CN108409836B - Polypeptide and application thereof - Google Patents
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- CN108409836B CN108409836B CN201810214130.3A CN201810214130A CN108409836B CN 108409836 B CN108409836 B CN 108409836B CN 201810214130 A CN201810214130 A CN 201810214130A CN 108409836 B CN108409836 B CN 108409836B
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Abstract
The invention belongs to the field of biomedicine, and particularly relates to a polypeptide and application thereof. The amino acid sequence of the polypeptide is as follows: DYHDPSLPTLRK (SEQ ID No. 1). The kit can be specifically combined with human colorectal cancer cells in a targeted manner, has no influence on human normal intestinal epithelial cells, has an identification effect on human pancreatic cancer cells, breast cancer cells and ovarian cancer cells, and has an important effect on the aspects of early diagnosis of the colorectal cancer, the pancreatic cancer, the breast cancer and the ovarian cancer, the research and development of targeted drugs and the like.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a polypeptide having a targeting effect on human colorectal cancer cells, pancreatic cancer cells, breast cancer cells or ovarian cancer cells and application thereof.
Background
Colorectal cancer is one of the most common malignant tumors of the digestive system, and according to 2016 big data research reports of the morbidity and mortality analysis of malignant tumors in China, 33.1 ten thousand new cases of colorectal cancer in China are reported every year, and the morbidity ranks fourth in all malignant tumors; 15.9 million people die of the disease every year, and the death rate is the fifth cause of cancer death. Colorectal cancer becomes the third most prevalent malignancy in the united states, with over 10.6 million people diagnosed with colorectal cancer each year. Unlike our country, despite the high incidence of colorectal cancer in the united states, mortality rates decline year by year, largely due to the increasing number of colorectal cancers discovered at an early stage, and thus the chances of being cured. The 5-year survival rate for patients with early stage colorectal cancer reaches 90%, while metastatic or advanced stage cancer is only 10%. In clinical practice, people generally consider that early diagnosis is expected to realize personalized treatment and is helpful for improving long-term outcome.
Currently, colorectal cancer screening modalities, such as fecal occult blood tests, fecal immunochemical tests, and colonoscopy, may not be optimal, and these methods may fail to detect neoplastic lesions or perform unnecessary endoscopic diagnosis. And dysplastic lesions may be indistinguishable from inflammation-related epithelial regeneration. In addition, adverse events such as perforation and the like may occur during colonoscopy. Therefore, early colorectal cancer detection and development of sensitive diagnostic technology, and targeted therapy is the focus of research.
The existing anticancer drugs mainly comprise five types of metal anticancer drugs, anticancer active components extracted from natural products, anticancer drug targeting preparations, genetic engineering drugs, nano controlled-release anticancer drugs and the like, and the drugs usually have the defects of large toxic and side effects, large drug dosage, easy generation of acquired drug resistance and the like, and influence on the treatment of tumors.
The phage display technology is an important technology for screening the interaction between molecules in the field of molecular biology. The main principle of phage display technology is that the target gene or the gene coding protein and polypeptide is cloned to the proper position of phage surface protein gene by means of gene engineering technology, and is made to express on the phage surface along with the amplification of phage DNA. Then, we use the target cell or target molecule to screen the phage by subtraction, finally screen the target phage which can be combined with the target cell or target molecule specificity from the phage peptide library, and sequence the DNA, then get the corresponding polypeptide coding sequence. The technology realizes the connection between the genotype and the phenotype of the protein or polypeptide, has the characteristics of simple and convenient operation and high-throughput detection, becomes an efficient means for screening the specific binding peptide of the tumor cells, and provides a new direction for the research of the early detection of the tumor and the target vector of the drug therapy.
Disclosure of Invention
The invention aims to provide a polypeptide which can be specifically combined with a human colorectal cancer cell in a targeted manner, has no influence on a human normal intestinal epithelial cell, has an identification effect on a human pancreatic cancer cell, a human breast cancer cell and a human ovarian cancer cell, has an important effect on aspects of early diagnosis of colorectal cancer, research and development of targeted drugs and the like, and also has an important effect on early diagnosis of pancreatic cancer, breast cancer and ovarian cancer and research and development of targeted drugs.
