CN108640976B

CN108640976B - Polypeptide specifically bound with human colon cancer cells

Info

Publication number: CN108640976B
Application number: CN201810385293.8A
Authority: CN
Inventors: 于丽凤; 魏敏杰; 赵琳; 贾真; 卫倩
Original assignee: China Medical University
Current assignee: China Medical University
Priority date: 2018-04-26
Filing date: 2018-04-26
Publication date: 2021-04-09
Anticipated expiration: 2038-04-26
Also published as: CN108640976A; WO2019205867A1

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a polypeptide specifically binding to colon cancer cells and application thereof. The polypeptide is the amino acid sequence of any one of the following (1): GLTSMRYHSVIV (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 of the invention can be specifically combined with colon cancer cells, has no specific action with normal intestinal epithelial cells, has obvious action effect and provides reliable scientific basis for clinical treatment.

Description

Polypeptide specifically bound with human colon cancer cells

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a polypeptide specifically binding to colon cancer cells SW480 and application thereof.

Background

Colon cancer is one of the most common digestive tract malignancies, second to gastric and esophageal cancer in incidence. The good hair age is generally over 50 years old, more men than women, and the ratio of men to women is 2: 1. The cause of colon cancer is not clear, and mainly comprises environmental factors, genetic factors, precancerous diseases and the like. Domestic investigation shows that the survival rate of early colon cancer after operation reaches 90-95%, but the early colon cancer is developed to the middle and late stages when more than 80% of patients are actually diagnosed in China, and the early diagnosis rate is only 10-15%; the survival rate of advanced colon cancer is only 5%. Therefore, early diagnosis and early treatment can obviously improve the prognosis of the colon cancer patient.

Meanwhile, early symptoms of colon cancer are mostly not noticed by patients, and diseases such as dysentery and enteritis are often treated when patients seek medical treatment, and once toxic symptoms or obstruction symptoms appear and abdominal masses are touched, the early symptoms are not early, so that the development of sensitive diagnosis technology and the early diagnosis are one of important research points.

The existing anticancer drugs generally have the defects of large toxic and side effects, large drug dosage, easy generation of acquired drug resistance and the like, which can affect the treatment of patients. Targeted therapy is a therapeutic modality that, at the cellular molecular level, targets an already well-defined oncogenic site. Corresponding therapeutic drugs can be designed, and the drugs entering the body can specifically select carcinogenic sites to combine and act, so that tumor cells are specifically killed, and normal tissue cells around the tumor cannot be affected, so that molecular targeted therapy is another important research point of cancer treatment.

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 to screen the phage by subtraction, finally screen the target phage which can combine with the target cell specificity from the phage polypeptide library, and sequence its 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 colon cancer cells in a targeted manner, has no influence on normal intestinal epithelial cells, and plays an important role in the aspects of early diagnosis of colon cancer, development of targeted drugs and the like.

The experiment takes human normal intestinal epithelium HIEC cells as a contrast, adopts human colon cancer SW480 cells to carry out subtraction screening on a phage display polypeptide library, selects positive phage clones which can be specifically combined with colon cancer cells by a blue-white screening test, and verifies the specificity of the combination of the phage and the colon cancer 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 colon cancer, artificially synthesizing the fluorescence-labeled positive polypeptide, and verifying the combination of the fluorescence-labeled polypeptide and human colon cancer cells, thereby providing an experimental basis for early diagnosis and targeted treatment of colon cancer.

In order to achieve the purpose, the invention adopts the following technical scheme: a polypeptide that specifically binds to a human colon cancer cell, the polypeptide being any of: (1) the amino acid sequence of the polypeptide is: GLTSMRYHSVIV (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 combination on tumor cells and specific combination with the tumor cells.

The tumor cell is a colon cancer cell.

The application of the polypeptide in preparing a tumor diagnosis kit, wherein the kit comprises the polypeptide or the polypeptide conjugate.

