CN108640976A - A kind of polypeptide with human colon cancer cell specific binding - Google Patents
A kind of polypeptide with human colon cancer cell specific binding Download PDFInfo
- Publication number
- CN108640976A CN108640976A CN201810385293.8A CN201810385293A CN108640976A CN 108640976 A CN108640976 A CN 108640976A CN 201810385293 A CN201810385293 A CN 201810385293A CN 108640976 A CN108640976 A CN 108640976A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- colon cancer
- cancer cell
- drug
- specific binding
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Public Health (AREA)
- Biophysics (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Genetics & Genomics (AREA)
- Veterinary Medicine (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention belongs to biomedical sectors, and in particular to a kind of polypeptide and application thereof having specific binding to colon cancer cell.The polypeptide is following arbitrary（1）The amino acid sequence of polypeptide is：GLTSMRYHSVIV（SEQ ID No.1）,（2）（1）In the peptide molecule by missing, be inserted into or replace one or several amino acid and with（1）The peptide molecule has the polypeptide derivative of identical biological function.The polypeptide of the present invention can be specifically bound with colon cancer cell, and with normal enterocyte without specific effect, and function and effect are apparent, and reliable scientific basis is provided for clinical treatment.
Description
Technical field
The invention belongs to biomedical sectors, and in particular to a kind of to have the more of specific binding to Colon Carcinoma
Peptide and application thereof.
Background technology
Colon cancer is one of most common malignant tumor of digestive tract, and incidence is only second to gastric cancer and the cancer of the esophagus.Good hair year
Age, male was more than women generally at 50 years old or more, and the ratio between men and women is 2:1.The pathogenic factor of colon cancer is still not clear, and mainly has ring
Disease etc. before border factor, inherent cause, cancer.National survey shows, early stage cancer patients survival rate reaches 90-95%, but
The patient that China actually exceeds 80% has developed to middle and advanced stage when making a definite diagnosis, early diagnostic rate is only 10-15%；And Advanced Colon Cancer
Survival rate then be only 5%.Therefore, early diagnosis, early treatment, can be obviously improved colorectal cancer patients more after.
Meanwhile the early symptom of colon cancer mostly be patient note that see a doctor when also often with " dysentery ", " enteritis " etc. disease at
Reason, therefore non-early stage develops sensitive diagnostic techniques when poisoning symptom or obstruction occur and touching abdomen block, from
And accomplish that early diagnosis is one of research emphasis.
Existing anticancer drug usually has that toxic side effect is larger, drug dose is big, easy tos produce the drug resistance day after tomorrow etc. and lacks
Point, this can impact the treatment of patient.Targeted therapy is on cellular and molecular level, for explicitly carcinogenic position
The therapeutic modality of point.Can design corresponding medicine, drug enter can specifically select carcinogenic site being in vivo combined and
It has an effect, keeps tumor cell specific dead, without involving the normal tissue cell around tumour, so molecular targeted control
Treatment is the another research emphasis for the treatment of of cancer.
Display technique of bacteriophage is a kind of technology of important screening intermolecular interaction of molecular biology field.Phagocytosis
The cardinal principle of body display technology is that the gene of target gene or coding protein and polypeptide is cloned by technique for gene engineering
On the appropriate location of phage surface protein gene, allows its amplification with phage DNA and express in phage surface, due to
The compatibility of foreign gene and phage gene, the expression product polypeptide or protein of foreign gene still can keep its original
Space structure and corresponding biological activity.Then, we carry out subtrahend screening using target cell to bacteriophage, finally from phagocytosis
The purpose bacteriophage that can be combined with target cell specificity is filtered out in body peptide library, and its DNA is sequenced, you can is obtained
The coded sequence of corresponding polypeptide.This technology realizes the contact between protein or polypeptide gene type and phenotype, and has
Have it is easy to operate, can high-throughput detection the characteristics of, to the efficient means as screening tumor cell specific binding peptide, be
The targeting vector research of the early detection and drug therapy of tumour provides new direction.
Invention content
The purpose of the present invention is to provide a kind of polypeptides, specific can be combined with colon cancer cell targeting, and to normal intestines
Epithelial cell does not influence, this plays an important roll in the early diagnosis of colon cancer and the research and development of targeted drug etc..
