CN102020710A - Novel mutant EN-46 of human epidermal growth factor - Google Patents
Novel mutant EN-46 of human epidermal growth factor Download PDFInfo
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- CN102020710A CN102020710A CN 201010271518 CN201010271518A CN102020710A CN 102020710 A CN102020710 A CN 102020710A CN 201010271518 CN201010271518 CN 201010271518 CN 201010271518 A CN201010271518 A CN 201010271518A CN 102020710 A CN102020710 A CN 102020710A
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
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Abstract
The invention belongs to the technical field of bioengineering and provides a novel mutant EN-46 of a human epidermal growth factor, in particular to a mutant EN-46 and a nucleic acid sequence of the mutant EN-46 as well as a vector containing the nucleic acid sequence and engineering bacteria containing the vector. The invention also provides a method for preparing C-end frameshift mutant of the human epidermal growth factor, in particular to a method for preparing the mutant EN-46, a composition containing the mutant and application of the composition in preparing medicines, health products or cosmetics. The mutant can achieve complete soluble expression in escherichia coli, the permeability of a cell membrane is enhanced, and expression products are completely secreted in an extracellular culture medium and entirely have the biologicalactivity of the human epidermal growth factor.
Description
Technical field
The present invention relates to a kind of human epidermal growth factor, be specifically related to a kind of human epidermal growth factor's mutant, Preparation Method And The Use.
Background technology
There is a peptide species in discovery mouse submandibular gland such as U.S. biologist professor Cohen in the period of the 1960-1963, has short widely cel l proliferation, and naming is Urogastron (epidermalgrowth factor is called for short EGF).Shortly after that, scientist has found the similar activity material in people's urine, is called as human epidermal growth factor (hEGF).Research afterwards confirms that further Urogastron extensively is present in people and the mammal body, is that a molecular weight of being made up of 53 amino acid is 6222 micromolecule polypeptide, and iso-electric point is 4.6.Mouse and people's Urogastron has the homology of height.Urogastron has biologic activity widely, takes place and aspect such as fetal development has vital role in cell growth and metabolism, cytodifferentiation and contrary differentiation and in form.
After the seventies in last century, people have carried out a large amount of research to structure and the function of hEGF, discovery influences the sequence that the active core of hEGF position only comprises at 3 pairs of disulfide linkage of intramolecularly, and N-holds before 6 halfcystines and aminoacid deletion or the change afterwards of 42 halfcystines of C-end do not have substantial influence to its biologic activity.
Confirm that now Urogastron is tissue injury reparation and the important molecular basis of human body regeneration, has been widely used in the clinical medicine applied research since the nineties in last century.China takes the lead in realizing the hEGF large-scale industrial production in the world, and official approval is used for all kinds of wounds of clinical treatment as medicine.
The hEGF suitability for industrialized production mainly adopts gene recombination technology at present, and most of is gene host bacterium with intestinal bacteria.Because intestinal bacteria are not modified and mechanism of secretion after not possessing perfect albumen, foreign gene (comprising the hEGF gene) expression product usually is present in the cell paste with insoluble inclusion body form.This brings great trouble for the downstream aftertreatment, and cell needs fragmentation, and inclusion body needs cracking, sex change and repeatability, could obtain activated product at last.Expression product the phenomenon of mispairing usually can occur at sex change, renaturation process, and the last yield of product is extremely low, and quality also is difficult to control.This defective has become the technology barrier of escherichia expression system maximum in the recombinant protein industrialization process.
Summary of the invention
The purpose of this invention is to provide a kind of human epidermal growth factor C-end phase shift mutant, especially mutant EN-46 and nucleotide sequence thereof, and the carrier and the engineering bacteria that comprise this nucleotide sequence.
Another object of the present invention provides preparation human epidermal growth factor C-end phase shift mutant, the especially preparation method of mutant EN-46.Be specifically related to a kind of expressed sequence with PCR random mutation technique construction human epidermal growth factor C-end phase shift mutant EN-46.
The present invention also provides composition and the application in preparation medicine, healthcare products or makeup thereof that contains human epidermal growth factor C-end phase shift mutant EN-46.
The invention has the advantages that this mutant, especially mutant EN-46 can realize solubility expression completely in host cell especially intestinal bacteria, the saturating property enhancing of cell membrane, expression product all is secreted in the outer substratum of born of the same parents, is convenient to the extraction separation purifying, is easy to suitability for industrialized production.Host cell especially this mutant polypeptide of escherichia coli expression has human epidermal growth factor's biologic activity fully, the short cell fission and the proliferation activity of concrete natural human Urogastron.
Below the present invention is done detailed description.
