WO2019205867A1

WO2019205867A1 - Polypeptide specifically binding to human colon cancer cells

Info

Publication number: WO2019205867A1
Application number: PCT/CN2019/079772
Authority: WO
Inventors: 于丽凤; 魏敏杰; 赵琳
Original assignee: 中国医科大学
Priority date: 2018-04-26
Filing date: 2019-03-27
Publication date: 2019-10-31
Also published as: CN108640976B; CN108640976A

Abstract

Disclosed are a polypeptide that specifically binds to colon cancer cells and a use thereof. The polypeptide is any of the following: (1) a polypeptide having the amino acid sequence: GLTSMRYHSVIV (SEQ ID No. 1), and (2) a polypeptide derivative for which one or several amino acids in the polypeptide molecule of (1) has undergone deletion, insertion, or displacement and which has the same biological function as that of the polypeptide molecule of (1). The polypeptide of the present invention is capable of specifically binding to colon cancer cells without specific action with normal intestinal epithelial cells.

Description

a polypeptide that specifically binds to human colon cancer cells

Technical field

The invention belongs to the field of biomedicine, and particularly relates to a polypeptide which specifically binds to colon cancer cell SW480 and uses thereof.

Background technique

Colon cancer is one of the most common malignant tumors of the digestive tract, and its incidence is second only to gastric cancer and esophageal cancer. The age of good hair is generally over 50 years old, males are more than women, and the ratio of male to female is 2:1. The cause of colon cancer is not clear, mainly environmental factors, genetic factors, precancerous diseases. Domestic surveys show that the survival rate of early colon cancer surgery is 90-95%, but in China more than 80% of patients have developed into the middle and late stage of diagnosis, the early diagnosis rate is only 10-15%; and the advanced colon cancer The survival rate is only 5%. Therefore, early diagnosis and early treatment can significantly improve the recovery of colon cancer patients.

At the same time, the early symptoms of colon cancer are not paid attention to by the patients. When they seek medical treatment, they are often treated with diseases such as “dysentery” and “intestinitis”. Once symptoms of poisoning or obstruction and touching the abdominal mass are not early, development of sensitive diagnosis Technology, so early diagnosis is one of the research priorities.

Existing anticancer drugs usually have disadvantages such as large toxic and side effects, large dosage of drugs, and prone to acquired drug resistance, which may affect the treatment of patients. Targeted therapy is the treatment of established cancer sites at the cellular level. The corresponding therapeutic drug can be designed, and the drug enters the body to specifically select the carcinogenic site to combine and act to cause the tumor cell to specifically die without affecting the normal tissue cells surrounding the tumor, so the molecular targeted therapy is cancer treatment. Another research focus.

Phage display technology is an important technique for screening intermolecular interactions in the field of molecular biology. The main principle of phage display technology is that the gene of interest or the gene encoding the protein and polypeptide is cloned into the appropriate position of the phage surface protein gene by genetic engineering technology, and it is expressed on the surface of the phage with the amplification of phage DNA, due to the foreign gene. In contrast to the phage gene, the expression product polypeptide or protein of the foreign gene can maintain its original spatial structure and corresponding biological activity. Then, we use the target cells to perform subtractive screening on the phage, and finally select the phage which can specifically bind to the target cells from the phage polypeptide library, and sequence the DNA to obtain the coding sequence of the corresponding polypeptide. This technology enables the connection between genotypes and phenotypes of proteins or peptides, and has the characteristics of simple operation and high-throughput detection, thus becoming an efficient means for screening tumor cell-specific binding peptides, for early detection of tumors and Targeted vector research in drug therapy offers new directions.

Summary of the invention

The object of the present invention is to provide a polypeptide which can specifically bind to colon cancer cells without affecting normal intestinal epithelial cells, which plays an important role in the early diagnosis of colon cancer and the development of targeted drugs.

In this experiment, human normal intestinal epithelial HIEC cells were used as control. Human phage display peptide library was used for subtractive screening of human phage display peptide library. Blue-white screening test was used to select positive phage which can specifically bind to colon cancer cells. The specificity of phage binding to colon cancer was verified by ELISA. Then, using Escherichia coli as a vector, the purified phage is amplified and the DNA is extracted for sequencing, thereby obtaining a coding sequence of a polypeptide capable of specifically binding to colon cancer, and synthesizing a fluorescently labeled positive polypeptide for fluorescent labeling-polypeptide and human The verification of the binding effect of colon cancer cells provides an experimental basis for early diagnosis and targeted therapy of colon cancer.

