CN100334114C - Novel fusion protein production and uses - Google Patents
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- CN100334114C CN100334114C CNB2005100854972A CN200510085497A CN100334114C CN 100334114 C CN100334114 C CN 100334114C CN B2005100854972 A CNB2005100854972 A CN B2005100854972A CN 200510085497 A CN200510085497 A CN 200510085497A CN 100334114 C CN100334114 C CN 100334114C
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Abstract
The present invention relates to the production and the application of a novel fusion protein, particularly to a human acid fibroblast growth factor variant fusion protein and a preparing method thereof. The fusion protein as a fusion body of acid fibroblast growth factor variants and thioredoxin can be used in the fields of wound healing, treatment, etc.
Description
Technical field:
The present invention relates to a kind of biotech drug, particularly relate to human acid fibroblast growth factor and hand over body fusion rotein and preparation method thereof, described fusion rotein is the syzygy of human acid fibroblast growth factor variant and Trx, and this fusion rotein can be applicable to fields such as therapeutic wound healing.
Background technology:
Human acid fibroblast growth factor (Human acidic fibroblast growth factor is called for short: aFGF or haFGF) is one of member of human inner cell's growth factor family.Can promote the mitotic division and the differentiation of various kinds of cell such as epithelial cell, neurocyte.
AFGF helps vascularization and injury repairing, has the potential clinical value aspect treatment chronic soft tissue ulcer and venous congestion, the glycosuria germ leg ulcer of foot; Jefferson medical college and Merck Sharp ﹠amp; The Dohme research laboratory has finished aFGF jointly and has promoted the research work that blood vessel is repaired, and confirms that aFGF is preventing postangioplasty because the angiostenosis that vascellum tunica interna incrassation causes is effective in cure; AFGF has neurotrophic effect, and in the retinal ganglial cells damage, aFGF has special cytothesis effect.U.S. Georgetown university research personnel sum up and think aFGF in the treatment of central nervous system injury, have the function of the injured neurons kept, stimulation revascularization; Spain Ramony Cajal medical college has confirmed that aFGF has provide protection to brain damage, can prevent the neurone degraded, and can significantly improve the survival rate of the damaged animal of spinal cord; The German Research personnel find that aFGF plays an important role for the healing of myocardium micronecrosis; The experiment of Japan Okayama Univ. shows that aFGF can induce HGF synthetic in a large number in vivo, and the latter plays important physiological function for liver regeneration.
But the aFGF original shape is stable poor, in clinical and practical application many restrictions is arranged.Sophisticated aFGF albumen is made up of 140 amino acid, and 6 amino acid of its N end disappearance still have activity, and the ED50 value of BALB/c 3T3 cell proliferation is about 40pg/ml, also has the 10 μ g aFGF of report to be enough to activate vascular endothelial cell.AFGF albumen iso-electric point is 5.8, molecular weight 16Kd (10).3 halfcystines (Cys16, Cys83, Cys117) are arranged in the primary structure of aFGF molecule, but do not have interchain disulfide bond, in aqueous environment, show as single chain protein.Researchs such as Thomas replace Cys16, Cys83, Cys117 with Serine respectively individually or simultaneously, promptly form S16, S16.S83, S16.S117, S83, S83.S117, S117, mutant such as S16.S83.S117 found that the biologic activity of these aFGF mutant obviously reduces the heparin dependency.
Intestinal bacteria thioredoxin (Tioredoxin), the reduction of catalysis disulfide linkage and exchange, and the oxidation of free thiol group.This albumen is expressed in colibacillus periplasm, is essential for effective formation of disulfide linkage in the colibacillus periplasm.Promote proteinic refolding external, the present invention finds unexpectedly that with human acid fibroblast growth and intestinal bacteria thioredoxin amalgamation and expression its stability and biologic activity obviously improve.