One, obtains the mutant nucleic acid molecule with PCR random mutation technology
When utilizing the Taq archaeal dna polymerase to carry out DNA amplification in vitro (PCR), base mispairing, disappearance or increase can occur, thereby cause occurring in the PCR product various mutant.This sudden change be fully at random, without any rule, disappearance or increase a base and will make original template DNA begin the dna sequence dna generation phase shift mutation of back during dna replication dna from disappearance or this base of increasing.People usually utilize the characteristics of this fallibility of PCR to make up the range gene mutant in experimental study, to study the influence that sudden change is caused the biology of gene function.Utilize above-mentioned principle, it is the PCR amplification in vitro that template is carried out that the present invention adopts the hEGF gene DNA of synthetic, obtains human epidermal growth factor's mutant.
Described human epidermal growth factor's mutant comprises human epidermal growth factor C-end phase shift mutant; Preferably, comprise all amino acid phase shift mutants behind 42 halfcystines of human epidermal growth factor C-end.
The amino acid no purpose increased after described human epidermal growth factor's mutant also comprised 42 halfcystines of C-end, replaced or lack one or more, as was no more than 11, preferably was no more than 8,5, more preferably no more than 4, and still kept its biologic activity; Preferably, comprise 42 halfcystines of C-end after amino acid number reduce, be reduced to 10 by 11 original amino acid, 8,5,4,3,2 or 1; Most preferred, C has appearred in the sequence of this mutant behind 42 halfcystine codons of coding hEGF C-end
127The disappearance of base and cause the phase shift mutation of aminoacid sequence generation subsequently, terminator codon TGA occurs in advance, and the amino acid number behind 42 halfcystines of C-end is reduced to 4 by 11, and aminoacid sequence is by original C ys
42Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg sports Cys
42Ser Thr ValThr, molecular weight is reduced to 5,036 dalton by 6,222 original dalton, this mutant called after human epidermal growth factor mutant EN-46.
The nucleotide sequence of the preferred mutant EN-46 of the present invention and normal people's Urogastron and aminoacid sequence contrast are as follows:
HEGF nucleotide sequence AAT TCC GAC TCT GAA TGC CCG CTG TCT CAC GAC GGT TAC TGC CTA CAC GAT GGT
HEGF aminoacid sequence Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly
EN46 nucleotide sequence AAT TCC GAC TCT GAA TGC CCG CTG TCT CAC GAC GGT TAC TGC CTA CAC GAT GGT
GTT?TGC?ATG?TAT?ATC?GAA?GCT?CTG?GAC?AAA?TAC?GCG?TGC?AAC?TGT?GTT?GTT?GGT?TAC?ATC?GGT?GAA?CGT
Val?Cys?Met?Tyr?Ile?Glu?Ala?Leu?Asp?Lys?Tyr?Ala?Cys?Asn?Cys?Val?Val?Gly?Tyr?Ile?Gly?Glu?Arg
GTT?TGC?ATG?TAT?ATC?GAA?GCT?CTG?GAC?AAA?TAC?GCG?TGC?AAC?TGT?GTT?GTT?GGT?TAC?ATC?GGT?GAA?CGT
HEGF nucleotide sequence TGC CAG TAC CGT GAC CTG AAA TGG TGG GAA CTG CGT
42 at hEGF C-end
Aminoacid sequence Cys behind the Gelucystine
42Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg
EN46 nucleotide sequence TGC AGT ACC GTG ACC TGA AATGGTGGGACTGCGTTAATAA
42 at EN-46 C-end
Aminoacid sequence Cys behind the Gelucystine
42Ser Thr Val Thr Stop
Sequencing result shows that nucleotide sequence and aminoacid sequence are identical before 42 halfcystines of mutant EN-46 and normal hEGF C-end, but the aminoacid sequence behind 42 halfcystines of C-end is then different fully with amino acid number.The aminoacid sequence of described mutant EN-46 is shown in sequence table 2, and its nucleotide sequence is shown in sequence table 1.This mutant can be realized solubility expression completely in host cell, expression product all is secreted in the outer substratum of born of the same parents.This mutant polypeptide of expressing in host cell has human epidermal growth factor's biologic activity fully.Wherein said host cell is intestinal bacteria.
Two, make up the expression vector plasmid that contains mutant
The mutant gene that obtains according to the method described above is connected with escherichia coli outer membrane protein leading peptide (ompA) gene, places T7 promotor (T7pro) and terminator (rrnBT
1T
2) between, construct the expression vector plasmid that contains mutant.
Described carrier is characterized in that containing the controlling element handled that the mutant EN-46 that is suitable for described human epidermal growth factor expresses, that link with described nucleic acid molecule in the purpose host cell.
Three, the engineering strain that efficiently expresses with expression vector plasmid transformed host cell and screening
Change the above-mentioned expression vector plasmid that builds over to host cell with ordinary method, be preferably intestinal bacteria.The transformant that contains above-mentioned expression framework can filter out the bacterium colony of the mutant that efficiently expresses.
