratum, can increase the neuronic ability that Nurrl cytodifferentiation becomes tyrosine hydroxylase positive to express, and be transplanted to monkey Parkinson disease model, study of behaviour and function assessment etc. all have remarkable result for the treatment of; Have and report that the SNP polymorphism of FGF20 and MAOB gene exists interaction, the two participates in the biological bypass of Dopamine HCL equally, significant in the genetic mechanism of PD.It is expected to, FGF-20 has huge potential applicability in clinical practice in the treatment of degenerative neural disease such as treatment parkinsonism, A Er extra large black disease etc.(Scott?WK?et?al.JAMA，2001，286(18)：2239-2244；Jeffers?M?et?al.Cancer?Res，2001，61(7)：3131-3138；de?Mena?L?et?al.Neurosci?Lett，2010，479(1)：22-25；Rideout?HJ?et?al.Neurochem，2003，84(4)：803-813：Tabarés-Seisdedos?R?et?al.Mol?Psychiatry，2009，14(6)：563-589；Shigeki?Ohmachi?et?al.Biochemical?and?Biophysical?Research?Communications，2000，277：355-360；Takagi?Y?et?al.J?Clin?Invest，2005，115(1)：102-109；Gao?X?et?al.Ann?Hum?Genet，2008，72(pt2)：157-162.)

The effect of FGF-20 in tumorigenesis and migration.Can there is specific binding with Tyrosylprotein kinase receptor FGFR2 α (IIIb) of cell surface, FGFR2 α (IIIc), FGFR2 β (IIIb) and FGFR3 α (IIIc) when unconventionality expression in FGF-20, cause cell DNA to synthesize and cell proliferation by MAPK approach.It is worth noting, FGF-20, compared with other family members, also has vasculogenesis source property, participates in tumor neovasculature formation, plays an important role in the developing of tumour.The research such as Jeffers shows, the expression vector carrying FGF-20 is proceeded to NIH 3T3 cell, finds that cellular form there occurs change, and subsequently by the NIH3T3 cell infusion nude mice of stable transfection, the tumour of growth fast all appears in result; Chamorro etc. have done rightabout experiment, namely adopt RNA perturbation technique to close FGF-20 genetic expression, find that RK3E cell clone significantly reduces, and non-anchor qualitative growth characteristics disappears, and occurs contact inhibition.Further experiment confirms that FGF-20 transcriptional level is subject to that closely-related Wm/B-catenin signal transduction pathway occurs with many human malignancies and regulates, and illustrates that FGF-20 expresses and is formed with substantial connection with tumour.Confirm at present, equal unconventionality expression FGF-20 in most human tumor cell line, wherein overexpression in lung cancer, colorectal cancer and stomach cancer cell, and may be the principal element participating in cancer pathology process, with the infiltration of tumour, shift closely related, the target molecule that can be used as tumor diagnosis and therapy has broad prospects.(Engstrom?W?et?al.An?ticancer?Res，2006，26(5A)：3307-3310；Polnaszek?N?et?al.C?ancer?Res，2003，63(18)：5754-5760；Jeffers?M?et?al.Cancer?Res，2001，61(7)：3131-3138；Chamorro?MN?et?al.EMBO?J，2005，24(1)：73-84；Katoh?M?et?al.Onco?l?Rep，2005，14(1)：287-290；Mario?N?Chamorro?et?al.The?EMBO?Journal，2005，24：73-84.)

Because the discovery of FGF-20 is for degenerative neural disease, tissue repair and tumour aspect important in inhibiting, the prospect mouth benefit of its research and clinical application receives the concern of people.FGF-20 is as novel somatomedin, and relevant report is less both at home and abroad at present.The application's patent, by the screening of gene optimization, expression vector and Host Strains, and then obtains colibacillary efficient expression strain; The mode of flow feeding is utilized to establish the high-density culture technology of mass-producing; Optimize inclusion body cleaning, sex change and refolding method, by cation exchange column chromatography, heparin column chromatography (IIeparin Sepharose CL-6B) or metal ion-chelant column chromatography (Chelating Sepharose Fast Flow) and combination thereof, final acquisition purity of protein is more than 95% and have the FGF-20 recombinant protein of high biologic activity, for carrying out FGF-20 dependent interaction Mechanism Study further and new drug development is laid a good foundation.

