CN102115495A - Preparation method and application of protein drug for collagen targeted therapy of hyperplastic scar - Google Patents
Preparation method and application of protein drug for collagen targeted therapy of hyperplastic scar Download PDFInfo
- Publication number
- CN102115495A CN102115495A CN 201010604351 CN201010604351A CN102115495A CN 102115495 A CN102115495 A CN 102115495A CN 201010604351 CN201010604351 CN 201010604351 CN 201010604351 A CN201010604351 A CN 201010604351A CN 102115495 A CN102115495 A CN 102115495A
- Authority
- CN
- China
- Prior art keywords
- leu
- lys
- glu
- asn
- gln
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the field of gene engineering and relates to a targeted hIL-10 recombinant protein called as CBD-IL-10 for short and the application of the CBD-IL-10. The recombinant protein is a recombinant protein expressed by a CBD polypeptide combined by human interlenkin10, hIL-10 and collagen specificity after the fusion in a gene level, and a new protein is generated after recombination. The protein has the anti-virus and anti-tumor activities as well as the immune function same as the hIL-10; and more importantly, the CBD-IL-10 can be combined with the collagen specificity in a targeted way during the wound healing process and can prevent and inhibit the formation of scars in the application of the drug for treating the hyperplastic scar and keloid.
Description
Technical field
The invention belongs to gene engineering technology field, the body's immunity that is specifically related to field of immunology regulates and the human interleukin 10 in the recombination and expression techniques field of gene (human interlenkin 10 hIL-10) merges the expressed recombinant protein in back and this recombinant protein is used with collagen specificity bonded CBD polypeptide at gene level.
Background technology
After the skin injury healing, scar still continues propagation, can develop into hypertrophic cicatrix.Hypertrophic cicatrix is given prominence to surface, and out-of-shape is uneven, flush hyperemia, and quality is real tough.Cusalgia and scratchiness are arranged, increase in envrionment temperature, excited, or symptom aggravation when taking food pungent irritable food.Hyperplasia often continues several months or the degeneration variation takes place a few Nian Houcai gradually, shows as rising height and lowers, and it is dark that color is changeed, and hyperemia is disappeared, deliquescing.Some finally can calm down, and the sufferings symptom also greatly alleviates or disappears.The hyperplasia scar, the good wound that only reaches corium in lesion depths of sending out as dark 2 degree burns with cut the skin donor site surface of a wound etc. of thick middle thickness sheet, also sees darker wound and operative incision once in a while.
The pathological tissue difference of hyperplasia scar and mature scar only is thickening of scar deep collegen filament, irregular arrangement, or be helicoid, or be wound in cord-like.Studying carefully it and form reason, is that extraordinary the continuing of the anabolism of collagen protein carries out because of collagen protein over-deposit in the scar tissue, surpasses catabolic speed, in considerable time, forms collegen filament in a large number and finally forms the hyperplasia scar.
Genetically engineered is a brand-new biotechnology science that is born in 1970's on molecular biology and molecular genetics comprehensive development basis.Discovery mouse Th2 cell strains such as Fiorentino produced a kind of new cytokine in 1989, can suppress transcribing of Th1 cell strain cytokines mRNA, be called cytokine synthesis inhibitory factor(CSIF) (cytokine synthesis inhibitory factor, CSIF), the same year the called after interleukin 10 (interlenkin 10, IL-10).The people is ripe, and the IL-10 molecule is 160 amino-acid residues, molecular weight 18.7kDa, pI8.1.Interleukin 10 (IL-10) is the important immunomodulating cytokines that the human body various kinds of cell produces, has biologic activity widely, comprise immunosuppression, anti-inflammatory and immunomodulatory properties, main biological function is restriction and stops inflammatory reaction, regulate the differentiation and the propagation of panimmunity cell, also have simultaneously the characteristic that opsonic immunity stimulates, remove the particle of infection and non-infection.Inside and outside and experimentation on animals show IL-1 inflammation, cancerate and autoimmune disorder aspect all brought into play critical function, be applied to treat acute and chronic inflammatory disease, autoimmune disorder such as inflammatory enteritis, rheumatoid arthritis, crohn, multiple sclerosis, psoriatic etc. clinically.
But, also there is not effective solution at present at hypertrophic cicatrix.