In order to achieve the above object, the present invention adopts the following technical scheme: a polypeptide which specifically binds to human colon cancer cells, and the polypeptide is any of the following: (1) the amino acid sequence of the polypeptide is: GLTSMRYHSVIV (SEQ ID No. 1), ( 2) A polypeptide derivative which has undergone deletion, insertion or substitution of one or several amino acids in the polypeptide molecule of (1) and which has the same biological function as the polypeptide molecule of (1).

The polypeptide has targeted binding to tumor cells and specifically binds to tumor cells.

The tumor cells are colon cancer cells.

The use of a polypeptide in the preparation of a tumor diagnostic kit comprising the polypeptide or polypeptide conjugate.

A polypeptide for specifically binding to human colon cancer cells for use in the preparation of a medicament for treating colon cancer, the medicament comprising the polypeptide and a pharmaceutically active ingredient, or comprising the polypeptide and a delivery vehicle. The medicament is any pharmaceutically therapeutically acceptable dosage form, and the preferred dosage form of the medicament is an injectable preparation.

The drug is any pharmacologically acceptable dose.

Compared with the prior art, the invention has the advantages that the phage display technology of the invention has simple operation, high-throughput panning, high efficiency, and can screen mimic epitopes, display polypeptides or proteins and genes contained in the phage. The connection of the password and the ease of purification of the recombinant phage. We use the peptides screened by the cell display technology to specifically bind to colon cancer cells, but have no specific effect on normal intestinal epithelial cells, and the effect is obvious, which provides a reliable scientific basis for clinical treatment.

DRAWINGS

Figure 1 ELISA results showing the OD ₄₀₅ results of the affinity of clone 1-20 positive phage clones and human colon cancer SW480 cells.

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

Figure 3 is a graph showing the sequencing results of the positive phage clone No. 8.

Figure 4 is a graph showing the results of cellular immunofluorescence of FITC-GV12 and HIEC, SW480 cells.

Fig. 5 is a flow chart showing the results of flow cytometry of FITC-GV12 binding to HIEC and SW480 cells. A is the binding ability result of HIEC and PBS; B is the binding ability result of HIEC and the targeting polypeptide FITC-GV12; C is the binding ability result of SW480 and PBS; D is the combination of SW480 and the targeting polypeptide FITC-GV12 Capability results map.

detailed description

The following description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes can be made by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and scope of the present invention are intended to be included within the scope of the present invention.

Example 1

Experimental material

1.1 phage peptide library, cells and host bacteria.

Phage 12 peptide library, E. coli ER2738, human colon cancer SW480 cells, human normal intestinal epithelial HIEC cells.

1.2 experimental reagents

L-15 medium, trypsin, FITG-labeled rabbit anti-mouse, fetal bovine serum, yeast powder, peptone, agar powder, tetracycline stock solution, Tween-20, bovine serum albumin BSA, M13 phage single-strand DNA extraction kit, IPTG, X-gal, PEG-8000, TMB.

1.3 experimental working fluid

1×PBS, LB liquid medium, LB-Tet solid plate, top agar, IPTG/X-gal working solution, IPTG/X-gal plate, PEG-NaCl, TBS buffer, 0.1% TBST, 0.5% TBST, 4 Preparation of % paraformaldehyde fixative, TBS-NaN3 solution, 3% BSA blocking solution, sodium iodide buffer, TE buffer, TMB working solution, tetracycline stock solution.

2. Experimental methods

2.1 Cultivation of E. coli.

(1) Resuscitation of E. coli: Take the E. coli glycerol cryopreservation solution from the -80 °C refrigerator, take a small amount of scribing on the LB/Tet solid plate with the inoculating loop, and then pour the LB/Tet solid plate at 37 °C. Incubate overnight in an electrothermal incubator and pick a single colony when using.

(2) Culture of Escherichia coli: 10 ml of LB/Tet liquid medium was added to a 15 ml centrifuge tube, and a single colony of Escherichia coli was picked and added thereto. Then, the centrifuge tube was placed in a constant temperature oscillator and cultured overnight, and the relevant experiment was carried out when the OD ₆₀₀ value of the bacterial liquid was 0.5.