Use of a polypeptide that specifically binds to human colon cancer cells in the preparation of a medicament comprising said polypeptide and a pharmaceutically active ingredient, or comprising said polypeptide and a delivery vehicle, for the treatment of colon cancer. 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 and therapeutically 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 polypeptide screened by the thallus display technology can be specifically combined with colon cancer cells, has no specific action with normal intestinal epithelial cells, has obvious action effect and provides reliable scientific basis for clinical treatment.

Drawings

FIG. 1 shows the OD of ELISA for identifying the affinity of No. 1-20 positive phage clones with human colon cancer SW480 cells₄₀₅And (5) a result chart.

FIG. 2 is a diagram showing the results of immunofluorescence experiments on cells with affinity of No. 8 positive phage clones to human normal intestinal epithelial HIEC cells and human colon cancer SW480 cells.

FIG. 3 is a graph showing the sequencing results of the number 8 positive phage clone.

FIG. 4 is a graph showing the results of cellular immunofluorescence of FITC-GV12 in targeted combination with HIEC and SW480 cells.

FIG. 5 is a graph showing the results of flow cytometry on the binding ability of FITC-GV12 to HIEC and SW480 cells. A is a graph of the binding capacity result of HIEC and PBS; b is a graph of the binding capacity of HIEC to the targeting polypeptide FITC-GV 12; c is a graph of the binding capacity results of SW480 and PBS; d is a graph showing the binding capacity of SW480 and the targeting polypeptide FITC-GV 12.

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, E.coli ER2738, human colon cancer SW480 cells, and human normal intestinal epithelium HIEC cells.

1.2 Experimental reagents

L-15 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: and (3) 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, scribing 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 in use.

(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. Then placing the centrifuge tube in a constant temperature oscillator for culturing overnight until OD of the bacterial liquid₆₀₀The correlation experiment was carried out at a value of 0.5.

2.2 subtractive selection of phage display polypeptide libraries.

(1) Preparing cells: firstly, pretreating a 6-hole culture plate by poly-lysine, taking human colon cancer SW480 cells and human normal intestinal epithelial HIEC cells, respectively treating the cells by trypsin, then spreading the cells in the cells, culturing the cells until the cells are attached to the wall successfully and the growth state is good, and then screening the cells.

(2) Preparing a bacterial liquid: on the screening day, the Escherichia coli ER2738 is inoculated into 20ml of LB/Tet liquid culture medium, placed in a constant temperature oscillator at 37 ℃ for shake culture, and the OD of the bacterial liquid is waited₆₀₀At a value of 0.5, the eluted phage was amplified and screened.

(3) Serum-free culture: the cell culture medium is sucked off, washed 1 time by PBS, added with a serum-free culture medium and placed in a constant temperature cell culture box with 5 percent CO2 at 37 ℃ 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 culture medium is sucked, the plate is placed on a clean paper towel upside down, residual culture medium is removed by forceful throwing, human colon cancer SW480 cells and human normal intestinal epithelium HIEC cells are sealed by a culture medium containing 1% BSA, and the cells are placed in a constant temperature cell culture box of 37 ℃ and filled with 5% CO2 for 1 h.

(6) Adsorption, 10. mu.l of the original polypeptide library was added to 990. mu.l of 0.5% BSA/PBS buffer to dilute the phage to 1.5X 10¹¹pfu/ml, adding the pfu/ml into the sealed human normal intestinal epithelial HIEC cells, adsorbing the phage capable of being combined with the human normal intestinal epithelial HIEC cells at 37 ℃ for 1h, and reserving the supernatant.

(7) And (3) combining, namely incubating the adsorbed phage supernatant and the human colon cancer SW480 cells 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 top agar is evenly paved on a preheated IPTG/X-gal flat plate, after the mixture is condensed, the top agar is placed in an electric heating constant temperature incubator with 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 an escherichia coli liquid and a bacteriophage to be amplified according to a ratio of 1:100, placing the mixture at 37 ℃, and violently shaking the mixture in a constant temperature oscillator for 4.5 hours to obtain an amplification solution of the bacteriophage.