The experiment uses human normal intestinal epithelial cells as a control, adopts human colorectal cancer cells HCT116 to carry out decrement screening on a phage display peptide library, selects positive phage clones which can be specifically combined with the colorectal cancer cells by a blue-white screening test, and verifies the combination specificity of the phage and the colorectal cancer cells by an ELISA test. And then taking escherichia coli as a carrier, amplifying and purifying the phage, extracting DNA of the phage, sequencing to obtain a coding sequence of the polypeptide capable of being specifically combined with the colorectal cancer cells, artificially synthesizing a fluorescence-labeled positive polypeptide, and verifying the combination effect of the fluorescence-labeled polypeptide and the human colorectal cancer cells and tissues, thereby providing an experimental basis for early diagnosis and targeted treatment of the colorectal cancer.
In order to achieve the purpose, the invention adopts the following technical scheme: a polypeptide which is (1) a polypeptide having an amino acid sequence which is: DYHDPSLPTLRK (SEQ ID No.1) and (2) a polypeptide derivative which has one or more amino acids deleted, inserted or substituted in the polypeptide molecule of (1) and has the same biological function as the polypeptide molecule of (1).
The polypeptide has targeting effect on tumor cells and is specifically combined with the tumor cells.
The tumor cell is human colorectal cancer cell, human pancreatic cancer cell, human breast cancer cell or human ovarian cancer cell.
The application of the polypeptide in preparing a tumor diagnosis kit, wherein the kit comprises the polypeptide or the polypeptide conjugate.
The application of the polypeptide in preparing a medicament for treating tumors, wherein the medicament comprises the polypeptide and a medicament active ingredient, or comprises the polypeptide and a medicament delivery carrier. The medicament is any pharmaceutically and therapeutically acceptable dosage form, and the preferred dosage form of the medicament is an injection preparation.
The medicament is in any pharmaceutically acceptable dose.
Compared with the prior art, the invention has the following effects: the invention has the advantages of simple operation, high flux panning, high efficiency, capability of screening simulation epitope, connection of displayed polypeptide or protein and gene code contained in the phage, easy purification of recombinant phage and the like by using the phage display technology. The peptide screened by the thallus display technology can be specifically combined with human colorectal cancer cells, has no specific effect with normal intestinal epithelial cells, and has obvious effect. And experiments prove that the peptide also has targeting effect on human pancreatic cancer cells, human breast cancer cells and human ovarian cancer cells.
Drawings
FIG. 1 shows the sequencing result of positive phage DNA which binds specifically to HCT 16.
FIG. 2 shows the targeting binding effect of FITC-DK12 to human normal intestinal epithelial cells HIEC and human colorectal cancer cells HCT116, SW 620.
FIG. 3 shows the targeting binding effect of FITC-DK12 to human ovarian epithelial cells HOSEpiC and human ovarian carcinoma glandular cells OVCAR-3, SK-OV-3.
FIG. 4 shows the targeting binding of FITC-DK12 to human embryonic pancreatic tissue-derived cells CCC-HPE-2 and human pancreatic cancer cells MIAPaCa-2.
FIG. 5 shows the targeting binding effect of FITC-DK12 to human mammary epithelial cells MCF-10A and human breast cancer cells MCF-7 and MDA-MB-23110A.
FIG. 6 shows the targeted binding of FITC-DK12 to human colorectal cancer and paraneoplastic tissues.
FIG. 7 shows the targeting binding of FITC-DK12 to HCT116, a tumor-bearing murine human colorectal cancer cell.
Detailed Description
The following description is of the preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Example 1
1. Experimental Material
1.1 phage peptide libraries, cells and host bacteria.