Summary of the invention
The object of the present invention is to provide a kind of collagen targeted hIL-10 recombinant protein (being called for short CBD-IL-10) and clinical application thereof, this recombinant protein had both kept the biologic activity of hIL-10, increased hIL-10 target in vivo again, not only give this albumen and had activity with same antiviral, the antitumor and body's immunity of hIL-10, it can combine what is more important with the collagen specificity target in wound healing process, thereby prevention suppresses the formation of scar.
For achieving the above object, the technical solution used in the present invention is: a kind of target CBD-IL-10 recombinant protein that is adopted in the preparation of collagen targeted therapy hypertrophic cicatrix protein drug, and its aminoacid sequence is as follows:
H
2N- Met?Ser?Pro?Gly?Gln?Gly?Thr?Gln?Ser?Glu?Asn?Ser?Cys?Thr?His?Phe?Pro?Gly?Asn?Leu?Pro?Asn?Met?Leu?Arg? Asp?Leu?Arg?Asp?Ala?Phe?Ser?Arg?Val?Lys?Thr?Phe?Phe?Gln?Met?Lys?Asp?Gln?Leu?Asp?Asn?Leu?Leu?Leu?Lys? Glu?Ser?Leu?Leu?Glu?Asp?Phe?Lys?Gly?Tyr?Leu?Gly?Cys?Gln?Ala?Leu?Ser?Glu?Met?Ile?Gln?Phe?Tyr?Leu?Glu? Glu?Val?Met?Pro?Gln?Ala?Glu?Asn?Gln?Asp?Pro?Asp?Ile?Lys?Ala?His?Val?Asn?Ser?Leu?Gly?Glu?Asn?Leu?Lys? Thr?Leu?Arg?Leu?Arg?Leu?Arg?Arg?Cys?His?Arg?Phe?Leu?Pro?Cys?Glu?Asn?Lys?Ser?Lys?Ala?Val?Glu?Gln?Val? Lys?Asn?Ala?Phe?Asn?Lys?Leu?Gln?Glu?Lys?Gly?Ile?Tyr?Lys?Ala?Met?Ser?Glu?Phe?Asp?Ile?Lys?Ile?Asn?Tyr
Wherein: H
2N-' represents protein amino terminal (N-terminal), and ' COOH ' represents protein carboxyl terminal (C-terminal).
Above-mentioned targeted hIL-10 recombinant protein is in the application as treatment hypertrophic cicatrix and keloid disease medicament.
Collagen protein over-deposit in the scar tissue, and CBD polypeptide (TKKTLRT, 7 peptides) can specificity incorporating collagen albumen, is collagen protein specificity bonded peptide sequence.With the CBD targeted peptide and the IL-10 amalgamation and expression of collagen protein specific combination, both can reduce dosage and treatment cost, alleviate the toxic side effect that medication brings; What is more important can make IL-10 medicine " target " arrive site of action in the body in vivo and effectively bring into play pharmacological action simultaneously.The present invention compared with prior art, the present invention is merged the recombinant protein that express the back to hIL-10 and collagen specificity bonded CBD polypeptide at gene level by genetic engineering means, formed a kind of new albumen (CBD-IL-10) after the reorganization, and given this albumen and have activity with same antiviral, the antitumor and body's immunity of hIL-10; What is more important CBD-IL-10 can combine with the collagen protein special target in wound healing process, thereby prevention suppresses the formation of scar.
Description of drawings
Fig. 1 is the sequence table of IL-10 aminoacid sequence.
Fig. 2 is the recombinant protein aminoacid sequence table of target CBD-IL-10.