The present invention adopts gene engineering method, and efficiently expressing recombinant human aFGF variant fusion rotein engineering strain is produced a large amount of reorganization aFGF variant fusion roteins, and worked out liquid and the freeze-dried preparation that is suitable for clinical therapeutic wound healing application in intestinal bacteria.
Summary of the invention:
The invention provides a kind of human acid fibroblast growth factor variant fusion rotein, this albumen is composed as follows:
The segmental fusion rotein of bacterium thioredoxin and haFGF, wherein the haFGF fragment is that natural haFGF fragment or cysteine residues (as Cys16, Cys83, Cys117) partly or entirely sport the amino acid that can not cause that the fusion rotein space structure changes; Amino acid after the sudden change is Gly, Ser, Ala preferably, and most preferred is Ser, and the aminoacid sequence after the most preferred sudden change is seen sequence table 2.
The present invention also provides the preparation method of fusion rotein of the present invention, and this method may further comprise the steps:
A. the gene DNA segment of synthetic coding people aFGF aminoacid sequence, in this fragment, the cysteine residues of aFGF gene (cys) partly or entirely sports Gly, Ser, Ala, and most preferred is Ser;
B. the gene fragment that obtains among the step a. is inserted 3 ' end of bacterium thioredoxin (Tioredoxin) gene on the expression vector;
The expression vector recombinant plasmid dna that c. will contain the Tioredoxin-aFGF antigen-4 fusion protein gene transforms suitable intestinal bacteria; The preferred expression system of the present invention is an escherichia expression system, wherein preferred pET32a/BL21 (DE3); Also can select the pThioHisA/Top10 system;
D. this genetic engineering bacterium is prepared highly purified Tioredoxin-aFGF fusion rotein through fermentation, extraction, purification procedures;
E. the Tioredoxin-aFGF fusion rotein that purifying is obtained is prepared into stable liquid or freeze-dried preparation, for clinical use.
The dna sequence dna of Tioredoxin-aFGF fusion rotein of wherein encoding is seen sequence table 1
The aminoacid sequence of wherein most preferred Tioredoxin-aFGF fusion rotein is seen sequence table 2.
Be prepared into pharmaceutical composition with fusion rotein of the present invention as active constituents of medicine of the present invention also comprising, said composition can add some medicine acceptable carriers as required, can adopt the technology of pharmaceutics routine techniques to prepare said composition, composition of the present invention can be oral, also can parenteral administration, preferred compositions is that the injection form of unitary dose is as lyophilized injectable powder.Of the present inventionly in making medicine, be with a wide range of applications and vast market prospect.
Advantage of the present invention mainly shows:
1. recombinant human aFGF is very big to intestinal bacteria toxicity, therefore the expression in intestinal bacteria is very low, usually just can detect with Western blot, even also can only reach the expression amount (Jaye of 10mg/L after optimizing, M.Burgess, N, W.1987.J.Biol.Chem 262 (34): 16612-16617), we express in the fusion rotein mode and expression amount are reached account for whole bacterial protein more than 20%, and every liter of fermented liquid can obtain more than the pure product 200mg.
2. stability is higher than the aFGF of prototype aFGF and sudden change far away.37 ℃, 1 hour postprecipitation of prototype precipitates after more 4 hours fully, non-activity, and the Tioredoxin-aFGF fusion rotein has a little precipitation with this understanding after 4 hours, its specific activity is 1.2 * 10
5U/mg
Description of drawings:
Fig. 1: expression vector pET32a+ and multiple clone site structural representation thereof
Fig. 2: construction of recombinant plasmid operation chart:
Fig. 3: genetic engineering bacterium is induced back Tioredoxin-aFGF Expression of Fusion Protein electrophoretogram at IPTG
Fig. 4: the electrophoretogram of the Tioredoxin-aFGF fusion rotein behind the purifying
Embodiment:
Further specify the present invention by the following examples, but not as limitation of the present invention.