Phage 12 peptide library, Escherichia coli E.coli ER2738, human normal intestinal epithelial cells HIEC, human colorectal cancer cells HCT116 and SW620, human ovarian epithelial cells HOSEpic, human ovarian adenocarcinoma cells OVCAR-3 and SK-OV-3, human embryonic pancreatic tissue-derived cells CCC-HPE-2, human pancreatic cancer cells MIA PaCa-2, human mammary epithelial cells MCF-10A, human breast cancer cells MCF-7 and MDA-MB-231, tumor-bearing mice, human colorectal cancer tissues.
1.2 Experimental reagents
DEME culture medium, RPMI-1640 culture medium, trypsin, FITG labeled rabbit anti-mouse, fetal bovine serum, yeast powder, peptone, agar powder, tetracycline stock solution, Tween-20 (tween-20), bovine serum albumin BSA, M13 phage single-stranded DNA extraction kit, IPTG, X-gal, PEG-8000 and TMB.
1.3 Experimental working fluid
1 XPBS, LB liquid culture medium, LB-Tet solid plate, top agar, IPTG/X-gal working solution, IPTG/X-gal plate, PEG-NaCl, TBS buffer solution, 0.1% TBST, 0.5% TBST, 4% paraformaldehyde fixing solution, TBS-NaN3 solution preparation, 3% BSA blocking solution, sodium iodide buffer solution, TE buffer solution, TMB working solution and tetracycline storage solution.
2. Experimental methods
2.1 cultivation of E.coli.
(1) And (3) recovery of escherichia coli: taking out the frozen stock solution of the Escherichia coli glycerol from a refrigerator at the temperature of-80 ℃, taking a small amount of the frozen stock solution by using an inoculating loop, streaking the small amount of the frozen stock solution on an LB/Tet solid plate, then inversely placing the LB/Tet solid plate in an electrothermal constant temperature incubator at the temperature of 37 ℃ for overnight culture, and picking out a single colony when the frozen stock solution is used.
(2) And (3) culturing escherichia coli: 10ml LB/Tet broth was added to a 15ml centrifuge tube and single colonies of E.coli were picked and added. And then placing the centrifugal tube in a constant temperature oscillator for culturing overnight, and carrying out related experiments when the OD600 value of the bacterial liquid is 0.5.
2.2 subtractive selection of phage display peptide libraries.
(1) Preparing cells: firstly, pretreating a 6-hole culture plate by poly-lysine, taking human colorectal cancer cells HCT116 and human normal intestinal epithelial cells HIEC, respectively treating with trypsin, then spreading the cells in the cells, culturing until the cells are attached to the wall successfully and the growth state is good, and then screening.
(2) Preparing a bacterial liquid: on the screening day, the Escherichia coli ER2738 is inoculated into 20ml LB/Tet liquid culture medium, and placed in a constant temperature oscillator at 37 ℃ for shake culture, and when the OD600 value of the bacterial liquid is 0.5, the bacterial liquid is used for amplifying, screening and eluting the phage.
(3) Serum-free culture: the cell culture medium was aspirated off, washed 1 time with PBS and serum-free medium was added, and placed in a 37 ℃ thermostatted cell incubator with 5% CO2 for 1 h.
(4) And (3) washing, namely sucking off the confining liquid, washing 5 times by using 0.1% TBST (tert-butyl-tert-butyl) slightly, rotating each time to wash the bottoms and the edges of the micropores, and spin-drying. 0.5% TBST and 1.0% TBST were used for screening up to rounds 2 and 3, respectively.
(5) And (3) cell blocking, namely sucking off the cell culture medium, pouring the plate on a clean paper towel, forcibly patting and throwing to remove the residual culture medium, blocking the human colorectal cancer cells HCT116 and the human normal intestinal epithelial cells HIEC by using the culture medium containing 1% BSA, and placing in a 37 ℃ constant temperature cell incubator filled with 5% CO2 for 1 h.