Preferably, described intestinal bacteria are E.coli JM109 (DE3), the bacterium colony called after E.coli EN-46 that contains high expression level EN-46 that filters out.
Four, the cultivation of engineering bacteria and abduction delivering
(1) preparation is fermented with common LB substratum, pH7.0-8.5;
(2) engineering bacteria is seeded in the described substratum, 37 ℃ of cultivations;
(3) be cultured to OD
600When reaching 3.0-4.0, add final concentration 12.5-100 μ gIPTG/ml abduction delivering, temperature regulation to 30 ℃ is regulated pH 7.0-8.5 therebetween;
(4) after abduction delivering 4-18 hour, stop fermentation, fermented liquid is collected supernatant liquor by membrane filtration.
Collect supernatant liquor and thalline and carry out the SDS-PAGE gel electrophoresis simultaneously.The result shows that the expression product that major part is tried bacterium colony all is secreted in the substratum with solubility, finds no in the thalline to express band.
Select the highest bacterium colony of expression amount and amplify fermentation test, further study its optimum process condition.Express stable single bacterium colony through continuous 3 batch fermentations proof, be prepared into the work seed, be stored in-20 ℃ of refrigerators.Described work seed is that engineering strain is E.coli EN-46.
This project bacterial classification has been delivered Chinese typical culture collection center (place: Wuhan City, Wuhan University) preservation, and the engineering bacterial strain of preservation is intestinal bacteria EN-46 Escherichia coli EN-46,
Preservation date: on July 19th, 2010, deposit number is CCTCC M 2010178.
Five, separation and purification mutant
Adopt micro-filtration, hydrophobic chromatography, anionresin and gel-filtration array mode separation and purification mutant.Preferred adopt following simple three-step approach: catch mutant in the fermented liquid with hydrophobic chromatography earlier, adopt anion-exchange chromatography separation and purification mutant again, adopt gel permeation chromatography to be further purified mutant at last.Through simple three step operations, can obtain purity greater than 95%, mutant with human epidermal growth factor's biologic activity, total recovery can reach more than 70%.
Described mutant is preferably mutant EN-46.
Six, the composition that contains mutant
A kind of composition comprises the human epidermal growth factor's who treats significant quantity mutant and derivative or vehicle or thinner or auxiliary substance.Described composition comprises pharmaceutical composition, Halth-care composition, perhaps make-up composition.Wherein treat significant quantity and be meant the dosage of biologic activity of performance mutant.Described mutant comprises the mutant of aforesaid various ways, preferably mutant EN-46.Described derivative is meant the multiple modified forms that comprises this mutant, comprises that the PEG of different molecular weight modifies body.Its consumption can be according to patient's age, body weight, and the severity of disease is adjusted.The per daily dose of wherein said mutant EN-46 is generally 0.05-5 μ g/cm
2
Described pharmaceutical composition, the mutant that comprises described human epidermal growth factor is preferably mutant EN-46 and acceptable accessories and forms, and described auxiliary material comprises the thinner of pharmaceutical field routine, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, lubricant etc.
The dosage form of this pharmaceutical composition comprises conventional oral preparations and non-gastrointestinal preparation.Oral preparations comprises, solid preparation, and as capsule, tablet, pill, granule; Liquid preparation is as syrup etc.Non-intestinal drug delivery agent comprises injection, spray, and ointments etc. also comprise freeze-dried powder.Comprise some novel drug-delivery preparations in addition, as slow, controlled release preparation, microemulsion formulation, targeting drug administration preparation etc.Described human epidermal growth factor's mutant is preferably mutant EN-46.Above-mentioned various formulation all can be according to the ordinary method preparation of pharmaceutical field.
Healthcare products and cosmeceutical composition and preparation thereof can comprise auxiliary material and dosage form thereof conventional in the field, can not elaborate at this according to preparation method's preparation conventional in the field.Seven, mutant is in preparation medicine, the purposes in healthcare products and the makeup
The mutant, especially the mutant EN-46 that prepare by the inventive method have human epidermal growth factor's biologic activity preferably, comprise short cell fission and proliferation activity with natural human Urogastron.Its traumatic wound that can heal is rapidly and efficiently repaired various skin injuries, reduces the formation of scar, to treating skin burn and other damages clinically unusual effect is arranged.In addition, this sudden change physical efficiency promotes the propagation of Eponychium cell, and the energy delaying decrepitude of skin promotes the effect that skin cells is repaired.Therefore human epidermal growth factor's mutant EN-46 and comprise human epidermal growth factor's mutant EN-46 can be at interior composition at the preparation medicine, application in healthcare products or the makeup, be included in preparation and promote application in the various medicine for healing wound, smooth away wrinkles and repair application in medicine such as acne or the makeup in preparation.The inventor has confirmed EN-46 to the promoting healing effect of the acute and chronic surface of a wound and in the application aspect the aesthetic medicine by specific embodiment, and result of treatment is remarkable.