The structure of recombinant expressed human acid fibroblast growth factor variant antigen-4 fusion protein gene engineering bacteria
(1) the double-stranded DNA gene of synthetic coding people aFGF 1-140 amino-acid residue, and, introduce an Ala residue at prototype haFGF N-terminal in 5 ' end adding NcoI site, adding the SalI site at 3 ' end, sequence is as follows:
5’…catggctttcaacctgccgccgggtaactacaaaaaaccgaaactgctgtactcctccaacggtggtcac
ttcctgcgtatcctgccggacggtaccgttgacggtacccgtgaccgttccgaccagcacatccagctgcagctg
tccgctgaatccgttggtgaagtttacatcaaatccaccgaaaccggtcagtacctggctatggacaccgacggt
ctgctgtacggttcccagaccccgaacgaagaatccctgttcctggaacgtctggaagaaaaccactacaacacc
tacatctccaaaaaacacgctgaaaaaaactggttcgttggtctgaaaaaaaacggttcctccaaacgtggtccg
cgtacccactacggtcagaaagctatcctgttcctgccgctgccggtttcctccgactaagtcgac…3’
The gained dna fragmentation is cloned among the pUC18 with the NcoI/SalI site and preserves, called after pUC18aFGF.
(2) with the synthetic gene clone to suitable expression vector, preferred expression vector such as pET32a (purchase) from novagen biotech company, its structure is seen accompanying drawing 1:
Host bacterium BL21 (DE3) is available from U.S. novagen company.
(3) structure, conversion, the enzyme of the structure of genetic engineering bacterium and screening expression plasmid are cut evaluation and are operated according to conventional molecular cloning, and the pET32aFGF expression plasmid is in the screening of escherichia coli expression and efficient expression engineering strain: (accompanying drawing 2)
To obtain transformant behind the expression plasmid pET32aFGF transformed into escherichia coli BL21 (DE3), picking list colony inoculation is in LB substratum (Amp
+100 μ g/mL), 37C, 220rpm, IPTG abduction delivering, SDS-PAGE electrophoresis detection.
There is the obvious expression band at the place at the 45000D molecular weight.(see figure 3)
The purifying of rh-aFGF variant fusion rotein
With the fermented liquid of fermentation culture gained, centrifugal (7000rpm 5min), collects thalline and (25mmol/L Tris, 0.15mol/L NaCl pH8.0) wash bacterium three times, remove the substratum residue with damping fluid through freezing large vol whizzer.
(1mmol/L EDTA pH8.0) suspends evenly, the ice bath cooling for 25mmol/L Tris, 0.15mol/LNaCl with damping fluid in 20% ratio (w/v) with the fermentation thalline of centrifugal collection.Regulate the operating pressure of APV1000 refiner and break bacterium.
Thalline is after the high-pressure homogenization fragmentation, and (9000rpm 30min) obtains rough inclusion body to pass through high speed centrifugation again.Adopt " Guanidinium hydrochloride of 7mol/L, 50mmol/L Tris, pH8.0 " dissolving damping fluid as the rhaFGF inclusion body.The rhaFGF inclusion body suspends with above-mentioned damping fluid according to the ratio of weight in wet base in 1/5 (w/v), and stirring at room was dissolved about 12 hours, then through the centrifugal collection supernatant of 9000rpm, 30min.
With dilution method renaturation rhaFGF, renaturation buffer is: 25mmol/L Tris, 2mol/L urea, 0.15mol/LNaCl, 1mmol/L EDTA, pH8.0.Renaturation 48 hours is carried out ultrafiltration and concentration then and (25mmol/LTris, 0.05mol/L NaCl pH8.0) dialyses to damping fluid.