(6) And (3) adsorbing, namely taking 10 mu l of the original peptide library, adding the original peptide library into 990 mu l of 0.5% BSA/PBS buffer solution, diluting the phage to be 1.5 multiplied by 1011pfu/ml, adding the diluted phage into the blocked human normal intestinal epithelial cell HIEC, adsorbing the phage capable of being combined with the human normal intestinal epithelial cell HIEC at 37 ℃ for 1h, and taking the supernatant.
(7) And (3) combining, namely incubating the phage supernatant after adsorption with the human colorectal cancer cell HCT116 for 1 h.
(8) And (4) washing, namely discarding the unbound phage, and pouring the microporous plate on a clean paper towel to be forcibly flapped to remove the residual solution. The plates were washed 5 times with 0.1% TBST as described above.
(9) Elution 1ml of 0.2M Glycine-HCl (pH2.2)1mg/ml BSA eluent was added, slowly shaken on ice for 10min, and then the eluent was aspirated and transferred to 150. mu.l of a previously prepared neutralization solution (1M Tris-HCl, pH 9.1).
(10) The above procedure was repeated 2 times.
2.3 measurement of phage titer.
Preheating an IPTG/X-gal flat plate in an electrothermal constant-temperature incubator at 37 ℃; taking out a proper amount of top agar, heating in a microwave oven, taking out after the top agar is completely melted, and subpackaging 3ml in 10ml centrifuge tubes; after the phage to be screened is diluted in equal proportion, 10 mul and 200 mul of escherichia coli liquid are taken to be fully mixed and react for 5min, then the mixture is added into 3ml of top agar, then the mixture is evenly paved on a preheated IPTG/X-gal flat plate, after condensation, the mixture is placed in an electric heating constant temperature incubator at 37 ℃ for overnight, and the titration result is observed.
2.4 amplification and purification of phage.
(1) Amplification of phage: adding 20ml of LB/Tet liquid culture medium into a conical flask, then adding escherichia coli liquid and phage to be amplified according to a ratio of 1:100, and placing the mixture in a constant temperature oscillator at 37 ℃ to shake vigorously for 4.5 hours to obtain phage amplification liquid.
(2) And (3) purifying the phage: centrifuging the phage amplification solution obtained by the above steps at 4 ℃ at 12000r/min for 10min, taking the supernatant, adding 1/6 volumes of PEG-NaCl, precipitating overnight, centrifuging at 12000r/min for 15min, discarding the supernatant, dissolving the precipitate with TBS buffer solution, adding 1/6 volumes of PEG-NaCl again, and incubating on ice for 1 h. Centrifuging at 14000r/min for 15min at 4 deg.C, discarding supernatant, dissolving the obtained precipitate with TBS-NaN3, and storing in refrigerator at 4 deg.C.
2.5 extraction and sequencing of Positive phage DNA
(1) Adding 100ul iodide buffer solution into the purified phage precipitate, adding 250ul absolute ethanol, mixing well, and acting at room temperature for 20 min.
(2) Centrifuging: 4 ℃, 14,000rpm, 10min, discard the supernatant.
(3) Cleaning: the precipitate was washed with 500ul 70% ethanol, centrifuged briefly and dried in vacuo.
(4)30ul TE (10mM Tris-HCl, pH5.0,1mM EDTA) buffer solution, and resuspending the precipitate, preparing DNA sequencing template solution, and sending to Shanghai for biological sequencing.
2.6 cellular immunofluorescence assay
(1) Cell plating: human normal intestinal epithelial cells HIEC, human colon cancer cells HCT116 and SW620, human ovarian epithelial cells HOSEpic, human ovarian adenocarcinoma cells OVCAR-3 and SK-OV-3, human embryonic pancreatic tissue-derived cells CCC-HPE-2, human pancreatic cancer cells MIA PaCa-2, human mammary epithelial cells MCF-10A, human breast cancer cells MCF-7 and MDA-MB-231 were plated in a six-well plate for use.
(2) Fixing: fix with 4% paraformaldehyde for 15 min.
(3) And (3) sealing: 4% paraformaldehyde was discarded, washed 2 times with PBS, and blocked with 3% BSA/PBS at 37 ℃ for 30 min.