Summary of the invention

The object of this invention is to provide a kind of easy and produce efficiently the method for FGF-20.

The present invention also aims to provide the carrier containing coding FGF-20 gene or plasmid.

Another object of the present invention is to provide expression vector for the method and host cell.

For achieving the above object, first the native sequences of FGF-20 is optimized according to intestinal bacteria Preference by the present invention, under the prerequisite not changing its aminoacid sequence, and the full length sequence of design and synthesis rhFGF-20.The invention provides the expression vector of screening and optimizing and the combination of host cell simultaneously, as BL21 (DE3) plysS, Transetta (DE3) host cell of pET3a, pET3c, pET28a expression vector and correspondence, and construct the engineering strain of high efficiency stable expression.

Further; the invention provides a kind of method of Restruction hFGF-2 0; by Optimal Medium, controling parameters and feed supplement feed-batch process, establish the high-density cultivation method of mass-producing, obtain high purity and active rhFGF-20 finally by column chromatographic isolation and purification.Specifically comprise the steps:

(1) by improved seed substratum engineering bacteria activated and increase, to be seeded in fermentor tank by optimal control parameter and to adopt the mode of flow feeding to carry out the high-density culture of mass-producing, optimizing induction starting time and induction time high expression foreign protein FGF-20;

(2) optimize cracked solution and carry out bacterial cell disruption by high pressure homogenizer, centrifugal the abandoning of low-temperature and high-speed takes back precipitation completely; Optimize inclusion body cleaning, sex change and refolding method and step, and carry out initial gross separation purifying by hollow-fibre ultrafiltration device;

(3) carry out fine separation purifying by column chromatography, specifically comprise: cation exchange column chromatography (SP Sepharose Fast Flow or CM Sepharose Fast Flow), heparin column chromatography (Heparin Sepharose CL-6B) or metal ion-chelant column chromatography (Chelating Sepharose Fast Flow) and combination thereof.

Described high-density cultivation method is:

(1) medium optimization in tank: reduce concentration of substrate, Optimal Medium compatibility forms, and as reduced carbon source content in substrate, adding the nutritive ingredients such as inorganic salt, trace element, growth promotion factor provides full nutrition, and protects the genetic stability of plasmid;

(2) Optimization about control parameter: inducing the front and rear key control parameter of induction by optimizing, as temperature, pH value, dissolved oxygen, revolution and induction starting time and induction time etc., obtaining high yield and the rational production technique of economic target;

(3) flow feeding method: the nutritive ingredient optimizing flow feeding, various flows is adopted to add feeding strategy, as speed change feed supplement, DO-stat method or pH-stat method, reduce substrate to engineering bacteria growth and the suppression of Product formation, extend the logarithmic growth cycle of engineering bacteria, effective raising output and expression amount, improve equipment effective rate of utilization high, reduce production cost, and optimal control is achieved to fermenting process.

The present invention has the following advantages:

(1) by being optimized FGF-20 nucleotide sequence and the combination of suitable expression vector and host cell, the engineering strain of high efficiency stable expression is obtained;

(2) by setting up high-density cultivation method, the expression level of FGF-20 can be made to reach more than 25% of bacterial protein, and thalline output reaches more than 70g/L fermented liquid;

(3) foreign protein is expressed with inclusion bodies, expression amount high and broken with purge process in be not easily degraded; Further established inclusion body cleaning, sex change renaturation and column chromatography method, simplify purification step, improve albumen and activity yield.Protein yield reaches more than 150mg/L fermented liquid, and purity of protein is higher than more than 95%.

(4) FGF-20 activity determination method is established.Biologic activity is the important indicator of reflection recombinant proteins and effect, and the present invention establishes the activity determination method carrying out FGF-20 proliferation with NIH3T3 cell strain.Experimental result is typical S type curve, and has dose-dependently within the specific limits, demonstrates good repeatability and reliability.