Embodiment
The present invention is merged the recombinant protein (CBD-IL-10) that express the back to hIL-10 and collagen specificity bonded CBD polypeptide at gene level by genetic engineering means.Described hIL-10, its aminoacid sequence is as follows:
H
2N-Met?Ser?Pro?Gly?Gln?Gly?Thr?Gln?Ser?Glu?Asn?Ser?Cys?Thr?His?Phe?Pro?Gly?Asn?Leu?Pro?Asn?Met?Leu?Arg?Asp?Leu?Arg?Asp?Ala?Phe?Ser?Arg?Val?Lys?Thr?Phe?Phe?Gln?Met?Lys?Asp?Gln?Leu?Asp?Asn?Leu?Leu?Leu?Lys?Glu?Ser?Leu?Leu?Glu?Asp?Phe?Lys?Gly?Tyr?Leu?Gly?Cys?Gln?Ala?Leu?Ser?Glu?Met?Ile?Gln?Phe?Tyr?Leu?Glu?Glu?Val?Met?Pro?Gln?Ala?Glu?Asn?Gln?Asp?Pro?Asp?Ile?Lys?Ala?His?Val?Asn?Ser?Leu?Gly?Glu?Asn?Leu?Lys?Thr?Leu?Arg?Leu?Arg?Leu?Arg?Arg?Cys?His?Arg?Phe?Leu?Pro?Cys?Glu?Asn?Lys?Ser?Lys?Ala?Val?Glu?Gln?Val?Lys?Asn?Ala?Phe?Asn?Lys?Leu?Gln?Glu?Lys?Gly?Ile?Tyr?Lys?Ala?Met?Ser?Glu?Phe?Asp?Ile?Lys?Ile?Asn?Tyr?Ile?Glu?Ala?Tyr?Met?Thr?Met?Lys?Ile?Arg?Gln-COOH；
Described collagen specificity bonded CBD targeted peptide, its aminoacid sequence is as follows:
H
2N-Thr Lys Lys Thr Leu Arg Thr-COOH, ' H
2N-' represents protein amino terminal (N-terminal), and ' COOH ' represents protein carboxyl terminal (C-terminal); Connect by introducing the flexible Linker of Gly Gly Gly Gly Ser between hIL-10 and the CBD targeted peptide, can not influence 26S Proteasome Structure and Function separately after the protein expression.
The aminoacid sequence of target CBD-IL-10 after the fusion is as follows:
H
2N-Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Lys Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Gln Gly Gly Gly Gly Ser Thr Lys Lys Thr Leu Arg Thr-COOH,‘H
2N-' represents protein amino terminal (N-terminal), and ' COOH ' represents protein carboxyl terminal (C-terminal).
The expression of embodiment 1:CBD-IL-10, purifying and Determination of biological activity
(1) CBD-IL-10
The acquisition of gene
1. the extraction of total RNA
Human peripheral separates mononuclearcell through lymphocyte separation medium, and PBS washing 2 times is resuspended in 10% RPMI RPMI-1640, and adding final concentration is the ConA of 10g/L, puts 5% CO2 incubator, cultivates 48h, collecting cell for 37 ℃.With reference to the method for RNA extraction agent box, extracted total RNA ,-80 ℃ of preservations are standby.
2. the extraction of total RNA and the amplification of IL-10 cDNA
Extract and the counter-rotating test kit with the RNA of Takara company, by the explanation extracted total RNA, carry out RT-PCR reaction: total RNA 500ng, 5 * reverse transcription Buffer2 μ l, oligo dT 0.5 μ l, 6 mer, 0.5 μ l, AMV reversed transcriptive enzyme 0.5 μ l, DEPC water adds to 10 μ l.37 ℃ of incubation 30min, back 85 ℃ of incubation 3 min obtain cell cDNA.
(2) structure of carrier, expression and evaluation thereof
1.
pThe structure of MD18-T cloning vector
By the habitual codon design of intestinal bacteria CBD gene order, primer and corresponding restriction enzyme site and linker (G
4S), synthetic following 2 primers F 1, F2.
CBD targeted peptide aminoacid sequence is as follows: H2N-Thr Lys Lys Thr Leu Arg Thr-COOH;
CBD gene order: 5 ' accaaaaaaacccttcgcacc3 ';
F1:?5’catatgagcccaggccagggcacccagtctgagaacag3’；
F2:5’gtcgactcaggtgcgaagggtttttttggtagaaccaccaccaccgtttcgtatcttcattgtcatgtag3’；
With F1, F2 is primer, is template with cDNA, thereupon by 95 ℃ of 30s, and 58 ℃ of 30s, 72 ℃ of 1min, 30 circulations are extended 12min for back 72 ℃, obtain the PCR product, and product reclaims and is connected into
pThe MD18-T carrier carries out enzyme and cuts evaluation and sequencing, obtains
pThe MD18T-IL10-CBD recombinant cloning vector.
2.
pThe structure of ET-IL10-CBD expression vector
Correct pMD18T-IL10-CBD recombinant cloning vector and protokaryon check order
pET-22b (+) expression vector respectively through NdeI/SalI double digestion, reclaims the purpose fragment and connects, and connects product and transforms BL21 (DE3), and the picking clone reclaims plasmid equally, and enzyme is cut evaluation, further identifies through order-checking, is built into
pThe engineering bacteria of ET-IL10-CBD expression vector.