2.2 Subtraction screening of phage display polypeptide libraries.

(1) Preparing cells: The 6-well culture plate was pretreated with poly-lysine, and human colon cancer SW480 cells and human normal intestinal epithelial HIEC cells were taken and treated with trypsin and then cultured until the cells were successfully labeled. Screening was performed after the wall was in good growth.

(2) Preparation of bacterial liquid: On the day of screening, Escherichia coli ER2738 was inoculated into 20 ml LB/Tet liquid medium, and placed in a 37 ° C constant temperature shaker for shaking culture. When the OD ₆₀₀ value of the bacterial liquid was 0.5, it was used for expansion. Increase the screening of eluted phage.

(3) Serum-free culture: The cell culture medium was aspirated, washed once with PBS, and then added to serum-free medium, and placed in a 37 ° C constant temperature cell incubator with 5% CO 2 for 1 h.

(4) Washing: The blocking solution was aspirated and washed gently with 0.1% TBST for 5 times, each time being rotated so that the bottom and edges of the micropores were washed and dried. 0.5% TBST and 1.0% TBST were used for the second and third rounds of screening, respectively.

(5) Closing the cells: Aspirate the cell culture medium, place the plate on a clean paper towel, remove the remaining medium, and block the human colon cancer SW480 cells and human normal intestinal epithelium HIEC with 1% BSA medium. The cells were placed in a 37 ° C thermostatic cell incubator with 5% CO 2 for 1 h.

(6) Adsorption: 10 μl of the original peptide library was added to 990 μl of 0.5% BSA/PBS buffer, the phage was diluted to 1.5×10 ¹¹ pfu/ml, and added to the closed human normal intestinal epithelial HIEC cells. At 37 ° C for 1 h, the phage which can bind to human normal intestinal epithelial HIEC cells was adsorbed, and the supernatant was taken.

(7) Binding: The adsorbed phage supernatant was incubated with human colon cancer SW480 cells for 1 h.

(8) Washing: Discard the unbound phage, place the microplate on a clean paper towel and pat it vigorously to remove the remaining solution. The plate was washed 5 times with 0.1% TBST as described above.

(9) Elution: Add 1 ml of 0.2 M Glycine-HCl (pH 2.2) 1 mg/ml BSA eluate, shake slowly for 10 min on ice, then aspirate the eluate and transfer to 150 μl of the previously prepared neutralized solution ( 1M Tris-HCl, pH 9.1).

(10) Repeat the operation twice as described above.

2.3 Determination of titer of phage.

The IPTG/X-gal plate was preheated in an electrothermal incubator at 37 ° C; the appropriate amount of top agar was taken out and heated in a microwave oven, and after it was completely melted, it was taken out, and 3 ml was dispensed in each 10 ml centrifuge tube; After the phage was diluted in a ratio, 10 μl and 200 μl of E. coli were mixed and reacted for 5 min, then added to 3 ml of top agar, and then evenly spread on preheated IPTG/X-gal plate, after condensation. The titration results were observed at 37 ° C in an electrothermal incubator overnight.

2.4 Amplification and purification of phage.

(1) Amplification of phage: 20 ml of LB/Tet liquid medium was added to the Erlenmeyer flask, then E. coli bacteria solution and phage to be amplified were added at 1:100, placed at 37 ° C, and shaken vigorously in a constant temperature oscillator 4.5 h, an phage amplification solution is obtained.

(2) Purification of phage: The phage amplification solution obtained by the above procedure was centrifuged at 4 ° C, 12000 r/min for 10 min, and the supernatant was added, and 1/6 volume of PEG-NaCl was added to precipitate overnight, and then centrifuged at 12000 r/min for 15 min, discarded. The supernatant was removed, the pellet was dissolved in TBS buffer, 1/6 volume of PEG-NaCl was again administered, and incubated on ice for 1 h. After centrifugation at 4 ° C, 14000 r / min for 15 min, the supernatant was discarded, and the obtained precipitate was dissolved in TBS-NaN 3 and stored in a refrigerator at 4 ° C.