(4) FITC-DK12 incubation: after wiping the blocking solution, FITC-DK12 was added at 37 ℃ for 1 h.
(5) DAPI staining: washing with PBS 3 times, adding 100 μ l DAPI, and standing at room temperature for 15min
(6) And (4) sealing, namely sealing after washing for 3 times by PBS.
2.7 tissue immunofluorescence assay
(1) Baking slices: 60 ℃ for 4-6 h
(2) Slice dewaxing to water: xylene I15 min → xylene II 15min → absolute ethyl alcohol I5 min → absolute ethyl alcohol II 5min → 95% ethyl alcohol 5min → 85% ethyl alcohol 5min → 75% ethyl alcohol 5min → distilled water 5 min.
(3) And (3) sealing: blocking with 3% BSA/PBS at 37 ℃ for 30 min.
(4) FITC-DK12 incubation: after wiping the blocking solution, FITC-DK12 was added at 37 ℃ for 1 h.
(5) DAPI staining: washing with PBS 3 times, adding 100 μ l DAPI, and standing at room temperature for 15min
(6) And (4) sealing, namely sealing after washing for 3 times by PBS.
2.8 tumor-bearing mouse experiment
(1) Carrying out tumor loading: the nude mice used for the experiment are aged four weeks, and human colon cancer cells HCT116 are inoculated in the armpits to form tumors.
(2) Imaging: the polypeptides were prepared into a 100. mu.M/ml solution in PBS for future use, 0.1ml of FITC-DK12 polypeptide solution was administered to the tail vein of mice bearing tumors, and 10 minutes later, the solution was imaged on a small animal imager.
3. Results of the experiment
The sequencing result of the target phage DNA aiming at human colorectal cancer cell HCT116 is shown in figure 1, and a polypeptide sequence is translated according to the principle of triple codon: DYHDPSLPTLRK (SEQ ID No.1) (DK 12).
The polypeptide FITC-DK12 is fluorescently labeled, the targeted binding effect of the polypeptide with human colorectal cancer cells, ovarian cancer cells, pancreatic cancer cells and breast cancer cells is detected by an immunofluorescence staining experiment, then the targeted binding capability of the polypeptide and human colorectal cancer tissues is further verified by the immunofluorescence staining experiment, and the targeted action of the polypeptide and human colorectal cancer cells HCT16 is verified by a tumor-bearing mouse. As shown in FIG. 2, the polypeptide sequence can be combined with human colorectal cancer cells HCT116 and SW620 in a targeting way, and has weak combination ability to normal intestinal mucosa epithelial cells HIEC, and the two have significant difference; meanwhile, the polypeptide sequence can also be combined with human ovarian adenocarcinoma cells OVCAR-3 and SK-OV-3 in a targeted manner, but the combination capability of the polypeptide sequence to human normal ovarian epithelial cells HOSEpic is weaker, and the polypeptide sequence and the HOSEpic have significant difference (see figure 3); the polypeptide sequence can also be combined with human pancreatic cancer cells MIAPaCa-2, human breast cancer cells MCF-7 and MDA-MB-231 in a targeted mode, the combination capability of human embryonic pancreatic tissue source cells CCC-HPE-2 and human mammary epithelial cells MCF-10A is weak, and the two are obviously different (see figures 4 and 5); further experiments confirmed that FITC-DK12 was able to specifically target human colorectal cancer tissue, was weak in binding to paracarcinoma tissues (see FIG. 6), and had targeting binding ability to HCT116, a tumor-bearing mouse human colorectal cancer cell (see FIG. 7).