Chelating Sepharose Fast Flow purifying Trx-aFGF:
Use the Chelating Sepharose Fast Flow of gel as Pharmacia, use 50mM Tris.Cl, pH8.0,0.5M NaCl damping fluid pre-equilibration pillar, not conjugated protein behind the last sample with the level pad wash-out, use 50mM Tris.Cl, pH8.0,1M NaCl washes foreign protein, uses 50mM Tris.Cl, pH8.0,50mM imidazoles wash-out purpose fusion rotein.The gained fusion rotein is replaced into 20mMPB with hyperfiltration process with damping fluid, pH7.2,0.4M NaCl.
Heparin affinitive layer purification Trx-aFGF
Use the Heparin Sepharose 6 Fast Flows of gel as Pharmacia, use 20mMPB, pH7.2,0.4MNaCl damping fluid pre-equilibration pillar, not conjugated protein behind the last sample with the level pad wash-out, use 20mMPB, pH7.2,0.6MNaCl washes foreign protein, uses 20mMPB, pH7.2,1M NaCl wash-out purpose fusion rotein.(accompanying drawing 4)
Secondly get the rhaFGF fusion rotein stoste of assay approval, at first adding human albumin to final concentration is 1%, with dilution buffer liquid A (25mmol/L Tris, 1% human albumin, pH8.0) electric conductivity value of rhaFGF fusion rotein stoste is diluted to 18.0mS/cm; Use then dilution buffer liquid B (25mmol/L Tris, 0.15mol/L NaCl, 1% human albumin, pH8.0) with the concentration dilution of rhaFGF fusion rotein in the sample to 0.2mg/ml, use the degerming of 0.22um membrane filtration at last, be rhaFGF fusion rotein work in-process.
1.0ml/ props up with the aseptic subpackaged one-tenth of the work in-process of assay approval, more than 8 hours, carries out freeze-drying through-40 ℃ of pre-freezes then, promptly gets freeze dried rhaFGF fusion rotein goods.
The liquid of Tioredoxin-aFGF fusion rotein and freeze-dried preparation
External application recombination human acidic mechanocyte cell growth factor (fusion rotein) freeze-dried preparation prescription is:
Tris 3.02mg
NaCl 9mg
Human albumin 10mg
Prop up freeze-drying after the packing by 1.0ml/, promptly every finished product contains 1 * 10
5IU (or 0.2mg) rhaFGF, 9mg NaCl, 3.02mg Tris and 10mg human albumin.
Embodiment 4
The physical and chemical property determining of Tioredox-aFGF
1. the mensuration of stoste purity
Measure by the HPLC method, chromatography column is TSK-GEL G2000 SW (XL), and the result shows that its purity reaches more than 95%.
2. molecular weight determination
The molecular weight of measuring is 32968.49, and is consistent with theoretical guess value.
3. Determination of biological activity (mtt assay)
But fibroblast growth factor differential stimulus fibroblasts proliferation, differentiation, and it promotes fibroblast proliferation, differentiation, adopts Balb/c 3T3 cell strain, mtt assay to measure the biologic activity for preparing the rhaFGF fusion rotein in this research.Its specific activity 5 * 10
5U/mg.
4.rhaFGF terminal 15 determined amino acid sequences of fusion rotein N-, measurement result is consistent with expected results.
1 5 10 15
Ser-Asp-Lys-Ile-Ile-His-Leu-Thr-Asp-Asp-Ser-Phe-Asp-Thr-Asp
5.rhaFGP fusion rotein isoelectric point determination
Adopt leveling board PAGE electrophoretic method to detect.PI is 5.3
Embodiment 5
The study on the stability of rh-aFGF variant fusion rotein stoste and freeze-dried preparation
1. the stability of rh-aFGF variant fusion rotein stoste
This laboratory in 4 ℃, room temperature, 37 ℃, remakes determination of activity with the pure product separated deposit of aFGF of the aFGF of recombinant expressed prototype and amalgamation and expression.The result is as follows:
AFGF original shape and amalgamation and expression thermostability are relatively
Above result confirms to merge aFGF stability apparently higher than prototype aFGF.