3.CBD-IL-10 the expression of recombinant protein in intestinal bacteria
Above-mentioned engineering bacteria
pET-IL10-CBD/BL21 is inoculated in the LB substratum that contains penbritin, and 37 ℃ of shaking culture are spent the night, and next day, the inoculum size with 2% was inoculated in the same substratum, continues 37 ℃ of shaking culture to logarithmic growth mid-term, nutrient solution optical density(OD) OD
600Be 0.5 ~ 0.6 o'clock, add IPTG and continue to cultivate 4 ~ 5 hr abduction deliverings, centrifugal collection thalline.
4, the detection of CBD-IL-10 protein expression and evaluation
The yeast culture thing of abduction delivering is centrifugal, collect thalline, adopt the SDS-PAGE electrophoretic analysis expression product of 12 %, the IPTG inductor compares with inductor not, a newborn protein band of tangible engrain is arranged in the molecular weight corresponding position, expressing quantity accounts for 40% of bacterial protein, conforms to the molecular weight size of estimating fusion rotein.With albumen from the gel electrotransfer to pvdf membrane, conventional Western blot analyzes, ECL is luminous, visible one obvious colour developing band, molecular weight unanimity.Inducible strain not, standard protein and express that other band does not have positive reaction in the bacterium.
(3) cultivation of genetic engineering bacterium and high density fermentation
1. the preparation of first order seed
Single colony inoculation of selecting on LB (the containing penbritin) flat board contains penbritin in 10ml LB() in the liquid nutrient medium, 37 ℃ of 200 rpm shaking culture spent the night.
2. the cultivation of fermentation seed liquid
First order seed is inoculated in the LB liquid nutrient medium (containing penbritin) of an amount of improvement by 2% inoculum size, and similarity condition is cultivated the density of thalline down to OD
600For about 1.5-2.
3, the high density fermentation of genetic engineering bacterium
Inoculum size by 5% is inoculated in fermentation seed liquid and contains peptone, yeast extract, sodium-chlor, ammonium sulfate, sal epsom, phosphoric acid salt, glycine and glycerine etc. in the complex medium of main component, stirring velocity is 100-800rpm, dissolved oxygen is controlled at 20-40%, temperature is that feed supplement stream adds to OD under 37 ℃ of conditions
600Be about at 30 o'clock, adding final concentration is the IPTG of 1mmol ∕ L, the centrifugal collection thalline of the 4hr that ferments again.
(4) purifying of CBD-IL-10 fusion rotein
1. fusion rotein slightly carries
Get the thalline 30g of abduction delivering, with the PB (1.5ml1mol/LMgCl of 150mL 20mmol/L pH6.8
2,3.5mgDNase, an amount of EDTA) and cytoclasis instrument smudge cells, 4 ℃ are stirred cracking 3hr.Detect the fusion rotein of finding expression through polyacrylamide gel electrophoresis and be present in the cleer and peaceful precipitation, illustrate that the recombinant protein of abduction delivering exists with solubility and inclusion body form.4 ℃ of centrifugal 15min of 12000rpm collect supernatant, the dialysis tubing of packing into, and the PB dialysis 24-36hr of the 10mmol/L of usefulness pH6.8 changes liquid 2-3 time, centrifugal collection supernatant liquor.
2. the dissolving of inclusion body and renaturation
Split bacterium precipitation IL-10 inclusion body through washing, urea dissolving, slow renaturation under 4 ℃ of dilutions gradually at renaturation buffer.
3, the purifying of fusion rotein
Get cationic exchange matrix SP-Sepharose Fast Flow dress post, column volume 5.4 * 20cm, with the PB balance of 10mmol/L pH6.8, with the PB linear gradient elution of the 10mmol/L pH6.0 that contains 0.5 mol/L NaCl, collect main peak and promptly obtain fusion rotein behind the last sample.Be CBD-IL-10, aminoacid sequence as shown in Figure 2.
(5) biological activity determination of CBD-IL-10
The MC/9 cell derives from the mastocyte of mouse, growth and breeding under the collaborative stimulation of mouse IL-4 or IL-3 and mouse IL-10 or people IL-10, and cell quantity increases along with the increase of IL-10 concentration, specific as follows:
Give 100 μ l MC/9 cells (2 * 10
4Cell/ml) adds the CBD-IL-10 of mouse IL-4 20 U, people IL-10 standard substance 2 U or expression, and 37 ℃, the 5%CO2 incubator is cultivated 72hr, trypan blue dyeing, blood cell counting plate counting MC/9 viable count.Calculate the activity unit of IL-10 according to cell quantity, with control group mIL-4, the equal nonsignificance of negative supernatant liquor comparing difference (
p0.05), and the CBD-IL-10 of IL-10 standard substance and expression all can stimulate the propagation of MC/9 cell, in cell quantity and the monoclonal antibody and group, the equal significance of negative control group comparing difference (
p＜0.05).Therefore, the IL-10 of the solubility of escherichia coli expression and renaturing inclusion bodies all has biologic activity.Analyze through ELISA detection by quantitative and MC/9 cell activity, the specific activity of the solubility of raw product and renaturing inclusion bodies IL-10 is respectively 1.02 * 10
4U/mg and 4.1 * 10
2U/mg.