Description of drawings
The original collection of illustrative plates of order-checking of Fig. 1 mutant EN-46 DNA
Fig. 2 test tube is expressed the SDS-PAGE electrophoresis result of EN-46 test
1.EN-46 induce 4 hours culture supernatant
2.EN-46 induce 4 hours thalline ultrasonic disruption supernatants
3.EN-46 induce 4 hours thalline ultrasonic disruption precipitations
4.hEGF induce 4 hours culture supernatant
5.hEGF induce 4 hours thalline ultrasonic disruption supernatants
6.hEGF induce 4 hours thalline ultrasonic disruption precipitations
7. inclusion body hEGF (contrast)
Fig. 35 liter jar fermentor tanks are expressed the SDS-PAGE electrophoresis result of EN-46
1. 3 hours culture supernatant of abduction delivering
2. 3 hours thalline of abduction delivering
3. 18 hours culture supernatant of abduction delivering
4. 18 hours thalline of abduction delivering
5. standard hEGF
Fig. 4 EN-46 purifying sample SDS-PAGE electrophoresis result
1. the supernatant of fermented liquid behind 0.45 μ m micro-filtration
2. the wash-out sample (compound sample) after the hydrophobic chromatography post captures
3. the wash-out sample (peak point) after the hydrophobic chromatography post captures
4. the wash-out sample (peak point) after ion column separates
5. the wash-out sample (compound sample) after ion column separates
6. the consummate sample behind gel permeation chromatography
7. standard hEGF
The graphic representation of the short cell proliferation of Fig. 5 EN-46 and standard hEGF
Fig. 6 EN-46 is to scalding to the patient Chen's of dark II degree result of treatment
Left figure: before treat with EN-46 the scald back
Middle figure: scald the back and use EN-46 preparation for treating 7 days, be almost recovered
Right figure: scald the back and use EN-46 preparation for treating 14 days, return to one's perfect health
Fig. 7 conventional medicament is treated the patient with diabetic feet that two wheat harvesting periods are not got better to be changeed, and uses the result of treatment behind the EN-46 instead
Left figure: before the EN-46 treatment
In figure: after treating for 1 week with EN-46
Right figure: after treating for 2 weeks with EN-46
Fig. 8 EN-46 has obvious repair to the acne local damage
Left figure: before the EN-46 treatment
Right figure: with EN-46 treatment 1 week back rehabilitation, and trace not
Fig. 9 EN-46 has the effect of tangible elimination canthus wrinkle to experimenter A
Trier A:55 year, the women
Left figure: before the EN-46 on probation
Right figure: on probation after 1 month canthus wrinkle obviously shoal
Figure 10 EN-46 has the effect of tangible elimination canthus wrinkle to experimenter B
Trier B:45 year, the women
Left figure: before the EN-46 on probation
Right figure: canthus wrinkle completely dissolve after 1 month on probation
Specific embodiment
Below the present invention is illustrated, be intended to set forth optimum implementation of the present invention, but protection scope of the present invention is not limited to this embodiment and experimental example with embodiment and experimental example.
Entrust Invitrogen company's synthetic pcr primer thing (primer1 and primer2):
Primer1：5’-CAT?
CAT?ATG?AAA?AAG?ACA?GCT?ATC-3’
NdeI
Primer2：5’-CAT?
CTG?CAG?TTA?TTA?ACG?CAG?TCC?CAC-3’
PstI
In the PCR reaction system of 50 μ l, add:
H
2O 37μl
4dNTP (each 200 μ M) 4 μ l
Primer1(0.25μM) 1μl
Primer2(0.25μM) 1μl
Template DNA (ompA-EGF) 1 μ l
The PCR reaction:
94 ℃ of preheatings 5 minutes, follow procedure: 94 ℃, 30 seconds--50 ℃, 30 seconds--72 ℃, 30 seconds, react 25 circulations.
The PCR reaction
The structure of embodiment 2 mutant EN-46 expression vector plasmids
The NdeI/PstI endonuclease bamhi of PCR product is connected on Y2K0422 (NdeI/PstI) expression vector.The mutant gene EN-46 that obtains places between strong promoter T7 and the terminator rrnBT1T2.Y2K0422 is the efficient expression vector plasmid that our company makes up and preserves, and this expression vector plasmid has the copy number height, can suppress non-target protein after IPTG induces and express and make target protein obtain the feature of high expression level.This expression vector plasmid is also available to have identical or equivalent feature, commercially available expression vector of the same type with Y2K0422 expression vector plasmid and replaces.