2, the stability of rh-aFGF variant fusion rotein freeze-dried preparation
With freeze dried sample (specification: 1 * 10
5U/0.2mg) respectively in 2-8 ℃, room temperature and 37 ℃ of investigations that keep sample, and proportionately the relevant regulations in the product examine set pattern journey is examined the every quality index of sample.
(1) 2-8 ℃ of investigation that keeps sample: sample is placed 2-8 ℃ of refrigerator, be set in respectively and measure above index in March, June, September, December, 18 months, 24 months, 36 months etc.
(2) the room temperature investigation that keeps sample: sample is placed 25 ℃ of thermostat containers, be set in respectively and measure above index in March, June, September, December, 18 months, 24 months, 36 months etc.
(3) 37 ℃ of investigations that keep sample: sample is placed 37 ℃ of thermostat containers, be set in respectively January, February, March, etc. measure above index.
The result shows: 1, rh-aFGF (fusion rotein) lyophilized injectable powder has good stability when room temperature, 37 ℃ of preservations, deposits 3 months activity and does not see obvious decline.2, rh-aFGF (fusion rotein) lyophilized injectable powder is when low temperature (2-8 ℃) is preserved, and the investigation result that keeps sample showed that activity did not almost change in 18 months.
Embodiment 6
Rh-aFGF variant fusion rotein is used for trauma care
1, pharmacodynamic study
(1) the rhaFGF fusion rotein is to the effect of miniature pig wound repair
Adopt Beijing to produce under the miniature pig anesthesia and cause trauma model, rhaFGF with 30,60, three kinds of dosage of 90U/cm2 handle the surface of a wound, the next day, change dressings once, observed 14 days.The surface of a wound is taken pictures, transparent film is traced the weighing method record and hindered 4,8, the 14 days surface of a wound areas in back; Hinder the chamber volume with the water flood measurement; Normal dyeing after formalin fixed, primary part observation surface of a wound granulation tissue growth (comprising the capillary vessel growth, collagen deposition) changes with re-epithelialization, to estimate repairing effect.The result shows:
A, the surface of a wound area of handling through different treatment plans prolong with post burn and dwindle gradually, and rhaFGF 90U/cm2 dosage group result of treatment is more obvious.
B, the surface of a wound are hindered the chamber volume and prolonged with post burn and dwindle gradually, and be wherein more obvious with rhaFGF 90U/cm2 dosage group result of treatment.
As seen c, histological examination treat that to be sliced into fibroblast growth on the 8th day active, and quantity is many, and its capillary vessel plumule and inoblast quantity are significantly more than the blank group.Hindered back 14 days, the face of respectively forming all heals.
Can be drawn as drawing a conclusion by The above results: rhaFGF has the promotion repair to the miniature pig back knife wound surface of a wound.Show and promote the growth of surface of a wound granulation tissue, capillary vessel plumule to form and re-epithelialization.Show with 90U/cm2 dosage group and to promote that the wound repair effect is more obvious.
(2) the rhaFGF fusion rotein is to the repair of rabbit scald wound
Adopt Beijing to produce under the large ear rabbit anesthesia and cause dark II degree scald wound model, rhaFGF with 30,60, three kinds of dosage of 90U/cm2 handle the surface of a wound, the next day, change dressings once, observed 21 days.The surface of a wound is taken pictures, transparent film is traced the weighing method record and hindered 3,7,14, the 21 days surface of a wound areas in back; Wound was got wound tissue in 14,21 days, after 10% formalin fixed, and film-making, normal dyeing, the growth of primary part observation surface of a wound granulation tissue changes with re-epithelialization, to estimate repairing effect.The result shows:
A, respectively form the face area and prolong with post burn and dwindle gradually, rhaFGF 90U/cm2 dosage group result of treatment is more obvious.