2.5 enzyme-linked immunosorbent assay

(1) Preparation of 96-well plates of cells, plating rules: 16 holes of two rows of 96-well plates were respectively added with 100 μl × PBS as a blank group; then each of the 1, 2, 3, and 4 rows was plated according to a serpentine shape. Human normal intestinal epithelial HIEC cell suspension, each well of rows 5, 6, 7, and 8 are plated with 100 μ of human colon cancer SW480 cell suspension in a serpentine shape, and then the plated cell plate is placed in the pass 5 An ELISA experiment can be performed overnight in a 37 ° C cell incubator with % CO 2 .

(2) Fixation: 96-well plates with cells were taken overnight, and the liquid in the dry wells was washed three times with PBS, and then fixed with 4% paraformaldehyde for 20 minutes.

(3) Blocking: Remove the fixed 96-well plate, take the liquid in the dry well, wash it with PBS 3 times, then add 3% hydrogen peroxide, and block it in a constant temperature incubator at 37 °C for 30 min to block Endogenous peroxidase activity.

(4) Closure: The blocked 96-well plate was taken out, and the liquid in the dry well was photographed, washed with PBS three times, and then 3% BSA/PBS was added and blocked in a 37 ° C cell incubator for 1 h.

(5) Adding phage sample: The blocked 96-well plate was taken out, and the liquid in the dry well was photographed, and the purified positive phage was added and reacted in a constant temperature incubator at 37 ° C for 1 h.

(6) Addition of primary antibody: The 96-well plate after the reaction was taken out, and the liquid in the dry well was photographed, and after washing 3 times with PBS, 1:4000 M13 antibody was added, and the mixture was allowed to stand overnight at 4 °C.

Secondary antibody: The 96-well plate after the reaction was taken out, and the liquid in the dry well was photographed, washed 3 times with PBS, and the secondary antibody was added thereto, and reacted in a constant temperature incubator at 37 ° C for 30 minutes.

(7) Adding substrate TMB: The 96-well plate after washing the PBS three times was added to the TMB display agent in the dark, and was placed in a 37 ° C cell incubator for 15 min.

(8) Termination: The 96-well plate after the reaction was taken out, and the reaction was terminated by adding 2 M sulfuric acid.

(9) Measurement of results: A 96-well plate in which all reactions were completed was placed in a microplate reader, and its OD value was measured at 405 nm, and the results were saved and analyzed.

Immunofluorescence experiment of 2.6 cells and positive phage

(1) Cell plating: Human normal intestinal epithelial HIEC cells and human colon cancer SW480 cells were plated in a six-well plate for use.

(2) Fixation: fixed with 4% paraformaldehyde for 15 min.

(3) Blocking: 4% paraformaldehyde was discarded, washed twice with PBS, and blocked with 3% BSA/PBS at 37 ° C for 30 min.

(4) Incubation of positive phage: the blocking solution was wiped and the positive phage was added at 37 ° C for 1 h.

(5) DAPI staining: washing with PBS 3 times, adding DAPI 100 μl, room temperature, 15 min

(6) Covering: After washing 3 times with PBS, the film was sealed.

Extraction and sequencing of 2.7 positive phage DNA

(1) 100 ul of iodide buffer was added to the purified phage precipitate, and 250 ul of absolute ethanol was added thereto, and the mixture was thoroughly mixed, and allowed to stand at room temperature for 20 minutes.

(2) Centrifugation: 4 ° C, 14,000 rpm, 10 min, the supernatant was discarded.

(3) Washing: The precipitate was washed with 500 ul of 70% ethanol, briefly centrifuged, and dried under vacuum.

(4) The pellet was resuspended in 30 ul TE (10 mM Tris-HCl, pH 5.0, 1 mM EDTA) buffer to prepare a DNA sequencing template solution, which was sent to Shanghai Biotech for sequencing.

2.8 cell immunofluorescence experiment

(1) Preparation of cell plating: Slides were placed on a 6-well plate in advance, and human colon cancer SW480 cells and human normal intestinal epithelial HIEC cells were plated in a six-well plate for use.

(2) Fixation: fixed with 4% paraformaldehyde for 15 min.

(4) Incubation of FITC-GV12: Wipe the blocking solution and add FITC-GV12 at 37 ° C for 1 h.

(6) Covering: After washing 3 times with PBS, the film was sealed.

2.9 flow cytometry

(1) Preparation of cell plating: Human colon cancer SW480 cells and human normal intestinal epithelial HIEC cells were plated in a six-well plate for use.

(2) Blocking: Washed twice with PBS, blocked with 3% BSA/PBS at 37 ° C for 30 min.