PCR product (NdeI/PstI) double digestion fragment
The expression framework of mutant EN-46
Wherein:
T7pro is the T7 promotor
OmpA is the escherichia coli outer membrane protein leading peptide
EN-46 is a hEGF mutant of the present invention
RrnBT
1T
2It is a Transcription Termination subsequence
The original collection of illustrative plates of dna sequencing of this mutant EN-46 is seen Fig. 1, and the result shows, C occurred in the sequence behind 42 halfcystine codons of coding C-end
127The disappearance of base and cause the phase shift mutation of aminoacid sequence generation subsequently, termination codon TGA occurs in advance, and the amino acid number behind 42 halfcystines of C-end is reduced to 4 by 11.
1. the expression vector plasmid transforms the host bacterium
Change the above-mentioned plasmid that builds over to intestinal bacteria E.coli JM109 (DE3) with ordinary method, the transformant that contains above-mentioned expression framework is used to screen the bacterium colony of high expression level EN-46.
2. the cultivation of engineering bacteria and abduction delivering and expression product born of the same parents' evaluation
Select some transformant bacterium colonies and in test tube, express test.Express and test: peptone 10-15 with common LB substratum (g/1); Yeast extract 5-15; NaCl 5-10; Na
2HPO
40-10; Penbritin 0.1-0.2, initial pH7.0.
The inoculation test tube is cultured to OD at 37 ℃
600When reaching 3.0-4.0, added 100mM IPTG abduction delivering 3-4 hour, centrifugal, to collect supernatant and thalline and carry out the SDS-PAGE gel electrophoresis simultaneously, gel electrophoresis the results are shown in Figure 2.Electrophoresis result shows that the target product that EN-46 expresses all is secreted into culture supernatant in solvable mode, and supernatant behind the thalline ultrasonic disruption and precipitation all find no the target protein band and exist.And the normal hEGF that has identical expression framework in contrast has only a small amount of target product in culture supernatant, and most of expression product is present in the precipitation behind the ultrasonic disruption in the inclusion body mode.
3. select the highest bacterium colony of expression amount and amplify fermentation test
Engineering bacteria with screening in above-mentioned 1, preparation carries out fermentation test in 5 liters of jars.Ferment with common LB substratum (g/1): peptone 10-15; Yeast extract 5-15; NaCl 5-10; Na
2HPO
40-10; Penbritin 0.1-0.2, pH7.0-8.5.
Engineering bacteria is cultured to OD for 37 ℃
600When reaching 3.0-4.0, add final concentration 12.5-100 μ gIPTG/ml abduction delivering, temperature regulation to 30 ℃ is regulated between the pH7.0-8.5 therebetween.Induce sampling after 3-4 hour, centrifugal, collect supernatant and thalline.Continue abduction delivering and stopped fermentation in 10-14 hour, fermented liquid is collected supernatant, thalline with 0.45 μ m membrane filtration.The sample of twice collection in front and back carries out the SDS-PAGE gel electrophoresis, and gel electrophoresis the results are shown in Figure 3.5 liter jar fermentation test results reconfirm that EN-46 all is secreted in the substratum in the solubility mode after 18 hours at abduction delivering, and thalline finds no residual.
4. the fermented supernatant fluid of collecting behind the membrane filtration is waited until next step separation, purification process.
5. express stable single bacterium colony through continuous 3 batch fermentations proof, be prepared into the work seed lot, be stored in-20 ℃ of refrigerators.
The simple three-step approach separation and purification of embodiment 4 usefulness EN-46
The first step is caught EN-46 in the fermented liquid with hydrophobic chromatography
(1) ferment filtrate of collecting in application example 3 slowly adds 3M (NH
4)
2SO
4To final concentration be 0.3M, catch EN-46 with hydrophobic chromatoghaphy medium;
(2) hydrophobic chromatoghaphy medium: GE company produces Phenyl Sepharose 6 Fast Flow (highsub), chromatography column 0.3M (NH before the last sample
4)
2SO
4/ 20mM Tris (pH8.2) is balance fully;
(3) going up sample finishes and uses earlier lavation buffer solution: 0.3M (NH
4)
2SO
4/ 20mM Tris (pH8.2) thorough washing is to baseline, and then with 0.05M (NH
4)
2SO
4/ 20mM Tris (pH8.2) thorough washing is to baseline;
(4) use elution buffer: the EN-46 of 20mM Tris (pH8.2) elute captured, elutriant are used for the second step ion exchange chromatography and separate;
Second step was used anion-exchange chromatography separation and purification EN-46
(5) to be adjusted to final concentration respectively with 4M NaCl and 0.5M Tris (pH8.2) be 100mM NaCl/20mM Tris (pH8.2) to the hydrophobic chromatography elutriant that contains EN-46;
(6) anion-exchange chromatography medium: GE company produces Q Sepharose High Perfomance, uses the abundant balance of 100mM NaCl/20mM Tris (pH8.2) damping fluid before the last sample;
(7) go up sample and finish, use 380mM NaCl/20mM Tris (pH8.2) damping fluid thorough washing to baseline;
(8) with 650mM NaCl/20mM Tris (pH8.2) buffer solution elution, elutriant is used for the 3rd step gel permeation chromatography and is further purified;
The 3rd step was further purified EN-46 with gel permeation chromatography
(9) gel permeation chromatography medium: GE company produces Superdex30, uses fully balance of 20mM phosphoric acid buffer (pH7.6) before the last sample.