This experiment is control with people's normal intestinal epithelial HIEC cells, using humanized's colon cancer SW480 cells to phagocytosis
Body display peptide library carries out subtrahend screening, is picked out and can be specifically bound with colon cancer cell with blue and white screening experiment
Positive phage clones, the specificity for being used in combination ELISA experimental verifications bacteriophage to be combined with colon cancer.Then it is to carry with Escherichia coli
Body extracts its DNA after amplification purification bacteriophage and is sequenced to get to the polypeptide that can be specifically bound with colon carcinogenesis
Coded sequence, and the positive polypeptides of artificial synthesized fluorescent marker carry out fluorescent marker-polypeptide and human colon cancer cell combination
Verification, and then provide experiment basis for the early diagnosis and targeted therapy of colon cancer.
To achieve the goals above, the present invention adopts the following technical scheme that：It is a kind of to be specifically bound with human colon cancer cell
Polypeptide, which is following arbitrary：（1）The amino acid sequence of polypeptide is： GLTSMRYHSVIV（SEQ ID No.1）,（2）
（1）In the peptide molecule by missing, be inserted into or replace one or several amino acid and with（1）The peptide molecule
Polypeptide derivative with identical biological function.
The polypeptide has targeting to combine tumour cell, is combined with tumor cell specific.
The tumour cell is colon cancer cell.
Application of the polypeptide in preparing tumor diagnosis kit includes the polypeptide or polypeptide coupling in the kit.
A kind of polypeptide with human colon cancer cell specific binding is being prepared for treating the application in colon cancer drug, should
Drug includes the polypeptide and active constituents of medicine, or comprising the polypeptide and passs drug carrier.The drug is any drug
Therapeutically acceptable dosage form, the preferred dosage form of the drug are ejection preparation.
The drug is acceptable dosage in any pharmacotherapeutics.
Compared with prior art, effect of the invention is that：The present invention using display technique of bacteriophage have it is easy to operate,
The elutriation of high throughput, high efficiency can screen mimic epitope, displayed polypeptides or protein and it includes the bases inside bacteriophage
The advantages that being easy to purifying because of the connection of password, recombinant phage.We, which use, enables the polypeptide that phage display technique is filtered out
Enough and colon cancer cell is specifically bound, and with normal enterocyte without specific effect, and function and effect are apparent, are
Clinical treatment provides reliable scientific basis.
Description of the drawings
Fig. 1 is that ELISA identifies 1-20 positive phage clones and humanized's colon cancer SW480 cellular affinities
OD405Result figure.
Fig. 2 is No. 8 positive phage clones and people's normal intestinal epithelial HIEC cells and humanized's colon cancer SW480 cells
The cellular immunofluorescence experimental result picture of affinity.
Fig. 3 is the sequencing result figure of No. 8 positive phage clones.
Fig. 4 is the cellular immunofluorescence result figure that FITC-GV12 is combined with HIEC, SW480 cell-targeting.
Fig. 5 is the FCM analysis result figure of FITC-GV12 and HIEC, SW480 cell combination ability.A be HIEC with
The binding ability result figure of PBS；B is the binding ability result figure of HIEC and target polypeptide FITC-GV12；C is SW480 and PBS
Binding ability result figure；D is the binding ability result figure of SW480 and target polypeptide FITC-GV12.
Specific implementation mode
It is as described below to be merely a preferred embodiment of the present invention, it is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, any made by repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Embodiment 1
1. experiment material
1.1 phage peptide libraries, cell and host strain.
12 peptide library of bacteriophage, E. coli ER2738, humanized colon cancer SW480 cells, people's normal intestinal epithelial
HIEC cells.
1.2 experiment reagent
L-15 culture mediums, trypsase, FITG label rabbit-antis mouse, fetal calf serum, yeast powder, peptone, agar powder, tetracycline storage
Liquid storage, Tween-20（Tween-20), bovine serum albumin BSA, M13 phage single-chain DNAs extracts kit, IPTG, X-gal,
PEG-8000、TMB。
1.3 experimental work liquid
1 × PBS, LB liquid medium, LB-Tet solid plates, top agar, IPTG/X-gal working solutions, IPTG/X-gal are flat
Plate, PEG-NaCl, TBS buffer solution, 0.1%TBST, 0.5%TBST, 4% paraformaldehyde fixer, the preparation of TBS-NaN3 liquid, 3%
BSA confining liquids, sodium iodide buffer solution, TE buffer solutions, TMB working solutions, tetracycline store liquid.
2. experimental method
The culture of 2.1 Escherichia coli.