Embodiment 2:CBD-IL-10 suppresses the scar proliferation test
(1) hypertrophic cicatrix tissue
3 months forearm hypertrophic cicatrix tissue (pathology confirmation) behind the burn-healing, red, hard, exceed about 4 ~ 7 mm of normal skin.Use 30 of BALA/c-nu male mices, 6 ~ 8 ages in week, body weight 18 ~ 20g.
(2) preparation of the animal model of burn back hypertrophic cicatrix
Remove fatty tissue under the hypertrophic cicatrix, scar is cut into 6mm * 5mm * 3mm size tissue block, place the precooling DMEM nutrient solution of (containing penicillin, two anti-each 100 U/ml of Streptomycin sulphate), make a little otch at nude mice omoplate skin under the aseptic condition, the scar tissue fritter is implanted in subcutaneous, will not sews up.In 3 h, finish 30 animal models.Observe animal model and have or not infection, rejection, select stable scar model to use as the treatment animal.Scar modelling 20d, this moment, the hypertrophic cicatrix blood circulation was set up, and scar is stable.
(3) laboratory animal grouping
Experiment is divided into the heavy dose of treatment group of control group, hIL-10 standard group and CBD-IL-10, middle dosage treatment group, low dose of treatment group, the nude mice number is respectively 6 for every group, adopt the method for injection in the scar, IL-10 is 10 μ g/ml/ time, CBD-IL-10 is respectively 5,10 and 20 μ g/ml/ time, 200 μ l/ time 3 times/week, treated for 3 weeks altogether; Control group is only given PBS at every turn, and all the other are identical.Draw materials behind the drug withdrawal 3d, directly measure scar maximum diameter a and path b is converted into the scar volume with vernier callipers, formula is V=ab
2/ 2.Compare with control group, IL-10 and CBD-IL-10 treatment group all can obviously suppress the growth of scar (as table 1.
P＜0.05).
Scar volume change (mm before and after table 1 treatment
3)
| Group | Before the transplanting | After treating for 3 weeks |
| Control group | 90.0±0.5 | 70.0±3.1 |
| hIL-10 | 90.0±0.4 | 49.2±4.3* |
| The CBD-IL-10 low dose group | 90.0±0.5 | 49.5±5.8* |
| Dosage group among the CBD-IL-10 | 90.0±0.6 | 38.2±7.1 # |
| The CBD-IL-10 high dose group | 90.0±0.3 | 36.5±5.6 # |
*
P＜0.05,
# P＜0.01 vs control group
(4) sub-micro and Ultrastructural observation
After HE, the Masson dyeing, respectively organizing the scar tissue periphery under the light microscopic all has one deck fibrillar connective tissue coating to hold.A large amount of fibroblast-like cells are arranged in the scar tissue of control group, and pressing close to the coating place has blood vessel to exist, and collegen filament are thick, and the whirlpool shape is arranged.Fibroblast-like cells is rare in each treatment group scar tissue, and collagen is sparse, arrange more neat, in, heavy dose of treatment group is especially remarkable.
The submicroscopic structure characteristics: control group inoblast organoid is abundant, and rough surfaced endoplasmic reticulum is significantly expanded, and the visible thick collagenous fiber bundle of a matter is whirlpool shape, nodositas arrangement; More capillary vessel is arranged, and vascular endothelial cell growth is vigorous, protrudes in lumen of vessels, makes lumen of vessels be the inaccessible shape of part.Each treatment group shows as: the inoblast of more necrosis, apoptosis; Part inoblast and vascular endothelial cell then show as vacuolar degeneration, and part inoblast volume is less, and rough surfaced endoplasmic reticulum is not expanded, and karyoplasmic ratio increases, and are rendered as the characteristics of typical fibers cell; The interstitial collagen bundle is more sparse, arranges more neat.