SEQUENCE LISTING
<110> university of Chinese medical science
<120> a polypeptide and uses thereof
<130>1
<160>1
<170>PatentIn version 3.3
<210>1
<211>12
<212>PRT
<213> Artificial sequence
<400>1
Asp Tyr His Asp Pro Ser Leu Pro Thr Leu Arg Lys
1 5 10
Claims (7)
1. The application of the polypeptide in preparing a colon cancer, pancreatic cancer, breast cancer or ovarian cancer diagnostic kit is characterized in that the amino acid sequence of the polypeptide is as follows: DYHDPSLPTLRK (SEQ ID No. 1); the polypeptide has a targeting effect on colon cancer cells, pancreatic cancer cells, breast cancer cells or ovarian cancer cells, and is specifically combined with colon cancer cells, pancreatic cancer cells, breast cancer cells or ovarian cancer cells.
2. Use of a polypeptide according to claim 1 in the preparation of a diagnostic kit for colon, pancreatic, breast or ovarian cancer comprising said polypeptide.
3. The application of a polypeptide in preparing a medicament for treating colon cancer and pancreatic cancer is characterized in that the amino acid sequence of the polypeptide is as follows: DYHDPSLPTLRK (SEQ ID No. 1).
4. The use of the polypeptide of claim 3 in the preparation of a medicament for the treatment of colon cancer or pancreatic cancer, wherein the medicament comprises the polypeptide and a pharmaceutically active ingredient, or comprises the polypeptide and a delivery vehicle.
5. Use of a polypeptide according to claim 3 for the preparation of a medicament for the treatment of colon and pancreatic cancer, in any pharmaceutically and therapeutically acceptable dosage form.
6. The use of the polypeptide of claim 3 in the preparation of a medicament for the treatment of colon and pancreatic cancer, wherein the medicament is in the form of an injectable formulation.
7. Use of a polypeptide according to claim 3 for the preparation of a medicament for the treatment of colon and pancreatic cancer, in any pharmaceutically and therapeutically acceptable dose.
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| CN108409836B (en) * | 2018-01-08 | 2020-04-21 | 中国医科大学 | Polypeptide and application thereof |
| CN110330551B (en) * | 2019-08-05 | 2023-02-28 | 中国医科大学 | Pancreatic cancer specific binding peptide and preparation method and application thereof |
| CN112979757B (en) * | 2019-12-17 | 2024-02-13 | 沈阳医健生命科技有限责任公司 | Polypeptide of specific targeting human liver cancer cell |
| CN111253472B (en) * | 2020-04-02 | 2022-05-27 | 中国医科大学 | Novel polypeptide targeting multiple tumor cells and application thereof |
| WO2023184332A1 (en) * | 2022-03-31 | 2023-10-05 | 深圳先进技术研究院 | Ovary-targeting polypeptide and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105198964A (en) * | 2015-10-12 | 2015-12-30 | 国家纳米科学中心 | Tumor targeted polypeptide, and preparation method and application thereof |
| EP3042955A1 (en) * | 2013-09-06 | 2016-07-13 | The University of Tokyo | Use of rhoa in cancer diagnosis and inhibitor screening |
| CN106749527A (en) * | 2016-12-28 | 2017-05-31 | 南京农业大学 | Phage-displayed polypeptides that imidaclothiz antibody specificity is combined and application thereof |
| CN109942682A (en) * | 2017-12-20 | 2019-06-28 | 深圳先进技术研究院 | A kind of CMKLR1 antagonism polypeptide and its derivative and application |
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| CN108530520B (en) * | 2018-04-19 | 2021-02-19 | 福建师范大学福清分校 | Manganese ion binding peptide, screening method thereof and method for detecting affinity performance |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3042955A1 (en) * | 2013-09-06 | 2016-07-13 | The University of Tokyo | Use of rhoa in cancer diagnosis and inhibitor screening |
| CN105198964A (en) * | 2015-10-12 | 2015-12-30 | 国家纳米科学中心 | Tumor targeted polypeptide, and preparation method and application thereof |
| CN106749527A (en) * | 2016-12-28 | 2017-05-31 | 南京农业大学 | Phage-displayed polypeptides that imidaclothiz antibody specificity is combined and application thereof |
| CN109942682A (en) * | 2017-12-20 | 2019-06-28 | 深圳先进技术研究院 | A kind of CMKLR1 antagonism polypeptide and its derivative and application |
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