B, histological examination section are dyeed through HE, as seen rhaFGF 90U/cm2 dosage group is hindered back 14 days major parts and is covered by newborn epidermis, surface attachment necrotic tissue in a small amount, and the surface of a wound heals substantially, the blank group lingering section surface of a wound, the necrotic tissue that surface attachment is more.Hindered back 21 days, the surface of a wound of rhaFGF treatment all heals, and epidermal area is thicker, the still residual a small amount of surface of a wound of blank group.
Can be drawn as drawing a conclusion by The above results: rhaFGF has the promotion repair to the rabbit back scald wound.Show and promote that surface of a wound granulation tissue is grown, necrotic tissue comes off and re-epithelialization.Show with 90U/cm2 dosage group and to promote that the wound repair effect is more obvious.
2, pharmacological toxicology research
(1) the wound and wound surface model experiment of Sprague-Dawley rat
Animal subject was used the scissors cropping at the back before 24 hours, the depilation area is about 40cm2, and polishing with emery paper in the depilation district blood point occurs to skin but do not have hemorrhage, this product evenly is coated on depilation district, animal subject back, and fixing with the gauze wrapping, after 24 hours, remove residue with asking to wash.By 14 days continuous observed and recorded, animal subject was active, the mental status, movable unusual for seeing; Breathe steadily, do not see cyanosis, tic; Appetite and stool no abnormality seen.Tangible irritant reaction is not seen in the administration part.Duration of test does not have animal dead.
(2) rabbit percutaneous drug delivery experiment repeatedly
40 of rabbit are divided 4 groups at random, and 10 every group, male and female half and half.Skin is smeared rhaFGF, and dosage is respectively 1000,3000,6000U/cm2 and control group (water for injection), and every day, percutaneous drug delivery was 1 time, and successive administration 28 days continues after the drug withdrawal to observe for 4 weeks.9 of the general physical signs of experimental observation, 10 of hematological indices, 15 of blood biochemicals, pathological examination kind of organ-tissue surplus 20.The result shows, compares with control group, and each administration treated animal fur fairing of duration of test, the mental status is good, behavioral activity is normal, breathes steadily, and feed is normal, that weight increase, administration local skin are not seen is coarse, chap, thicken, fash or focus of infection, and damaged skin is not seen cicatrization yet.Electrocardiogram(ECG, hematology, blood biochemical, organ weights and organ coefficient etc. all fluctuate in normal range, do not see that the regularity with toxicology meaning changes.During drug withdrawal and drug withdrawal 2 week the back animals do not produce rhaFGF antibody.Each internal organs of pathological examination are not seen the abnormal change relevant with medicine, and the administration local skin is organized the no abnormality seen hyperplasia.
(3) guinea pig skin hypersensitive test
Cavy cut off back wool with scissors in being tried in preceding 24 hours, and the every side of unhairing area is about 3 * 3cm2, got to be subjected to test product 0.2ml to be coated in depilation district, animal left side, and is fixing, continues 6 hours, and the 7th day and the 14th day in kind repeats once; Last is given and to be tried after the thing sensitization 14 days, will be tried thing 0.2ml and be coated in depilation district, animal right side, removes after 6 hours and is tried thing, observes immediately, and observes the national response situation of skin once more in 24,48,72 hours.The result shows that rhaFGF does not have sensitization, and the sensitization rate is 0.0%, does not see that erythema and oedema appear in guinea pig back administration district.