(2) And (3) purifying the phage: centrifuging the phage amplification solution obtained in the previous step at 4 ℃ and 12000r/min for 10min, taking the supernatant, adding 1/6 volumes of PEG-NaCl to precipitate 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 ℃, discarding the supernatant, dissolving the obtained precipitate with TBS-NaN3, and storing in a refrigerator at 4 ℃.

2.5 enzyme-linked immunosorbent assay

(1) Preparation of cell 96-well plates, plating rule: two rows of 16 holes on the edge of a 96-well plate are respectively added with 100 mu l of PBS to be used as blank groups; then, 100 mu of human normal intestinal epithelial HIEC cell suspension is respectively paved on each pore in the

rows

1, 2, 3 and 4 according to a snake shape, 100 mu of human colon cancer SW480 cell suspension is respectively paved on each pore in the

rows

5, 6, 7 and 8 according to a snake shape, and then the paved cell plate is placed in a 37 ℃ cell constant temperature incubator filled with 5% CO2 overnight to carry out ELISA experiment.

(2) Fixing: the overnight plated 96-well plates were removed, rinsed dry, washed 3 times with PBS, and fixed with 4% paraformaldehyde for 20 min.

(3) Blocking: and (3) taking out the fixed 96-well plate, drying liquid in the well, washing the well for 3 times by using PBS (phosphate buffer solution), adding 3% hydrogen peroxide, and sealing the well in a 37 ℃ cell constant temperature incubator for 30min to block the activity of endogenous peroxidase.

(4) And (3) sealing: and (3) taking out the blocked 96-well plate, beating the liquid in the hole, washing the hole for 3 times by using PBS, adding 3% BSA/PBS, and sealing the hole for 1h in a 37 ℃ cell constant temperature incubator.

(5) Adding a phage sample: and (3) taking out the sealed 96-well plate, beating the liquid in the hole, adding the purified positive phage, and reacting for 1h in a 37 ℃ cell constant temperature incubator.

(6) Adding a primary antibody: and (3) taking out the reacted 96-well plate, beating the liquid in the well, washing the well for 3 times by using PBS, adding a 1: 4000M 13 antibody, and standing the well at 4 ℃ overnight.

Secondary antibody: and (3) taking out the reacted 96-well plate, beating the liquid in the well, washing the well for 3 times by using PBS, adding a secondary antibody, and reacting the mixture for 30min in a cell constant-temperature incubator at 37 ℃.

(7) Adding a substrate TMB: and (3) adding a TMB (Tetramethylbenzidine) display agent into a 96-well plate washed by PBS for 3 times under the condition of keeping out of the sun, and placing the plate in a 37-DEG C cell constant-temperature incubator for 15min in the absence of the sun.

(8) And (4) terminating: the reacted 96-well plate was removed and the reaction was stopped by adding 2M sulfuric acid.

(9) Measurement of results: the 96-well plate which has completed all reactions is placed in a microplate reader, the OD value is measured at 405nm, and the result is stored and analyzed.

2.6 immunofluorescence assay of cells with Positive phages

(1) Cell plating: human normal intestinal epithelial HIEC cells and human derived colon cancer SW480 cells were plated in six-well plates 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) And (3) positive phage incubation: after wiping the blocking solution, the positive phage 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 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.

2.8 cellular immunofluorescence assay

(1) Preparing cell plating: spreading a glass slide on a 6-well plate, and spreading human colon cancer SW480 cells and human normal intestinal epithelial HIEC cells in a six-well plate for later use.

(2) Fixing: fix with 4% paraformaldehyde for 15 min.

(4) FITC-GV12 incubation: after wiping the blocking solution, FITC-GV12 was added at 37 ℃ for 1 h.

(6) And (4) sealing, namely sealing after washing for 3 times by PBS.

2.9 flow cytometry

(1) Preparing cell plating: and (3) paving the human colon cancer SW480 cells and the human normal intestinal epithelial HIEC cells in a six-well plate for later use.

(2) And (3) sealing: PBS washing 2 times, with 3% BSA/PBS at 37 degrees C, blocking for 30 min.

(3) FITC-GV12 incubation: the blocking solution was discarded and FITC-GV12 was added and incubated at 37 ℃ for 30 min.