Embodiment 3: collagen specificity is in conjunction with evaluation
Utilize ELISA to identify: in the mouse tail procollagen and after, join in 96 orifice plates in 0.1mg/ hole, 4 ℃ are spent the night, PBST washing 3 times, every hole added under the BSA room temperature of 200ul 0.25% sealing 2 hours.Same TBST washing 3 times, each 3 parallel hole of CBD-IL-10 of adding 0.157-10 uM in the respective aperture, control group adds the IL-10 of same concentrations, hatches 1 hour PBS washing 3 times for 37 ℃.Added 50 ul mouse-anti IL-10 monoclonal antibody (1:1000 dilution) incubated at room 1 hour, the same washing 3 times adds 100 ul alkaline phosphatase labelled goat anti-mouse antibodies (1:10000 dilution), incubated at room 1 hour, the same washing 3 times.Every hole add 100 ul 2mg/ml phosphoric acid oil of mirbane (
p-NPP) room temperature 10 minutes, every hole adds the NaOH termination reaction of 100 ul 0.2M.Every hole is got 100 ul and is changed in another 96 orifice plate, 405nm microplate reader reading, and statistical results show as a result, the CBD-IL-10 group has significant collagen binding specificity (table 2)
。
<211>173
<212>?PRT
＜213〉artificial sequence
<173>?Met?Ser?Pro?Gly?Gln?Gly?Thr?Gln?Ser?Glu?Asn?Ser?Cys?Thr?His?Phe?Pro?Gly?Asn?Leu?Pro?Asn?Met?Leu?Arg?Asp?Leu?Arg?Asp?Ala?Phe?Ser?Arg?Val?Lys?Thr?Phe?Phe?Gln?Met?Lys?Asp?Gln?Leu?Asp?Asn?Leu?Leu?Leu?Lys?Glu?Ser?Leu?Leu?Glu?Asp?Phe?Lys?Gly?Tyr?Leu?Gly?Cys?Gln?Ala?Leu?Ser?Glu?Met?Ile?Gln?Phe?Tyr?Leu?Glu?Glu?Val?Met?Pro?Gln?Ala?Glu?Asn?Gln?Asp?Pro?Asp?Ile?Lys?Ala?His?Val?Asn?Ser?Leu?Gly?Glu?Asn?Leu?Lys?Thr?Leu?Arg?Leu?Arg?Leu?Arg?Arg?Cys?His?Arg?Phe?Leu?Pro?Cys?Glu?Asn?Lys?Ser?Lys?Ala?Val?Glu?Gln?Val?Lys?Asn?Ala?Phe?Asn?Lys?Leu?Gln?Glu?Lys?Gly?Ile?Tyr?Lys?Ala?Met?Ser?Glu?Phe?Asp?Ile?Lys?Ile?Asn?Tyr?Ile?Glu?Ala?Tyr?Met?Thr?Met?Lys?Ile?Arg?Gln?Gly?Gly?Gly?Gly?Ser?Thr?Lys?Lys?Thr?Leu?Arg?Thr。
Claims (2)
1. target CBD-IL-10 recombinant protein, its aminoacid sequence is as follows:
H
2N-Met?Ser?Pro?Gly?Gln?Gly?Thr?Gln?Ser?Glu?Asn?Ser?Cys?Thr?His?Phe?Pro?Gly?Asn?Leu?Pro?Asn?Met?Leu?Arg?Asp?Leu?Arg?Asp?Ala?Phe?Ser?Arg?Val?Lys?Thr?Phe?Phe?Gln?Met?Lys?Asp?Gln?Leu?Asp?Asn?Leu?Leu?Leu?Lys?Glu?Ser?Leu?Leu?Glu?Asp?Phe?Lys?Gly?Tyr?Leu?Gly?Cys?Gln?Ala?Leu?Ser?Glu?Met?Ile?Gln?Phe?Tyr?Leu?Glu?Glu?Val?Met?Pro?Gln?Ala?Glu?Asn?Gln?Asp?Pro?Asp?Ile?Lys?Ala?His?Val?Asn?Ser?Leu?Gly?Glu?Asn?Leu?Lys?Thr?Leu?Arg?Leu?Arg?Leu?Arg?Arg?Cys?His?Arg?Phe?Leu?Pro?Cys?Glu?Asn?Lys?Ser?Lys?Ala?Val?Glu?Gln?Val?Lys?Asn?Ala?Phe?Asn?Lys?Leu?Gln?Glu?Lys?Gly?Ile?Tyr?Lys?Ala?Met?Ser?Glu?Phe?Asp?Ile?Lys?Ile?Asn?Tyr?Ile?Glu?Ala?Tyr?Met?Thr?Met?Lys?Ile?Arg?Gln?Gly?Gly?Gly?Gly?Ser?Thr?Lys?Lys?Thr?Leu?Arg?Thr-COOH，
Wherein: H
2N-' represents protein amino terminal (N-terminal), and ' COOH ' represents protein carboxyl terminal (C-terminal).