(10) the ion exchange chromatography elutriant that contains EN-46 is directly used in sample, goes up sample 2ml at every turn, flows directly into 20mM phosphoric acid buffer (pH7.6) behind the last sample, per sample in molecular weight vary in size and make EN-46 obtain further separation and purification.
Through simple three step operations, can obtain purity greater than 95%, EN-46 with hEGF biologic activity, total recovery can reach more than 70%.Purifying sample SDS-PAGE electrophoresis result is seen Fig. 4.
Measure and use cell strain: Balb/3T3, be cultured to logarithmic phase with ordinary method.
(1) with DMEM substratum (containing 10% serum) the logarithmic phase cell is adjusted to 5-8x10
4Individual cell/ml, every hole adds 100 μ l cell suspensions in 96 orifice plates.37 ℃, 5%CO
2Overnight incubation;
(2) supernatant discarded adds DMEM substratum (containing 0.5% serum) 100 μ l, and 37 ℃, 5%CO
2Overnight incubation;
(3) supernatant discarded adds DMEM substratum (containing 0.5% serum and different concns EN-46) 100 μ l, and 37 ℃, 5%CO
2Cultivated 72 hours;
(4) every hole adds MTT solution (5.0mg/ml) 20 μ l, 37 ℃, 5%CO
2Incubation 5 hours, supernatant discarded adds 100 μ l10%SDS, and cracking is 20 minutes under the room temperature, measures extinction value (OD value) in wavelength 570nm;
(5) each weaker concn is made the balance controlled trial with standard hEGF simultaneously;
(6) be that X-coordinate, corresponding OD value are the ordinate zou mapping with EN-46, standard hEGF concentration, measure the maximum half proliferative amount of EN-46 and be no more than 10ng/ml, quite active with standard hEGF.
The EN-46 of table different concns and standard hEGF cultivate mtt assay detection OD after 5 days
570The result
(concentration unit: ng/ml)
The short cell-proliferation activity graphic representation of EN-46 and standard hEGF is seen Fig. 5.
It is the pharmaceutical composition of main active ingredient with EN-46 that this invention has disclosed a kind of.Said composition is a kind of liquid preparation, can be sprayed directly on the injured surface of a wound.Consisting of of said composition:, add the EN-46 of 0.5-10mg in the phosphate buffer solution of 20mM (pH6.5-7.0), 50-100g glycerine, 1-10g N.F,USP MANNITOL at 1000ml.
It is the composite skin care product of main active ingredient with EN-46 that this invention has disclosed a kind of.Said composition is a kind of gel preparation, can directly spread upon on the skin.Consisting of of said composition:, add the EN-46 of 0.5-5mg in the phosphate buffer solution of 20mM (pH6.5-7.0), 5-10g glycerine, 1-10g N.F,USP MANNITOL, 1-8g carbomer at 1000ml.
Embodiment 8 contains the external use liquid sprays of mutant EN-46
It is the liquid spray of main active ingredient with EN-46 that the present invention discloses a kind of.This sprays can be sprayed directly on the injured surface of a wound.Consisting of of this liquid spray:, add the EN-46 of 0.5-10mg in the phosphate buffer solution of 20mM (pH6.5-7.0), optimum addition 1-4mg at 1000ml; 50-100g glycerine, optimum addition 60-80g; 1-10g N.F,USP MANNITOL, optimum addition 6-8g.
Embodiment 9 contains the skin-care gel agent of mutant EN-46
It is the skin-care gel agent of main active ingredient with EN-46 that this invention has disclosed a kind of.This gelifying agent can directly spread upon on the skin, and it consists of: at 1000ml, add the EN-46 of 0.5-5mg in the phosphate buffer solution of 20mM (pH6.5-7.0), optimum addition is 0.8-2mg; 5-10g glycerine, optimum addition are 6-8g; 1-10g N.F,USP MANNITOL, optimum addition are 6-8g; 1-8g carbomer, optimum addition are 2-5g.
The effect that the sprays that embodiment 10 contains mutant EN-46 has the acceleration of wound reparation, improves healing quality
Observed the effect of EN-46 sprays (evenly spraying the surface of a wound every day 2 times) to trauma repair.Test-results shows that the EN-46 preparation all has tangible promotion epithelial growth, quickens the effect of wound healing all kinds of surface of a wound, and total effective rate surpasses 90%.