（1）The recovery of Escherichia coli：Escherichia coli glycerine frozen stock solution is taken out from -80oC refrigerators, is taken on a small quantity with oese
It lines on LB/Tet solid plates, then this LB/Tet solid plate is inverted in the electro-heating standing-temperature cultivator of 37oC and is cultivated
Overnight, picking single bacterium colony when use.
（2）The culture of Escherichia coli：10ml LB/Tet fluid nutrient mediums, picking large intestine bar are added in 15ml centrifuge tubes
Bacterium single bacterium colony is added thereto.Then centrifuge tube is placed in overnight incubation in constant temperature oscillator, waits for the OD of bacterium solution600When value is 0.5
It can carry out related experiment.
The subtrahend in 2.2 phage-displayed polypeptides libraries screens.
（1）Prepare cell：6 well culture plates are pre-processed through poly-lysine first, take humanized's colon cancer SW480 cells
With people's normal intestinal epithelial HIEC cells, respectively with being laid on after trypsin treatment wherein, culture is adherent to cell success and grows
It is screened after in good condition.
（2）Prepare bacterium solution：On the screening same day, Escherichia coli ER2738 is inoculated in 20ml LB/Tet fluid nutrient mediums, is set
The shake culture in 37oC constant temperature oscillators waits for the OD of bacterium solution600When value is 0.5, the bacteriophage for expanding screening elution.
（3）Free serum culture：Cell culture medium is sopped up, serum free medium is added after washing 1 time with PBS, is placed in and is connected with
1h in the 37oC constant temperature cell incubators of 5%CO2.
（4）Washing:Confining liquid is sopped up, is more lightly washed 5 times with 0.1%TBST, is both needed to rotate the bottom so that micropore every time
And edge is all washed, drying.0.5%TBST, 1.0%TBST are used respectively when being screened to the 2nd, 3 wheels.
（5）Closing cell:Cell culture medium is sopped up, tablet is inverted in the training for firmly getting rid of removal remaining on clean paper handkerchief
Base is supported, with culture medium closing humanized's colon cancer SW480 cells and people's normal intestinal epithelial HIEC cells containing 1%BSA, is placed in logical
There is 1h in the 37oC constant temperature cell incubators of 5%CO2.
（6）Absorption:10 μ l of original polypeptide library are taken, are added in 990 μ l, 0.5% BSA/ PBS buffer solution, by bacteriophage
It is diluted to 1.5 × 1011Pfu/ml, and add it in closed people's normal intestinal epithelial HIEC cells, 37oC 1h, it adsorbs
Supernatant can be left and taken with the bacteriophage of people's normal intestinal epithelial HIEC cell combinations.
（7）In conjunction with:Phage supernatants after absorption are incubated 1h jointly with humanized's colon cancer SW480 cells.
（8）Washing:Unbonded bacteriophage is discarded, microwell plate is inverted on clean paper handkerchief firmly to clap and be got rid of, with removal
The solution of remaining.0.1%TBST board-washings are used as stated above 5 times.
（9）Elution:0.2M Glycine-HCl (pH2.2) 1mg/mlBSA eluent 1ml are added, shake 10min slowly on ice,
Then eluent is sucked out and is transferred to 150 μ l neutralizers being ready in advance（1M Tris-HCl, pH9.1）In.
（10）According to above-mentioned steps repetitive operation 2 times.
The titer determination of 2.3 bacteriophages.
By the preheating of IPTG/X-gal tablets in the electro-heating standing-temperature cultivator of 37oC；Suitable top agar is taken out in microwave
It is heated in stove, is taken out after it melts completely, 3ml is dispensed in each 10ml centrifuge tubes；Bacteriophage to be screened is carried out etc.
After dilution, after taking 10 μ l to be sufficiently mixed with the Escherichia coli bacteria liquid of 200 μ l and react 5min, it is added in the top agar of 3ml,
Then it is equably layered on the IPTG/X-gal tablets of preheating, the electro-heating standing-temperature cultivator to be condensed for being placed on 37oC is stayed overnight, and is seen
Examine titration results.
The amplification and purifying of 2.4 bacteriophages.
（1）The amplification of bacteriophage：20mlLB/Tet fluid nutrient mediums are added in conical flask, then press 1:100 are added greatly
Enterobacteria bacterium solution and bacteriophage to be amplified are placed in 37oC, acutely shake 4.5h in constant temperature oscillator, obtain the amplification of bacteriophage
Liquid.