2. the described target CBD-hIL-10 of claim 1 recombinant protein is in the application as treatment hypertrophic cicatrix and keloid disease medicament.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010106043515A CN102115495B (en) | 2010-12-24 | 2010-12-24 | Preparation method and application of protein drug for collagen targeted therapy of hyperplastic scar |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010106043515A CN102115495B (en) | 2010-12-24 | 2010-12-24 | Preparation method and application of protein drug for collagen targeted therapy of hyperplastic scar |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102115495A true CN102115495A (en) | 2011-07-06 |
| CN102115495B CN102115495B (en) | 2012-07-04 |
Family
ID=44214434
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010106043515A Expired - Fee Related CN102115495B (en) | 2010-12-24 | 2010-12-24 | Preparation method and application of protein drug for collagen targeted therapy of hyperplastic scar |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102115495B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102836424A (en) * | 2012-09-25 | 2012-12-26 | 中国人民解放军第四军医大学 | Vaccine for preventing and treating scars and preparation method thereof |
| CN103446188A (en) * | 2013-09-02 | 2013-12-18 | 中国人民解放军第三军医大学第一附属医院 | Applications of TRADD (Tumor necrosis factor receptor associated death domain protein) gene over-expressed lentivirus for inhibiting formation of skin scars |
| CN113248569A (en) * | 2021-06-18 | 2021-08-13 | 中国科学院苏州纳米技术与纳米仿生研究所 | Leptin receptor affinity peptide and application thereof |
| CN114917413A (en) * | 2022-06-14 | 2022-08-19 | 健诺维(成都)生物科技有限公司 | Amniotic membrane loaded with recombinant polypeptide and preparation method thereof |
| US20230174623A1 (en) * | 2018-09-28 | 2023-06-08 | Massachusetts Institute Of Technology | Collagen-localized immunomodulatory molecules and methods thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001007059A1 (en) * | 1999-07-21 | 2001-02-01 | University Of Southern California | Matrix-targeted fusion polypeptides for tissue regeneration and wound healing |
| US6268348B1 (en) * | 1989-08-14 | 2001-07-31 | The Regents Of The University Of California | Synthetic compounds and compositions with enhanced cell binding |
| CN1865286A (en) * | 2005-05-17 | 2006-11-22 | 北京上地新世纪生物医药研究所有限公司 | Double function epidermal growth factor and its preparation method and uses |
| CN1869072A (en) * | 2006-05-24 | 2006-11-29 | 中国科学院遗传与发育生物学研究所 | Nerve growth factor combined with collagen specificity and its coading gene and application |
-
2010
- 2010-12-24 CN CN2010106043515A patent/CN102115495B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6268348B1 (en) * | 1989-08-14 | 2001-07-31 | The Regents Of The University Of California | Synthetic compounds and compositions with enhanced cell binding |
| WO2001007059A1 (en) * | 1999-07-21 | 2001-02-01 | University Of Southern California | Matrix-targeted fusion polypeptides for tissue regeneration and wound healing |
| CN1865286A (en) * | 2005-05-17 | 2006-11-22 | 北京上地新世纪生物医药研究所有限公司 | Double function epidermal growth factor and its preparation method and uses |
| CN1869072A (en) * | 2006-05-24 | 2006-11-29 | 中国科学院遗传与发育生物学研究所 | Nerve growth factor combined with collagen specificity and its coading gene and application |
Non-Patent Citations (2)
| Title |
|---|
| 《Molecular Cardiology》 20090323 Jing Zhang等 Collagen-Targeting Vascular Endothelial Growth Factor Improves Cardiac Performance After Myocardial Infarction 1776-1784 1-2 , * |
| 《中国博士学位论文数据库》 20101124 陈伟 碱性成纤维细胞因子靶向结合胶原支架材料促进膀胱重建的实验研究 1-14 1-2 , * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102836424A (en) * | 2012-09-25 | 2012-12-26 | 中国人民解放军第四军医大学 | Vaccine for preventing and treating scars and preparation method thereof |
| CN102836424B (en) * | 2012-09-25 | 2014-03-19 | 中国人民解放军第四军医大学 | Vaccine for preventing and treating scars and preparation method thereof |
| CN103446188A (en) * | 2013-09-02 | 2013-12-18 | 中国人民解放军第三军医大学第一附属医院 | Applications of TRADD (Tumor necrosis factor receptor associated death domain protein) gene over-expressed lentivirus for inhibiting formation of skin scars |