Accompanying drawing explanation

Fig. 1 pET3c-FGF20 recombinant vectors figure

Fig. 2 pET28a-FGF20 recombinant vectors figure

Fig. 3 FGF-20 fermentation expression SDS-PAGE electrophoretic analysis

Fig. 4 CM column chromatography SDS-PAGE electrophoretic analysis

Fig. 5 affinity column chromatography SDS-PAGE electrophoretic analysis

It is active that Fig. 6 mtt assay measures the short proliferative biological of FGF-20 albumen to NIH3T3 cell

Embodiment

The structure of embodiment 1FGF-20 albumen efficient expression engineering

According to hFGF-2 0 native sequences (accession number: BC098128.1) announced in GenBank and aminoacid sequence, be optimized according to e. coli codon Preference, under the condition not changing aminoacid sequence, the encoding sequence primer of design rhFGF-20.The full length nucleotide sequence of rhFGF-20 is obtained by the method for pcr amplification.Hold at the 5 ' end and 3 ' of rhFGF-20 preferred sequence and add initiator codon and terminator codon respectively, and introduce specific cleavage site Nde I and BamH I, rhFGF-20 gene and expression vector pET3a-c (or pET28a) is processed respectively again with NdeI and BamHI double digestion, corresponding fragment is reclaimed after 37 DEG C of enzymes cut 3h ~ 4h, connect with T4DNA ligase enzyme 16 DEG C and spend the night, build recombinant expression vector (see Fig. 1, Fig. 2).Connect product conversion and enter intestinal bacteria Transetta (DE3) (or BL21 (DE3) plysS), containing carrier resistance LB flat board on select positive colony and carry out the qualification of plasmid double digestion and bacterium colony PCR examines and determine, confirm expressed sequence correctly in insertion vector.Entrust gene sequencing company carry out forward, Inverse order sequence analysis measure, confirm cloned sequence and implementation sequence completely the same.Carry out shake-flask culture, measure target protein expression amount and account for total protein about 25% after induction, and carry out immunoblotting (Western Blot) and detect and present positive reaction, the recombinant exogenous protein expressed by confirmation is FGF-20.Carry out engineering bacteria construction test, continuous passage 50 times, detects its plasmid stability, structural stability and expression stability, and confirms the FGF-20 engineering strain obtaining high expression, inheritance stability, for next step test is had laid a good foundation.Goal gene inserts in the pET series expression vector containing the induction of Lac operon by this engineering bacteria, then transforms the e. coli host bacteria compatible with expression vector, and IPTG or lactose can be utilized to carry out abduction delivering.

The foundation of embodiment 2FGF-20 protein high density cultural method

In the present invention, the fermentation condition of engineering bacterium expression rhFGF-20 is not particularly limited.The fermentation condition of this area routine can be adopted.Usually, engineering bacteria with Tryptones, yeast powder, ammonium salt etc. for nitrogenous source, glucose, glycerine etc. are carbon source, and phosphoric acid salt is buffer system composition, and are aided with magnesium sulfate, sodium-chlor and trace element and add substratum based on VITAMIN; And added by the adjustment of controling parameters (pH, temperature, dissolved oxygen, stirring velocity etc.) and the stream of nutritive substance, glucose content in monitoring fermented liquid and tails assay, regulate product specific growth rate, reduce the accumulation of acetic acid, extend the logarithmic growth cycle, significantly improve thalline output and expression level.Be specially: FGF-20 engineering strain is seeded in LB substratum with 1: 200 ~ 300 and carries out activation preparation generation seed, cultivate 3h ~ 4h, treat A ₆₀₀reach about 0.8 ~ 1.0, the ratio in 1: 10 ~ 20 is seeded to carries out amplification preparation two generation seed containing in the right LB substratum of phosphate buffered, cultivates 8h ~ 10h, treats A ₆₀₀reach about 4 ~ 8, ratio in 1: 10 ~ 20 is seeded in the fermentor tank containing substratum in tank, with temperature 35 DEG C ~ 37 DEG C, pH value 6.8 ~ 7.0, agitation revolution 200rpm ~ 600rpm, DO > 25% fed-batch mode being aided with nutritive substance carries out cultivation 4h ~ 6h.Treat A ₆₀₀reach 20 ~ 25, stream adds people's inductor (IPTG) and induces to final concentration 1mM.After regulating induction temperature be 33 DEG C ~ 35 DEG C, pH value 7.0 ~ 7.2, agitation revolution 600rpm ~ 300rpm, DO > 30% be aided with the fed-batch mode that stream adds carbon source, nitrogenous source and phosphate buffer soln and carry out expression 4h ~ 6h.Low-temperature and high-speed is centrifugal, and thalline-20 DEG C is frozen, and sample carries out SDS-PAGE detection and measures expression amount (see Fig. 3).