（2）The purifying of bacteriophage：Phage amplification liquid 4oC, the 12000r/min that will be obtained through above-mentioned steps, centrifugation
10min takes after 1/6 volume PEG-NaCl precipitates overnights are added after supernatant, and 12000r/min centrifuges 15min, discards supernatant liquid, uses
TBS buffer solutions precipitate, and give 1/6 volume PEG-NaCl again, are incubated 1h on ice.4oC, 14000r/min, centrifugation
15min is discarded supernatant, and obtained precipitation TBS-NaN3 is dissolved and is placed on the preservation of 4oC refrigerators.
2.5 enzyme-linked immunosorbent assay
（1）Prepare 96 orifice plate of cell, bed board rule：96 orifice plate rims, two row, 16 holes are separately added into 100 μ l × PBS as blank
Group；Then each aperture of 1,2,3,4 rows is according to 100 μ people's normal intestinal epithelial HIEC cell suspensions of snakelike each paving, 5,6,7,8 rows
Each aperture according to 100 μ humanized's colon cancer SW480 cell suspensions of snakelike each paving, then the cell plates completed are placed in
ELISA experiments can be carried out overnight by being connected in the 37oC cell constant temperature incubators of 5%CO2.
（2）It is fixed：96 orifice plates for being covered with cell overnight are taken out, is patted dry in hole after liquid, is washed 3 times with PBS, be then added
4% paraformaldehyde fixes 20min.
（3）It blocks：96 orifice plates after fixing are taken out, is patted dry in hole after liquid, is washed 3 times with PBS, 3% peroxide is then added
Change hydrogen, 30min is closed in 37oC cell constant temperature incubators, to block the activity of endogenous peroxydase.
（4）Closing：96 orifice plates after blocking are taken out, is patted dry in hole after liquid, is washed 3 times with PBS, add 3%BSA/
PBS closes 1h in 37oC cell constant temperature incubators.
（5）Add Phage samples：96 orifice plates after closing are taken out, are patted dry in hole after liquid, the positive that purifying obtains is added
Bacteriophage reacts 1h in 37oC cell constant temperature incubators.
（6）Add primary antibody：96 orifice plates after reaction are taken out, pats dry in hole after liquid, 1 is added after washing 3 times with PBS:4000
M13 antibody, 4oC is overnight.
Secondary antibody：96 orifice plates after reaction are taken out, is patted dry in hole after liquid, is washed 3 times with PBS, add secondary antibody, it is thin in 37oC
30min is reacted in born of the same parents' constant incubator.
（7）Add substrate TMB：TMB developers are added in 96 orifice plates after PBS is washed 3 times under the conditions of being protected from light, and are protected from light and are placed in
15min in 37oC cell constant temperature incubators.
（8）It terminates：96 orifice plates after reaction are taken out, 2M sulfuric acid is added and terminates reaction.
（9）As a result measurement：96 orifice plates for completing total overall reaction are placed in microplate reader, its OD value is measured at 405nm,
It preserves result and is analyzed.
The immunofluorescence experiment of 2.6 cells and positive bacteriophage
（1）Plating cells：People's normal intestinal epithelial HIEC cells and humanized's colon cancer SW480 cells are laid in six orifice plates and are waited for
With.
（2）It is fixed：15min is fixed with 4% paraformaldehyde.
（3）Closing：4% paraformaldehyde is discarded, PBS is washed 2 times, and 30min is closed in 37 DEG C with 3%BSA/PBS.
（4）Positive bacteriophage is incubated：Positive bacteriophage, 37 DEG C of 1h are added after confining liquid is wiped.
（5）DAPI is dyed：Add 100 μ l of DAPI, room temperature, 15min after washing 3 times with PBS
（6）Mounting:After PBS washes 3 times, mounting.
The extraction and sequencing of 2.7 positive bacteriophage DNA
（1）100ul iodide buffer solutions are added in the phages of above-mentioned purifying, adds 250ul absolute ethyl alcohols, fills
Mixing, room temperature is divided to act on 20min.
（2）Centrifugation：4 DEG C, 14,000rpm, 10min abandon supernatant.
（3）Cleaning：Precipitation is washed with 70% ethyl alcohol of 500ul, is dried in vacuo after of short duration centrifugation.
（4）Precipitation is resuspended in 30ulTE (10mM Tris-HCl, pH5.0,1mMEDTA) buffer solution, and DNA sequencing template is made
Liquid is sent and Shanghai life work sequencing.