One of them case and result of treatment thereof: Chen, 4 years old, to be scalded to dark II degree by boiling water accidentally, therapeutic process only uses the EN-46 liquid preparation, and basic recovery from illness in the 7th day returns to one's perfect health after 14 days, and does not stay any vestige.The results are shown in Figure 6.
Embodiment 11 contains the repair of mutant EN-46 sprays to refractory wounds
Observed of the repair of EN-46 sprays to refractory wounds such as diabetic foot and severe pressure sores.Own control is tested before and after adopting treatment.The surface of a wound squirts the surface of a wound with mutant EN-46 preparation local uniform after conventional debridement, every day 2 times.Curative effect judging standard: healing is effective more than 30% in 4 weeks.Test-results shows that EN-46 is efficient up to more than 90% to the treatment diabetic foot, and curative ratio is up to 80%; To the curative ratio of severe pressure sore up to more than 80%.
One of them case and result of treatment: patient with diabetic feet, conventional medicament are treated the commentaries on classics of not getting better of two wheat harvesting periods, use the EN-46 preparation instead and return to one's perfect health after two weeks.The results are shown in Figure 7.
Embodiment 12 contains the application of mutant EN-46 gelifying agent aspect medical cosmetology
Observed and contained the application of mutant EN-46 gelifying agent aspect medical cosmetology.The result shows, use mutant EN-46 handle laser beautifying and the iatrogenic surface of a wound of various beauty treatment (incomplete statistics 1316 examples) healing time than control group shorten near half, do not stay or stay less scar after the healing.Part experimenter's result of treatment the results are shown in Figure 8,9,10.
Claims (24)
1. a human epidermal growth factor mutant is characterized by human epidermal growth factor C-end phase shift mutant.
2. the described human epidermal growth factor's of claim 1 mutant is characterized by all amino acid phase shift mutants behind 42 halfcystines of human epidermal growth factor C-end.
3. the described human epidermal growth factor's of claim 2 mutant, the aminoacid sequence that it is characterized in that this mutant is mutant EN-46 shown in sequence table 2.
4. a human epidermal growth factor mutant EN-46 is characterized in that nucleotide sequence is shown in sequence table 1.
5. the described mutant EN-46 of claim 4 is characterized in that this mutant gene can realize solubility expression completely in host cell, and expression product all is secreted in the outer substratum of born of the same parents.
6. the described mutant EN-46 of claim 5 is characterized in that this mutant polypeptide of expressing has human epidermal growth factor's biologic activity fully in host cell.
7. claim 5 or 6 described mutant EN-46 is characterized in that host cell is intestinal bacteria.
8. the carrier that contains the arbitrary described nucleic acid molecule of claim 4-7.
9. the described carrier of claim 8 is characterized in that containing the controlling element handled that the mutant EN-46 that is suitable for described human epidermal growth factor expresses, that link with described nucleic acid molecule in the purpose host cell.
10. the host cell that contains the described carrier of claim 8 or 9.
11. the described host cell of claim 10 is characterized by intestinal bacteria.
12. the described host cell of claim 11 is characterized in that intestinal bacteria are bacterial strain E.coliEN-46, is preserved in Chinese typical culture collection center, deposit number is CCTCC M2010178.
13. the preparation method of the arbitrary described human epidermal growth factor's of claim 3-12 mutant EN-46 is characterized in that comprising the steps:
(1) obtains mutant EN-46 nucleic acid molecule with PCR random mutation technology;
(2) make up the expression vector plasmid that contains mutant EN-46 nucleic acid molecule;
(3) engineering strain that efficiently expresses with expression vector plasmid transformed host cell and screening;
(4) fermentation culture of engineering strain;
(5) separation and purification mutant.
14. the described preparation method of claim 13 is characterized in that engineering strain is E.coliEN-46.
15. the described preparation method of claim 14 is characterized in that fermentation culture comprises the steps:
(1) preparation is fermented with common LB substratum, pH7.0-8.5;
(2) engineering bacteria is seeded in the described substratum, 37 ℃ of cultivations;
(3) be cultured to OD
600When reaching 3.0-4.0, add final concentration 12.5-100 μ g IPTG/ml abduction delivering, temperature regulation to 30 ℃ is regulated pH 7.0-8.5 therebetween;
(4) after abduction delivering 4-18 hour, stop fermentation, fermented liquid is collected supernatant liquor by membrane filtration.
16. the described preparation method of claim 15 is characterized in that the step of separation and purification comprises the employing hydrophobic chromatography, anionresin and gel-filtration array mode separation and purification mutant EN-46.
17. the described preparation method of claim 16 is characterized in that the step of separation and purification comprises:
The first step is caught mutant EN-46 in the fermented liquid with hydrophobic chromatography;
Second step was used anion-exchange chromatography separation and purification mutant EN-46;
The 3rd step was further purified mutant EN-46 with gel permeation chromatography.