Table 1 each stage substratum composition

The foundation of embodiment 3FGF-20 method for purifying proteins

1, bacterial cell disruption

After thalline room temperature being melted, be fully suspended in cell lysis buffer solution (20mM Tris-HCl, 2mM EDTA-2Na with 1: 20 (g: mL) ratio, 0.1M NaCl, Triton X-100,0.2% sodium deoxycholate, pH7.5), carry out high-pressure homogeneous.Crumbling method is that 200bar circulates 1 time, then with 800bar homogeneous 1 ~ 2 time, microscopy is without after whole E. coli within the scope of visual fields, and 20000rpm is centrifugal, 4 DEG C, and 30min, abandons supernatant, collecting precipitation.

2, inclusion body cleaning

Get above-mentioned centrifugal after precipitation, add 10 times amount lavation buffer solutions (20mM Tris-HCl, 2mM EDTA-2Na, 0.1M NaCl, Triton X-100,0.2% sodium deoxycholate, pH7.5) abundant agitator treating, after 10min ~ 15min, 20000rpm is centrifugal, 4 DEG C, 30min, abandon supernatant, collecting precipitation.Get precipitation again, add 10 times amount lavation buffer solutions (20mM Tris-HCl, 2mM EDTA-2Na, 0.1M NaCl, Triton X-100, pH7.5) abundant agitator treating, after 10min ~ 15min, 20000rpm is centrifugal, 4 DEG C, 30min, abandon supernatant, collecting precipitation, then repeated washing is once, obtains inclusion body.

3, inclusion body sex change

Take appropriate inclusion body, add 20 times amount (g/V) and dissolve damping fluid (20mM Tris-HCl, 8M urea, pH7.5), fully suspend, and 4 DEG C of dissolvings are spent the night under magnetic agitation condition.20000rpm is centrifugal, 4 DEG C, 30min, and the not molten thing of centrifugal segregation, obtains solubilization of inclusion bodies liquid.

4, renaturing inclusion bodies

Get solubilization of inclusion bodies liquid, under 4 DEG C of magnetic agitation conditions, slowly instillation 7 times of volume renaturation buffers (20mM Tris-HCl, pH7.5), be 1M to urea final concentration, renaturation process is about 15h ~ 18h.Renaturation is complete, and adopt hollow-fibre ultrafiltration device to carry out initial gross separation, the molecular weight cut-off adopted is 50KD.Get supernatant liquor, be renaturation solution.Sample determination protein content, and carry out SDS-PAGE and detect its purity.

5, column chromatography purification

(1) cationic exchange

Chromatography media: CM-Sepharose FF or SP-Sepharose FF

Buffered soln: solution A: 20mM Tris-HCl (pH6.5 ~ 7.5)+50mM NaCl

Solution B: 20mM Tris-HCl (pH6.5 ~ 7.5)+1.2M NaCl

Balance: with solution A balance 3 ~ 5 column volumes.

Loading: by the protein solution loading after renaturation.

Cleaning: with the solution A cleaning chromatography column of 3 ~ 5 column volumes after loading, balance to baseline.

Gradient: with 20 ~ 30 column volumes, solution B is adjusted to 100% from 0% liter after cleaning.

Collect: collect FGF-20 sample peak, measure protein content, and carry out SDS-PAGE and detect its purity.

(2) affinity chromatography

A.pET3a-c carrier

Chromatography media: Heparin Sepharose CL-6B

Buffered soln: solution A: 20mM Tris-HCl (pH6.5 ~ 7.5)+50mM NaCl

Solution B: 20mM Tris-HCl (pH6.5 ~ 7.5)+2.0M NaCl

Balance: with solution A balance 3 ~ 5 column volumes.