(4) Collecting cells: washing with PBS for 3 times, adding pancreatin to digest and collecting cells for upstream detection.

3. Results of the experiment

As shown in FIG. 1, 20 E.coli clones carrying phage polypeptides were randomly picked and analyzed by ELISA, and the results showed that the average absorbance value OD of SW480 in the experimental group was determined₄₀₅Average absorbance value OD with control HIEC₄₀₅The number of clones with a ratio of greater than 2 was 12, 1, 2, 5, 8, 9, 10, 12, 14, 15, 17, 18, 20. The 12 positive phages have stronger binding effect with SW480 cells of the human colon cancer and weaker binding effect with HIEC cells of the human normal intestinal epithelium. Then, 8 # positive phage is selected to perform an immunofluorescence staining experiment to further verify the targeted binding of the positive phage and human colon cancer cells. The result of the cell immunofluorescence experiment shows that the positive phageThe body can be specifically combined with human colon cancer cells SW480, but the combination ability with human normal intestinal epithelium HIEC cells is weaker, and the two have significant difference, further suggesting that the positive phage polypeptide has the function of targeted combination on human colon cancer cells (as shown in figure 2).

Then, 12 positive phage clones were amplified, purified, extracted, and subjected to DNA sequencing, and the sequencing results showed that 8 positive phage clones in total showed the same sequence, No.1, 2, 8, 10, 14, 17, 18, and 20, respectively. As shown in fig. 3, the polypeptide sequence was translated according to the triple codon principle: GLTSMRYHSVIV (SEQ ID No. 1) (GV 12).

Then, a green fluorescence labeled positive polypeptide FITC-GV12 is artificially synthesized, and the targeted combination of the polypeptide and human colon cancer cells SW480 is verified by adopting an immunofluorescence staining experiment and a flow cytometry technology respectively. As shown in FIG. 4, the binding ability of the polypeptide sequence to human normal intestinal epithelial HIEC cell is weak, the relative fluorescence intensity value is 1129.92 + -460.65, the binding ability to human colon cancer cell SW480 is strong, the relative fluorescence intensity value is 36429.45 + -4173.03, and the two have significant differenceP < 0.0001。

The flow cytometry experiments further confirmed the above results. The HIEC cells of human normal intestinal epithelium and SW480 cells of human colon cancer were respectively incubated with 10 μ M FITC-GV12 and then subjected to flow detection, and the percentage of positive cells was respectively 1.9% and 99.9%, and the two cells were significantly different (see FIG. 5). The polypeptide is proved to be capable of being specifically combined with colon cancer cells in a targeted mode, and has no influence on normal intestinal epithelial cells.

SEQUENCE LISTING

<110> university of Chinese medical science

<120> a polypeptide specifically binding to human colon cancer cells

<130> 1

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 12

<212> PRT

<213> unknown

<400> 1

Gly Leu Thr Ser Met Arg Tyr His Ser Val Ile Val

1 5 10

Claims

1. A polypeptide that specifically binds to human colon cancer cells, wherein the amino acid sequence of the polypeptide is SEQ ID No. 1: GLTSMRYHSVIV are provided.

2. The polypeptide of claim 1, wherein the polypeptide has targeted binding to tumor cells and specific binding to colon cancer cells.

3. Use of a polypeptide according to claim 1 which specifically binds to human colon cancer cells in the manufacture of a medicament for the treatment of colon cancer.

4. Use of a polypeptide that specifically binds to human colon cancer cells as defined in claim 1 in the preparation of a medicament for the treatment of colon cancer, wherein the medicament comprises said polypeptide and a pharmaceutically active ingredient, or comprises said polypeptide and a delivery vehicle.

5. Use of a polypeptide that specifically binds to human colon cancer cells for the preparation of a medicament for the treatment of colon cancer according to claim 4, wherein the medicament is in any pharmaceutically therapeutically acceptable dosage form.

6. Use of a polypeptide that specifically binds to human colon cancer cells for the preparation of a medicament for the treatment of colon cancer according to claim 4, wherein the medicament is in any pharmaceutically therapeutically acceptable dose.