Sequence table
＜110〉Peng Juan Zhao Quan
＜120〉a kind of manufacturing of new fusion protein and application
<160>2
<210>1
<211>906
<212>DNA
＜213〉artificial sequence
<400>1
atgagcgata?aaattattca?cctgactgac?gacagttttg?acacggatgt?actcaaagcg 60
gacggggcga?tcctcgtcga?tttctgggca?gagtggtgcg?gtccgtgcaa?aatgatcgcc 120
ccgattctgg?atgaaatcgc?tgacgaatat?cagggcaaac?tgaccgttgc?aaaactgaac 180
atcgatcaaa?accctggcac?tgcgccgaaa?tatggcatcc?gtggtatccc?gactctgctg 240
ctgttcaaaa?acggtgaagt?ggcggcaacc?aaagtgggtg?cactgtctaa?aggtcagttg 300
aaagagttcc?tcgacgctaa?cctggccggt?tctggttctg?gccatatgca?ccatcatcat 360
catcattctt?ctggtctggt?gccacgcggt?tctggtatga?aagaaaccgc?tgctgctaaa 420
ttcgaacgcc?agcacatgga?cagcccagat?ctgggtaccg?acgacgacga?caaggccatg 480
gctttcaacc?tgccgccggg?taactacaaa?aaaccgaaac?tgctgtactc?ctccaacggt 540
ggtcacttcc?tgcgtatcct?gccggacggt?accgttgacg?gtacccgtga?ccgttccgac 600
cagcacatcc?agctgcagct?gtccgctgaa?tccgttggtg?aagtttacat?caaatccacc 660
gaaaccggtc?agtacctggc?tatggacacc?gacggtctgc?tgtacggttc?ccagaccccg 720
aacgaagaat?ccctgttcct?ggaacgtctg?gaagaaaacc?actacaacac?ctacatctcc 780
aaaaaacacg?ctgaaaaaaa?ctggttcgtt?ggtctgaaaa?aaaacggttc?ctccaaacgt 840
ggtccgcgta?cccactacgg?tcagaaagct?atcctgttcc?tgccgctgcc?ggtttcctcc 900
gactaa 906
<210>2
<211>301
<212>PRT
＜213〉artificial sequence
<400>2
Met?Ser?Asp?Lys?Ile?Ile?His?Leu?Thr?Asp?Asp?Ser?Phe?Asp?Thr
5 10 15
Asp?Val?Leu?Lys?Ala?Asp?Gly?Ala?Ile?Leu?Val?Asp?Phe?Trp?Ala
20 25 30
Glu?Trp?Cys?Gly?Pro?Cys?Lys?Met?Ile?Ala?Pro?Ile?Leu?Asp?Glu
35 40 45
Ile?Ala?Asp?Glu?Tyr?Gln?Gly?Lys?Leu?Thr?Val?Ala?Lys?Leu?Asn
50 55 60
Ile?Asp?Gln?Asn?Pro?Gly?Thr?Ala?Pro?Lys?Tyr?Gly?Ile?Arg?Gly
65 70 75
Ile?Pro?Thr?Leu?Leu?Leu?Phe?Lys?Asn?Gly?Glu?Val?Ala?Ala?Thr
80 85 90
Lys?Val?Gly?Ala?Leu?Ser?Lys?Gly?Gln?Leu?Lys?Glu?Phe?Leu?Asp
95 100 105
Ala?Asn?Leu?Ala?Gly?Ser?Gly?Ser?Gly?His?Met?His?His?His?His
110 115 120
His?His?Ser?Ser?Gly?Leu?Val?Pro?Arg?Gly?Ser?Gly?Met?Lys?Glu
125 130 135
Thr?Ala?Ala?Ala?Lys?Phe?Glu?Arg?Gln?His?Met?Asp?Ser?Pro?Asp
140 145 150
Leu?Gly?Thr?Asp?Asp?Asp?Asp?Lys?Ala?Met?Ala?Phe?Asn?Leu?Pro
155 160 165
Pro?Gly?Asn?Tys?Lys?Lys?Pro?Lys?Leu?Leu?Tyr?Ser?Ser?Asn?Gly
170 175 180
Gly?His?Phe?Leu?Arg?Ile?Leu?Pro?Asp?Gly?Thr?Val?Asp?Gly?Thr
185 190 195
Arg?Asp?Arg?Ser?Asp?Gln?His?Ile?Gln?Leu?Gln?Leu?Ser?Ala?Glu
200 205 210
Ser?Val?Gly?Glu?Val?Tyr?Ile?Lys?Ser?Thr?Glu?Thr?Gly?Gln?Tyr
215 220 225
Leu?Ala?Met?Asp?Thr?Asp?Gly?Leu?Leu?Tyr?Gly?Ser?Gln?Thr?Pro
230 235 240
Asn?Glu?Glu?Ser?Leu?Phe?Leu?Glu?Arg?Leu?Glu?Glu?Asn?His?Tyr
245 250 255
Asn?Thr?Tyr?Ile?Ser?Lys?Lys?His?Ala?Glu?Lys?Asn?Trp?Phe?Val
260 265 270
Gly?Leu?Lys?Lys?Asn?Gly?Ser?Ser?Lys?Arg?Gly?Pro?Arg?Thr?His
275 280 285
Tyr?Gly?Gln?Lys?Ala?Ile?Leu?Phe?Leu?Pro?Leu?Pro?Val?Ser?Ser
290 295 300
Asp
301
Claims (6)
1. the fusion rotein of human acid fibroblast growth factor and Trx, its fusion rotein aminoacid sequence is the sequence of sequence 2 in the sequence table.