(3) FITC-GV12 incubation: After the blocking solution was discarded, FITC-GV12 was added, and the mixture was incubated at 37 ° C for 30 min.

(4) Collecting cells: After washing three times with PBS, trypsin digestion was performed to collect up-flow detection of the cells.

3. Experimental results

1, 20 randomly picked phages carrying polypeptides E. coli clones by cell ELISA showed experimental group mean absorbance of OD ₄₀₅ SW480 and the average absorbance values of the control group HIEC OD ₄₀₅ ratio of greater than There are 12 clones of 2, which are 1, 2, 5, 8, 9, 10, 12, 14, 15, 17, 18, 20. The above 12 positive phage have stronger binding effect to human colon cancer SW480 cells, but weaker binding to human normal intestinal epithelial HIEC cells. Therefore, positive phage No. 8 was selected for immunofluorescence staining to further verify the targeted binding of positive phage to human colon cancer cells. The results of immunofluorescence assay showed that the positive phage could specifically bind to human colon cancer cell SW480, but the binding ability to human normal intestinal epithelial HIEC cells was weak, and there was a significant difference between them, further suggesting that the positive phage polypeptide was on human colon. Cancer cells have the role of targeted binding (as shown in Figure 2).

Next, 12 positive phage clones were amplified, purified and extracted for DNA sequencing. The results showed that the sequencing results of a total of 8 positive phage showed the same sequence, which were 1, 2, 8, 10, 14, 17, 18, respectively. number 20. As shown in Figure 3, the polypeptide sequence was translated according to the principle of the triplet codon: GLTSMRYHSVIV (SEQ ID No. 1) (GV12).

Then, the green fluorescently-labeled positive polypeptide FITC-GV12 was artificially synthesized, and the targeted binding of the polypeptide to human colon cancer cell SW480 was verified by immunofluorescence staining and flow cytometry. As shown in Figure 4, the polypeptide sequence has weak binding ability to human normal intestinal epithelial HIEC cells, and the relative fluorescence intensity value is 1129.92±460.65, while the binding ability to human colon cancer cell SW480 is stronger, and the relative fluorescence intensity value is 36429.45±4173.03, there was a significant difference between the two P<0.0001.

The above results were further confirmed by flow cytometry experiments. Human normal intestinal epithelial HIEC cells and human colon cancer SW480 cells were incubated with 10 μM FITC-GV12, respectively, and flow-through detection. The percentage of positive cells was 1.9% and 99.9%, respectively, which were significantly different (Fig. 5). It was demonstrated that the polypeptide specifically binds to colon cancer cells and has no effect on normal intestinal epithelial cells.

Claims

A polypeptide which specifically binds to human colon cancer cells, characterized in that the polypeptide is any of the following:

(1) The amino acid sequence of the polypeptide is: GLTSMRYHSVIV (SEQ ID No. 1);

(2) A polypeptide derivative which has undergone deletion, insertion or substitution of one or several amino acids in the polypeptide molecule of (1) and which has the same biological function as the polypeptide molecule of (1).
The polypeptide specifically binding to human colon cancer cells according to claim 1, wherein the polypeptide has targeted binding to tumor cells and specifically binds to tumor cells.
The polypeptide for specifically binding to human colon cancer cells according to claim 2, wherein the tumor cells are colon cancer cells.
The use of a polypeptide which specifically binds to human colon cancer cells according to claim 1 for the preparation of a tumor diagnostic kit, characterized in that the polypeptide or polypeptide conjugate is contained in the kit.
The use of a polypeptide which specifically binds to human colon cancer cells according to claim 1 for the preparation of a medicament for the treatment of colon cancer.
The use of a polypeptide which specifically binds to human colon cancer cells according to claim 1 for the preparation of a medicament for treating colon cancer, characterized in that the medicament comprises the polypeptide and a pharmaceutically active ingredient, or comprises The polypeptide and the delivery vehicle described.
The use of a polypeptide which specifically binds to human colon cancer cells according to claim 5 for the preparation of a medicament for the treatment of colon cancer, characterized in that the medicament is any pharmacologically acceptable dosage form, the medicament A preferred dosage form is an injectable preparation.
The use of a polypeptide which specifically binds to human colon cancer cells according to claim 5 for the preparation of a medicament for the treatment of colon cancer, characterized in that the medicament is any pharmacologically acceptable dose.