| CN103446188B (en) * | 2013-09-02 | 2017-01-25 | 中国人民解放军第三军医大学第一附属医院 | Applications of TRADD (Tumor necrosis factor receptor associated death domain protein) gene over-expressed lentivirus for inhibiting formation of skin scars |
| US20230174623A1 (en) * | 2018-09-28 | 2023-06-08 | Massachusetts Institute Of Technology | Collagen-localized immunomodulatory molecules and methods thereof |
| CN113248569A (en) * | 2021-06-18 | 2021-08-13 | 中国科学院苏州纳米技术与纳米仿生研究所 | Leptin receptor affinity peptide and application thereof |
| CN113248569B (en) * | 2021-06-18 | 2021-10-12 | 中国科学院苏州纳米技术与纳米仿生研究所 | Leptin receptor affinity peptide and application thereof |
| WO2022262509A1 (en) * | 2021-06-18 | 2022-12-22 | 江苏独步生物科技有限公司 | Leptin receptor affinity peptide and use thereof |
| CN114917413A (en) * | 2022-06-14 | 2022-08-19 | 健诺维(成都)生物科技有限公司 | Amniotic membrane loaded with recombinant polypeptide and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102115495B (en) | 2012-07-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102115495B (en) | Preparation method and application of protein drug for collagen targeted therapy of hyperplastic scar | |
| CN101367873B (en) | Modified glucagon sample peptide-1analogue and modifying matter, and uses thereof | |
| CN101649311B (en) | Preparation method of human lysozyme-antibacterial peptide Catesbeianin-1 fusion protein and application of same on preventing and curing cow mastitis | |
| CN113186109A (en) | Saccharomyces cerevisiae expression long-acting recombinant fibronectin and application thereof in cosmetics | |
| CN103145851B (en) | Recombinant protein PACAP38-NtA, and coding gene and application thereof | |
| CN113501870A (en) | Application of milk-derived polypeptide and chimeric peptide thereof in preparation of medicine for promoting energy metabolism of fat cells | |
| CN101514229B (en) | Human interferon alpha derivative and polyethylene glycol modified substance thereof | |
| CN111269290B (en) | Preparation method of sturgeon anti-inflammatory peptide | |
| US20140286926A1 (en) | Method for treatment or prevention of allergic diseases | |
| CN102247589A (en) | Application of marine collagen peptide in preparing medicament and food for delaying skin aging | |
| CN101891826B (en) | Targeted hIL-10 recombinant protein and application thereof | |
| CN101433713B (en) | GnRH-PE mutant fusion protein and uses thereof | |
| CN102901826A (en) | New potential human cytokine TMEM98 and uses thereof | |
| CN101851620A (en) | Method for producing FGFs by utilizing edible medicinal fungus | |
| CN101580846A (en) | Human cytoglobin for preventing and curing cirrhosis and preparation method thereof | |
| CN101487020B (en) | Preparation and use of recombinant gold needle mushroom immunomodulatory protein | |
| CN1904041B (en) | Human recombination phospholipase D2, its preparation method and application in preparation medicine | |
| CN102180962A (en) | Tetraodon nigroviridis interferon IFNgamma1 and preparation method and application thereof | |
| TW200920387A (en) | Composition of mushroom beta-glucan for anti-cancer and preparation method thereof | |
| CN100535005C (en) | Long chain human insulin-like growth factor(LR3IGF-1) and its preparing process and application method | |
| CN110257390A (en) | Stichopus japonicus Bax gene, coding albumen and its cloning process, recombination stichopus japonicus Bax construction of genetic engineering methods and applications | |
| CN100500844C (en) | High secretion expression of recombination thymosin-alpha 1 in Escherichia coli and separation and purification therefor | |
| CN100371349C (en) | Recombination immune therapeutic protein and its expression method and application | |
| CN112010944B (en) | Peptide and method for in vitro amplification of CIK cells | |
| CN100556455C (en) | A kind of medicinal composition and application thereof that is used to prevent and treat type i diabetes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120704 Termination date: 20121224 |