18. a composition is characterized in that containing arbitrary described mutant EN-46 and vehicle or thinner or the auxiliary substance that comprises the human epidermal growth factor who treats significant quantity of claim 3-12.
19. the described composition of claim 18 comprises pharmaceutical composition, Halth-care composition, perhaps make-up composition.
20. the described composition of claim 19, the dosage form of wherein said pharmaceutical composition comprises oral preparations and non-gastrointestinal preparation.
21. the described composition of claim 20 is characterized in that pharmaceutical preparation is an injection, sprays, paste, capsule, tablet.
22. the arbitrary described human epidermal growth factor's of claim 3-21 mutant EN-46 is characterized in that in preparation medicine, the application in healthcare products or the makeup.
23. the described human epidermal growth factor's of claim 22 mutant EN-46, the application of its feature in the various medicine for healing wound of preparation promotion.
24. the described human epidermal growth factor's of claim 22 mutant EN-46, its feature smoothes away wrinkles and repairs the medicine of acne or the application in the makeup in preparation.
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| CN2010102715180A CN102020710B (en) | 2010-09-03 | 2010-09-03 | Novel mutant EN-46 of human epidermal growth factor |
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| CN2010102715180A CN102020710B (en) | 2010-09-03 | 2010-09-03 | Novel mutant EN-46 of human epidermal growth factor |
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| CN102020710B CN102020710B (en) | 2012-09-05 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719439A (en) * | 2012-05-29 | 2012-10-10 | 中山大学 | Mutant human epidermal growth factor gene, protein, preparation methods for mutant human epidermal growth factor gene and protein, and application of mutant human epidermal growth factor gene and protein |
| CN112175063A (en) * | 2020-10-28 | 2021-01-05 | 宁波博睿瀚达生物科技有限公司 | Process for preparing high-purity recombinant epidermal growth factor by high performance liquid chromatography |
| CN112209999A (en) * | 2020-10-28 | 2021-01-12 | 宁波博睿瀚达生物科技有限公司 | Method for rapidly separating pigment in recombinant epidermal growth factor fermentation liquid |
| WO2021077794A1 (en) * | 2018-10-25 | 2021-04-29 | 浙江大学 | Preparation and application of soluble tim-3 recombinant protein and mutant protein thereof |
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| CN1051198A (en) * | 1989-10-11 | 1991-05-08 | 皮特曼-穆尔澳大利亚有限公司 | Recombinant growth factors |
| CN1083371A (en) * | 1993-01-03 | 1994-03-09 | 周至惠 | Acne, folliculitis, erythra special nursing agent |
| CN1160582A (en) * | 1996-12-27 | 1997-10-01 | 暨南大学生物工程研究所 | External-use composite containing cell growth factor |
| CN101316618A (en) * | 2005-11-14 | 2008-12-03 | 株式会社大熊 | Sustained release film formulation for healing wound comprising epidermal growth factor |
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| CN1051198A (en) * | 1989-10-11 | 1991-05-08 | 皮特曼-穆尔澳大利亚有限公司 | Recombinant growth factors |
| CN1083371A (en) * | 1993-01-03 | 1994-03-09 | 周至惠 | Acne, folliculitis, erythra special nursing agent |
| CN1160582A (en) * | 1996-12-27 | 1997-10-01 | 暨南大学生物工程研究所 | External-use composite containing cell growth factor |
| CN101316618A (en) * | 2005-11-14 | 2008-12-03 | 株式会社大熊 | Sustained release film formulation for healing wound comprising epidermal growth factor |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719439A (en) * | 2012-05-29 | 2012-10-10 | 中山大学 | Mutant human epidermal growth factor gene, protein, preparation methods for mutant human epidermal growth factor gene and protein, and application of mutant human epidermal growth factor gene and protein |
| WO2021077794A1 (en) * | 2018-10-25 | 2021-04-29 | 浙江大学 | Preparation and application of soluble tim-3 recombinant protein and mutant protein thereof |
| CN112175063A (en) * | 2020-10-28 | 2021-01-05 | 宁波博睿瀚达生物科技有限公司 | Process for preparing high-purity recombinant epidermal growth factor by high performance liquid chromatography |
| CN112209999A (en) * | 2020-10-28 | 2021-01-12 | 宁波博睿瀚达生物科技有限公司 | Method for rapidly separating pigment in recombinant epidermal growth factor fermentation liquid |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102020710B (en) | 2012-09-05 |
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Denomination of invention: A novel mutant EN-46 of human epidermal growth factor Effective date of registration: 20231212 Granted publication date: 20120905 Pledgee: Shenzhen Branch of China Merchants Bank Co.,Ltd. Pledgor: SHENZHEN WATSIN GENETECH CO.,LTD. Registration number: Y2023980071039 |