2.8 cellular immunofluorescences are tested
（1）Prepare plating cells：Spread glass slide in advance in 6 orifice plates, just by humanized colon cancer SW480 cells, humanized
Normal enteric epithelium HIEC cells are laid in six orifice plates for use.
（2）It is fixed：15min is fixed with 4% paraformaldehyde.
（3）Closing：4% paraformaldehyde is discarded, PBS is washed 2 times, and 30min is closed in 37 DEG C with 3%BSA/PBS.
（4）FITC-GV12 is incubated：FITC-GV12,37 DEG C of 1h are added after confining liquid is wiped.
（5）DAPI is dyed：Add 100 μ l of DAPI, room temperature, 15min after washing 3 times with PBS
（6）Mounting:After PBS washes 3 times, mounting.
2.9 Flow Cytometry
（1）Prepare plating cells：Humanized colon cancer SW480 cells, humanized's normal intestinal epithelial HIEC cells are laid on six holes
It is for use in plate.
（2）Closing：PBS is washed 2 times, with 3%BSA/PBS in 37 DEG C, closes 30min.
（3）FITC-GV12 is incubated：FITC-GV12 is added after confining liquid is discarded, 37 DEG C, is incubated 30min.
（4）Collect cell：After washing 3 times with PBS plus collected by trypsinisation cell up flow type detects.
3. experimental result
As shown in Figure 1, the escherichia coli cloning of 20 carrying phage polypeptides of random picking, through cell ELISA, knot
Fruit shows the mean absorbance values OD of experimental group SW480405With the mean absorbance values OD of control group HIEC405The ratio between more than 2
Clone shares 12, is 1,2,5,8,9,10,12,14,15,17,18,20 respectively.Above-mentioned 12 positive bacteriophages and humanized
The effect of colon cancer SW480 cell combinations is stronger, and is acted on humanized's normal intestinal epithelial HIEC cell combinations then weaker.Then it selects
No. 8 positive bacteriophages are taken to carry out immunofluorescence dyeing experiment, further to verify the target of positive bacteriophage and human colon cancer cell
To combination.Cellular immunofluorescence experimental result shows that positive bacteriophage can be specifically bound with human colon cancer cell SW480,
And, the two significant difference weaker with humanized's normal intestinal epithelial HIEC cell combination abilities, further prompt positive phagocytosis
Body polypeptide has the function of that targeting combines to human colon cancer cell（As shown in Figure 2）.
Next its DNA sequencing is expanded, purified and is extracted to 12 positive phage clones, as a result display shares 8
The sequencing result of a positive bacteriophage shows same sequence, respectively 1,2,8,10,14,17,18, No. 20.As shown in figure 3,
According to the principle of codeword triplet, polypeptide sequence is translated：GLTSMRYHSVIV（SEQ ID No.1）（GV12）.
Then immunofluorescence dyeing experiment is respectively adopted in the positive polypeptides FITC-GV12 of artificial synthesized green fluorescent label
The polypeptide is verified with Flow Cytometry to be combined with the targeting of human colon cancer cell SW480.As shown in figure 4, the polypeptide sequence with
Humanized's normal intestinal epithelial HIEC cell combination abilities are weaker, and relative intensity of fluorescence value is 1129.92 ± 460.65, and and people
Colon Carcinoma binding ability is then relatively strong, and relative intensity of fluorescence value is 36429.45 ± 4173.03, and the two has significantly
Sex differernceP < 0.0001。
FCM analysis experiment further demonstrates the above results.People's normal intestinal epithelial HIEC cells and human colon carcinoma
SW480 cells carry out flow cytometer detection after being incubated respectively with 10 μM of FITC-GV12, and the percentage of positive cell is respectively
1.9% and 99.9%, there were significant differences for the two（Such as Fig. 5）.Prove that the polypeptide specific can be combined with colon cancer cell targeting, and it is right
Normal intestinal epithelial cell does not influence.
SEQUENCE LISTING
<110>Chinese Medical Sciences University
<120>A kind of polypeptide with human colon cancer cell specific binding
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 12
<212> PRT
<213>It is unknown
<400> 1
Gly Leu Thr Ser Met Arg Tyr His Ser Val Ile Val
1 5 10
Claims (8)
1. a kind of polypeptide with human colon cancer cell specific binding, which is characterized in that the polypeptide is following arbitrary：
（1）The amino acid sequence of polypeptide is： GLTSMRYHSVIV（SEQ ID No.1）；
（2）（1）In the peptide molecule by missing, be inserted into or replace one or several amino acid and with（1）Described
Peptide molecule has the polypeptide derivative of identical biological function.