2. the dna sequence dna of the fusion rotein of the claim 1 of encoding.
3. the dna sequence dna of claim 2 is sequences of sequence 1 in the sequence table.
4. the preparation method of the fusion rotein of claim 1 may further comprise the steps:
A. the dna fragmentation of human acid fibroblast growth factor aminoacid sequence in the synthetic coding claim 1;
B. the gene fragment that obtains among the step a. is inserted 3 ' end of bacterium thioredoxin (Tioredoxin) gene on the expression vector;
The expression vector recombinant plasmid dna that c. will contain the Tioredoxin-aFGF antigen-4 fusion protein gene transforms suitable intestinal bacteria; This genetic engineering bacterium is prepared highly purified Tioredoxin-aFGF fusion rotein through fermentation, extraction, purification procedures.
5. the preparation method of claim 4, wherein Shi Yi intestinal bacteria are pET32a.
6. the preparation method of claim 4 is wherein fermented, extraction, purification procedures be: genetic engineering bacterium is seeded in the suitable culture base cultivates, the zymophyte of centrifugal collection; Thalline obtains rough inclusion body through high speed centrifugation again after the high-pressure homogenization fragmentation; Dissolved inclusion body 12 hours with the Guanidinium hydrochloride damping fluid that contains 7mol/L, centrifugal then collection supernatant; With dilution method renaturation rhaFGF, renaturation 48 hours carries out ultrafiltration and concentration and damping fluid is dialysed removing Guanidinium hydrochloride then; Purifying at first adopts the affine exchange column chromatography purification of Chelating Sepharose Fast Flow Trx-aFGF, with the buffer solution elution purpose fusion rotein that contains imidazoles; Use Heparin Sepharose 6Fast Flow affinitive layer purification Tioredoxin-aFGF fusion rotein at last, with containing 1M NaCl wash-out purpose fusion rotein.
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| CN107961394B (en) * | 2017-10-27 | 2020-10-13 | 南开大学 | anti-HPV cervical wound-protecting dressing design based on ternary fusion protein |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1500810A (en) * | 1996-12-27 | 2004-06-02 | 暨南大学生物工程研究所 | Fibroblast growth factor-2 analogue, producing process and application thereof |
| CN1504573A (en) * | 2003-09-01 | 2004-06-16 | 广州暨南大学医药生物技术研究开发中 | Confluent protein capable of self-cracking into polypeptide with antibiosis and reparation function |
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2005
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1500810A (en) * | 1996-12-27 | 2004-06-02 | 暨南大学生物工程研究所 | Fibroblast growth factor-2 analogue, producing process and application thereof |
| CN1504573A (en) * | 2003-09-01 | 2004-06-16 | 广州暨南大学医药生物技术研究开发中 | Confluent protein capable of self-cracking into polypeptide with antibiosis and reparation function |
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