Loading: the FGF-20 protein solution loading that ion-exchange chromatography is collected.

Cleaning: with the solution A cleaning chromatography column of 3 ~ 5 column volumes after loading, balance to baseline.

Gradient: with 20 ~ 30 column volumes, solution B is adjusted to 100% from 0% after cleaning.

Collect: collect FGF-20 sample peak, measure protein content, and carry out SDS-PAGE, HPLC and detect its purity.

B.pET28a carrier

Chromatography media: Chelating Sepharose Fast Flow

Buffered soln: solution A: 20mM Tris-HCl (pH6.5 ~ 7.5)+50mM NaCl+20mM imidazoles

Solution B: 20mM Tris-HCl (pH6.5 ~ 7.5)+50mM NaCl+500mM imidazoles

Other are with above-mentioned a step.

Sample is through this two-step purifying, i.e. ion-exchange, affinity chromatography, purity is increased to more than 95% (see Fig. 4, Fig. 5).

Embodiment 4FGF-20 albuminous cell activity test method is set up

The biologic activity of genetically engineered recombinant protein is the important indicator of reflection drug quality and effect, therefore set up stable, sensitive activity determination method for the production of product and quality examination significant, be also the important indicator instructing clinical medicine dose simultaneously.The present invention promotes epithelium and the mitotic function of mesenchymal cell according to FGF-20 specificity, adopts cell proliferation method, selects mouse embryo fibroblasts (NIH3T3 cell) to be target cell, in conjunction with MTT colorimetric determination albumen In vitro biological activity.Experiment shows, do not containing in the nutrient solution of calf serum and rhFGF-20 albumen, NIH3T3 Growth of Cells is slow; And do not containing calf serum but adding in the nutrient solution of rhFGF-20 albumen, NIH3T3 Growth of Cells is obviously accelerated.Adopt full-automatic microplate reader to take 630nm as reference wavelength, measure absorbancy in 570nm place.Result shows, and the FGF-20 albumen of purifying, when 10ng/ml ~ 100ng/ml concentration, has significant proliferation to NIH3T3 cell, and is linearly correlated with.Compared with the FGF-20 standard substance of outsourcing, biologic activity is suitable, no difference of science of statistics (see Fig. 6).

Claims (5)

1. a provenance is to the prokaryotic expression carrier of pET3a-c or pET28a (+), and it contains the recombinant DNA of coding FGF-20, and the nucleotide sequence of wherein said coding FGF-20 is shown in shown in Nucleotide or aminoacid sequence table.

2. e. coli bl21 (DE3) plysS or Transetta (DE3) containing expression vector described in claim 1.

3. produce a method of FGF-20, by cultivating host cell according to claim 2, and add inductor abduction delivering FGF-20, and obtain highly purified FGF-20 albumen further by separation and purification.

4. method according to claim 3, is characterized in that, the method comprises the steps:

(1) by improved seed substratum engineering bacteria according to claim 2 activated and increase, to be seeded in fermentor tank by optimal control parameter and to adopt the mode of flow feeding to carry out the high-density culture of mass-producing, optimizing induction starting time and induction time high expression foreign protein FGF-20;

(2) optimize cracked solution and carry out bacterial cell disruption by high pressure homogenizer, centrifugal the abandoning of low-temperature and high-speed takes back precipitation completely; Optimize inclusion body cleaning, sex change and refolding method and step, and carry out initial gross separation purifying by hollow-fibre ultrafiltration device;

(3) carry out fine separation purifying by column chromatography, specifically comprise: cation exchange column chromatography (SP Sepharose Fast Flow or CM Sepharose Fast Flow), heparin affinity column chromatography (Heparin Sepharose CL-6B) or metal ion-chelant column chromatography (Chelating Sepharose Fast Flow) and combination thereof.

5. the FGF-20 of preparation according to claims 1 ~ 4, is characterized in that, can be applicable to the tissue repair of skin or mucosa injury, degenerative neural disease such as treatment Parkinson or A Er extra large black disease etc.Also can be prepared into different preparation, be applied to the treatment of biological beauty and burn and scald, dermal chronic ulcer.