2. a kind of polypeptide with human colon cancer cell specific binding according to claim 1, which is characterized in that the polypeptide
There is targeting to combine tumour cell, is combined with tumor cell specific.
3. a kind of polypeptide with human colon cancer cell specific binding according to claim 2, which is characterized in that described
Tumour cell is colon cancer cell.
4. a kind of polypeptide with human colon cancer cell specific binding as described in claim 1 is preparing tumor diagnosis kit
In application, which is characterized in that in the kit include the polypeptide or polypeptide coupling.
5. a kind of polypeptide with human colon cancer cell specific binding as described in claim 1 is being prepared for treating colon cancer
Application in drug.
6. a kind of polypeptide with human colon cancer cell specific binding as described in claim 1 is being prepared for treating colon cancer
Application in drug, which is characterized in that the drug include the polypeptide and active constituents of medicine, or comprising the polypeptide with
Pass drug carrier.
7. a kind of polypeptide with human colon cancer cell specific binding as claimed in claim 5 is being prepared for treating colon cancer
Application in drug, which is characterized in that the drug is acceptable dosage form in any pharmacotherapeutics, the preferred dosage form of the drug
For ejection preparation.
8. a kind of polypeptide with human colon cancer cell specific binding as claimed in claim 5 is being prepared for treating colon cancer
Application in drug, which is characterized in that the drug is acceptable dosage in any pharmacotherapeutics.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810385293.8A CN108640976B (en) | 2018-04-26 | 2018-04-26 | Polypeptide specifically bound with human colon cancer cells |
| PCT/CN2019/079772 WO2019205867A1 (en) | 2018-04-26 | 2019-03-27 | Polypeptide specifically binding to human colon cancer cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810385293.8A CN108640976B (en) | 2018-04-26 | 2018-04-26 | Polypeptide specifically bound with human colon cancer cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108640976A true CN108640976A (en) | 2018-10-12 |
| CN108640976B CN108640976B (en) | 2021-04-09 |
Family
ID=63747671
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810385293.8A Active CN108640976B (en) | 2018-04-26 | 2018-04-26 | Polypeptide specifically bound with human colon cancer cells |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN108640976B (en) |
| WO (1) | WO2019205867A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019205867A1 (en) * | 2018-04-26 | 2019-10-31 | 中国医科大学 | Polypeptide specifically binding to human colon cancer cells |
| CN111518171A (en) * | 2020-05-06 | 2020-08-11 | 中国医科大学 | Polypeptide targeting human liver cancer cells and application thereof |
| CN113527431A (en) * | 2020-04-15 | 2021-10-22 | 辽宁中健医药科技有限公司 | Polypeptide specifically targeting human colorectal cancer cells and application thereof |
| CN113925973A (en) * | 2020-07-14 | 2022-01-14 | 辽宁中健医药科技有限公司 | Polypeptide coupled drug, preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104086627A (en) * | 2014-05-29 | 2014-10-08 | 郑州大学 | PD-L1 IgV affinity peptide S10 with antitumor activity |
| CN104650190A (en) * | 2015-01-21 | 2015-05-27 | 陕西师范大学 | Polypeptide specifically bound on surface of hepatoma carcinoma cell |
| EP3117824A1 (en) * | 2015-07-17 | 2017-01-18 | BE Pharbel Manufacturing | Multilayered pharmaceutically active compound-releasing microparticles in a liquid dosage form |
| CN106967154A (en) * | 2017-03-24 | 2017-07-21 | 孟祥军 | The screening technique of human colon cancer cell specific binding polypeptide and its application |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102477083A (en) * | 2010-11-22 | 2012-05-30 | 大连创达技术交易市场有限公司 | Carcinoembryonic antigen specific binding peptide with targeting therapy and low-toxic side effects |
| CN102127153B (en) * | 2010-12-16 | 2012-11-14 | 陕西师范大学 | Caco-2 cell surface specific binding polypeptide and screening method thereof |
| CN108640976B (en) * | 2018-04-26 | 2021-04-09 | 中国医科大学 | Polypeptide specifically bound with human colon cancer cells |
-
2018
- 2018-04-26 CN CN201810385293.8A patent/CN108640976B/en active Active
-
2019
- 2019-03-27 WO PCT/CN2019/079772 patent/WO2019205867A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104086627A (en) * | 2014-05-29 | 2014-10-08 | 郑州大学 | PD-L1 IgV affinity peptide S10 with antitumor activity |
| CN104650190A (en) * | 2015-01-21 | 2015-05-27 | 陕西师范大学 | Polypeptide specifically bound on surface of hepatoma carcinoma cell |
| EP3117824A1 (en) * | 2015-07-17 | 2017-01-18 | BE Pharbel Manufacturing | Multilayered pharmaceutically active compound-releasing microparticles in a liquid dosage form |
| CN106967154A (en) * | 2017-03-24 | 2017-07-21 | 孟祥军 | The screening technique of human colon cancer cell specific binding polypeptide and its application |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019205867A1 (en) * | 2018-04-26 | 2019-10-31 | 中国医科大学 | Polypeptide specifically binding to human colon cancer cells |
| CN113527431A (en) * | 2020-04-15 | 2021-10-22 | 辽宁中健医药科技有限公司 | Polypeptide specifically targeting human colorectal cancer cells and application thereof |
| CN113527431B (en) * | 2020-04-15 | 2024-04-19 | 辽宁中健医药科技有限公司 | Polypeptide specifically targeting human colorectal cancer cells and application thereof |
| CN111518171A (en) * | 2020-05-06 | 2020-08-11 | 中国医科大学 | Polypeptide targeting human liver cancer cells and application thereof |
| CN113925973A (en) * | 2020-07-14 | 2022-01-14 | 辽宁中健医药科技有限公司 | Polypeptide coupled drug, preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2019205867A1 (en) | 2019-10-31 |
| CN108640976B (en) | 2021-04-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108610396A (en) | A kind of specific polypeptide of targeting human colon cancer cell | |
| CN108640976A (en) | A kind of polypeptide with human colon cancer cell specific binding | |
| CN111995675A (en) | Monoclonal antibody aiming at new coronavirus SARS-CoV-2 spinous process protein RBD region and application thereof | |
| CN108409836A (en) | A kind of polypeptide and application thereof | |
| CN108341854A (en) | A kind of novel polypeptide of cancer target and application thereof | |
| Zhang et al. | Panning and identification of a colon tumor binding peptide from a phage display peptide library | |
| CN104650190B (en) | The polypeptide that liver cancer cells surface specific combines | |
| CN102127153A (en) | Caco-2 cell surface specific binding polypeptide and screening method thereof | |
| CN104109207A (en) | Lung-targeted anti-surfactant protein A nano-antibody and preparation method thereof | |
| CN108752425A (en) | The method for building cell-penetrating peptide expression library using display technique of bacteriophage | |
| CN110330551A (en) | A kind of cancer of pancreas specific bond peptide and preparation method thereof and purposes | |
| CN101429234B (en) | Vaccine based on simulating human blood vessel endothelial cell growth factor VEGF epitope and preparation method thereof | |
| CN105037499B (en) | It is directed to people's histamine receptor 4 using phage antibody library（HR4）Epitope mimic peptide screening and its vaccine construction method | |
| CN112409447B (en) | Affinity short peptide for targeted recognition of annexin A2 and preparation method and application thereof | |
| CN105859846A (en) | Polypeptide having binding affinity to HPV16 E7 and application thereof | |
| CN109517036A (en) | Lung carcinoma cell specific binding polypeptide and preparation method thereof | |
| CN111518171B (en) | Polypeptide targeting human hepatoma cells and application thereof | |
| CN107353325A (en) | Folacin receptor alpha specific WHWTNWGKTSPA and its application | |
| CN113527430B (en) | Novel polypeptide for tumor cell specific targeting and application thereof | |
| CN113527429B (en) | Human liver cancer cell specific binding polypeptide and its use | |
| CN112979757B (en) | Polypeptide of specific targeting human liver cancer cell | |
| CN113527431B (en) | Polypeptide specifically targeting human colorectal cancer cells and application thereof | |
| CN111018951A (en) | Polypeptide targeting triple negative breast cancer cells and application thereof | |
| CN104047061A (en) | Schistosoma japonicum thioredoxin glutathione reductase Sj TGR single-domain antibody and preparation method thereof | |
| CN109021077A (en) | A kind of polypeptide and its preparation method and application with the acute myeloid leukemia cell of resistance to ATRA specific binding |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |