CN106967154B

CN106967154B - Screening method of human colon cancer cell specific binding polypeptide and application thereof

Info

Publication number: CN106967154B
Application number: CN201710183340.6A
Authority: CN
Inventors: 孟祥军; 田晓慧; 侯利丹; 祝丹希; 梁钰; 王建华
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-03-24
Filing date: 2017-03-24
Publication date: 2020-06-16
Anticipated expiration: 2037-03-24
Also published as: CN106967154A

Abstract

The invention relates to a colon cancer cell surface specific binding polypeptide and application thereof. The method comprises the following steps: incubating a clone strain obtained by amplifying a bacteriophage dodecapeptide library with colon cancer cells, discarding clone strains which are not combined with the colon cancer cells, and amplifying clone strains combined with the colon cancer cells after screening; step (2): circulating the step (1) for 4 times, continuously enriching the phage clone strain to obtain a clone strain with higher affinity with colon cancer cells, and repeatedly verifying the binding capacity in a liquid phase; and (3): and (3) incubating the phage clone strain in the step (2) with normal colon epithelial cells, and removing the non-specific binding clone strain. And (4) carrying out sequence determination on the positive clone strains, and respectively verifying the specificity of the polypeptide on colon cancer cells and tissues through immunofluorescence in the step (5). The invention screens the short peptide which is specifically combined with colon cancer cells by utilizing a phage display peptide library technology, and the short peptide is used as a molecular marker or a targeting vector and the like and is used for targeting treatment drugs.

Description

Screening method of human colon cancer cell specific binding polypeptide and application thereof

Technical Field

The invention relates to the field of medicine, in particular to a screening method of colon cancer cell surface specific binding polypeptide and application thereof.

Background

Colon cancer is one of common malignant tumors of digestive system, the morbidity is second to gastric cancer and esophageal cancer, the life health of human beings is seriously influenced, 44 thousands of confirmed cases of colorectal cancer are newly added every year in China at present, and 23 thousands of dead patients are killed every year. Early detection and early removal of precancerous lesions and focal cancerous lesions can effectively reduce the fatality rate of colorectal cancer. However, because the colon cancer develops slowly and the early symptoms are much and not obvious, 6 percent of people with no symptoms above 40 years old suffer from early intestinal cancer, 70 to 80 percent of patients with colon cancer have already entered the middle and late stage when diagnosed, and more than 25 percent of patients have metastasized cancer cells. The metastatic colon cancer has poor curative effect after operation, high recurrence rate after operation, and 5-year survival rate of patients who can not excise the tumor is almost 0. Although the drug targeting therapy has achieved great success, there are still many disadvantages such as large molecular weight of the antibody used for targeting, low penetration in tumor tissues, toxic effects on liver and bone marrow; the immunogenicity is strong; long preparation process and period, high price and the like. Therefore, the research of effective measures for early diagnosis and treatment of colon cancer becomes a hot problem in current scientific research.

The different characteristics of the tumor cells and normal cells provide possibility for searching molecules which are specifically targeted and combined with the tumor cells, and the phage display technology can obtain the polypeptide which is specifically combined with the tumor by affinity screening under the condition that the molecules on the surface of the tumor cells are unknown, thereby being beneficial to searching new targeted molecules for diagnosis and targeted treatment of the tumor.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a colon cancer cell surface specific binding polypeptide and application thereof in diagnosis and treatment of colon cancer.

The screening steps of the colon cancer cell surface specific binding polypeptide provided by the invention are as follows:

step (1): incubating random dodecapeptide library phage clone strains and colon cancer cells together by using a whole cell screening method, discarding clone strains which are not combined with the colon cancer cells, and amplifying the clone strains combined with the colon cancer cells;

step (2): circulating the step (1) for 4 times, enriching the phage clone strain combined with the colon cancer cells to the maximum extent to obtain a clone strain combined with the colon cancer cells with higher affinity, and amplifying;

and (3): and (3) incubating the amplified clone strain with normal colon epithelial cells, carrying out negative screening, and removing the non-specific clone strain combined with the normal cells.

And (4): and (4) carrying out sequence determination on the clone strain after final amplification, and analyzing a polypeptide sequence.

Preferably, in the step (2), screening and verification are carried out in a liquid phase, after 4 rounds of screening, multiple repeated tests are carried out to compare the average enrichment rate before and after each monoclonal is combined with colon cancer cells, the cell binding capacity of each monoclonal is detected, and the positive phage clone strain is determined according to the standard that the average enrichment rate is more than one order of magnitude higher than that of a negative control group.

Preferably, in step (4), the polypeptide sequence is immunofluorescent-verified in colon cancer cells and tissues.

Compared with the prior art, the invention has the following beneficial effects:

the invention creatively adopts the phage display technology, utilizes the means of genetic engineering to insert the exogenous peptide or protein gene into the specific protein gene of the phage, the polypeptide or protein coded by the exogenous gene is presented on the surface of the phage in the form of fusion protein, and the displayed polypeptide or protein can keep relative spatial structure and biological activity. The phage library is subjected to biopanning, namely the phage library is incubated with target molecules, clone strains which are not combined with the target molecules are washed away, the combined clone strains are collected, amplified and enriched, the process is repeatedly circulated for 3-5 times, and the phage clone strains which are specifically combined with the target molecules can be obtained. The corresponding polypeptide sequence was obtained by gene sequencing. The method is convenient and efficient, the screened polypeptide has small molecular weight, and the immune anaphylactic reaction of the traditional monoclonal antibody is effectively overcome. The polypeptide is specifically bound on the cell surface, is not bound with normal cells, can effectively distinguish colon cancer cells from normal cells, is expected to become a targeting molecule, and is used for diagnosis and targeted therapy of tumors.

Description of the drawings:

FIGS. 1A-C are graphs showing the survival time of non-transplanted and post-transplanted diabetic mice in the examples of the present invention;

FIG. 2 is a graph showing the results of IPGTT of mice with impaired glucose tolerance before and after transplantation in accordance with an embodiment of the present invention;

FIG. 3 is a graph showing the results of flow cytometry detection of the urine stem cell surface marker in the example of the present invention

Detailed Description

The invention will be further understood by reference to the following examples.

The first embodiment is as follows:

the screening method comprises the following steps:

the screening method of the colon cancer cell surface specific binding polypeptide comprises the following steps:

The polypeptide sequence is also verified by immunofluorescence.

Second, phage titer determination (Titration method)

Diluting the amplified phage supernatant by 100 times with a sterilized LB liquid culture medium without antibiotics, then respectively taking 100ul of each diluted titer in a centrifuge tube, uniformly mixing with XL1Blue bacterial liquid in a logarithmic growth phase of 500ul, adding 3ml of LB top agar which is kept warm in a constant temperature water bath kettle at 57 ℃, quickly pouring the mixture on an LB solid plate with tetracycline resistance after uniform mixing, placing the mixture in an incubator at 37 ℃ for overnight culture after slight solidification, counting the number of plaques the next day, and adjusting the titer of the phage (pfu/100ul) to the number of phage dilution times, namely the number of phage contained in each 100ul of phage supernatant, and determining the titer of the amplified phage supernatant according to the method, and then diluting the titer to a fixed titer for checking the cell binding capacity.

Thirdly, the method for screening out the clone strain combined with the colon cancer cell comprises the following steps:

and 4 rounds of circular screening are carried out by taking colon cancer fine COLO320HSR cells as target cells and human normal colon mucosal epithelial cells NCM460 as adsorbed cells, and the phage input amount of each round is kept. The specific screening procedure was as follows:

step (1): collecting 1 dish of COLO320HSR cells, collecting suspended cells therein at 3000rpm for 5min, resuspending with 1ml PBS, blowing adherent cells with 2ml PBS, placing in two EP tubes, dividing the resuspended suspended cells into two parts, mixing uniformly in the cells of the two EP tubes, at 6000rpm for 3min, washing for 1 time, discarding supernatant, removing supernatant, adding 500ul 4% paraformaldehyde into each tube, and fixing at room temperature for 20 min. Normal colonic epithelial cells NCM460 were treated in the same manner.

Step (2): washing twice with PBS at 10000rpm for 3min, removing supernatant, adding 5% skimmed milk 0.5ml per tube, sealing for 1h, centrifuging at 10000rpm for 3min, removing milk, resuspending the cells with PBS 0.5ml, and mixing in an EP tube.

And (3): the titer after dilution in the Titray titer assay described above was 10¹²Taking 50ul of reserved samples from 100ul of phage for titer determination, adding 1ml of phage with the same titer into the fixed COLO320HSR cells, gently blowing and beating the cells and the phage uniformly, then shaking the suspension at low speed for 1h at room temperature, after the phage and the COLO320HSR cells are fully combined, 8000rp, centrifuging for 3min to discard the supernatant, then adding 1ml of 1% PBST (containing 1% Tween-20) for re-suspension, 8000rpm, centrifuging for 3min, repeatedly washing the precipitate by the same method for 4 times, and washing off the phage which is not specifically combined or is not combined with the cells in the precipitate.

And (4): and (3) resuspending and uniformly mixing the precipitate with 0.5ml of XL1Blue bacterial liquid, culturing on a shaker at 37 ℃ for 1h, centrifuging at 1000rpm for 5min, collecting supernatant, transferring the supernatant into the fixed and sealed normal colon epithelial cells NCM460, uniformly resuspending and shaking at room temperature for 1h, 10000rpm for 5min, and centrifuging to collect supernatant of 500ul in total.

And (5): 50ul of the suspension was taken out for titer determination, and 1.5ml of XL1Blue bacterial liquid in logarithmic phase was added to the remaining portion, mixed well and shaken at 37 ℃ overnight.

And (6): taking out the phage-bacterium liquid mixed solution amplified overnight, centrifuging at 8000rpm for 20min to remove bacterial precipitate, collecting supernatant, adding PEG8000/NaCl according to 1/4 volume of the supernatant, standing on ice for 3 hr, centrifuging at constant temperature of 4 deg.C at 13000rpm after phage in the liquid is fully precipitated, removing supernatant, and resuspending the precipitate with 0.5ml PBS.

And (7): the titer of phage supernatants added to cells before each round of selection, the overnight phage supernatants left after selection, the overnight amplified phage supernatants collected after selection by PEG8000, and the phage enrichment rate after each round of selection (titer of phage supernatants not overnight after selection)/(volume of phage titer added to cells before selection) were determined separately under the same experimental conditions according to the Titration titer assay.

And (8): repeating the process 1-7 times for 3-5 times, continuously enriching the positive clone strains combined with colon cancer cells (figure 1A), selecting 21 clone strains, and repeatedly verifying the combination and the capability of the 21 clone strains and COLO320HSR in a liquid phase for many times, wherein the average enrichment rate of the clone strains No. 2#, No. 5#, No. 8# and No. 19# is obviously higher than that of a negative control group by more than one order of magnitude, and thus determining the positive phage clone strains (figure 1B).

Fourth, DNA sequence determination and analysis of positive phage clones

Rapid purification of sequencing templates

The method is very rapid, does not require phenol or chromatography, and can produce sufficiently pure templates for manual or automated dideoxy sequencing.

Step (1): after plaque amplification centrifugation, 500. mu.l of phage-containing supernatant was transferred to a fresh centrifuge tube.

Step (2): add 200. mu.l PEG/NaCl, reverse mix, and let stand at room temperature for 10 min.

And (3): centrifuge for 10min and discard the supernatant.

And (4): centrifuge briefly and carefully aspirate the residual supernatant.

And (5): the pellet was resuspended in 100. mu.l iodide buffer and 250. mu.l ethanol was added. Incubate at room temperature for 10 min. A short incubation at room temperature precipitates single-stranded phage dna (ssdna) while most of the phage proteins remain in solution.

And (6): centrifuge for 10min and discard the supernatant. Precipitating with 70% ethanol, and vacuum drying.

And (7): the pellet was resuspended in 30. mu.l of TE [10mM Tris-HCl (pH8.0),1mM EDTA ].

And (8): 5. mu.l of the above template solution³⁵S or³³P-labeled manual dideoxy sequencing, or dye-labeled automatic cycle dideoxy sequencing. The amount of template is increased or decreased according to the sequencing method.

Phage single-stranded DNA sequencing: 5ul of the corresponding template solution of the above positive clones were taken, and the sequence of the polypeptide was analyzed by sequencing (FIG. 1C).

Fifthly, verifying specificity of synthesized polypeptide by immunofluorescence

1. The specificity of the polypeptide is verified on the cell, and the steps are as follows

Step (1): the previously washed, autoclaved coverslip was placed into a 24-well plate.

Step (2): taking colon cancer cells COLO320HSR in good growth state and human normal colon mucosal epithelial cells NCM460, preparing cell suspension by using RPMI-1640 culture medium containing 10% fetal calf serum, counting cells, and adding into a 24-hole culture plate according to 8000 holes.

And (3): placing in incubator, culturing continuously until cell fusion reaches 80%, taking out the pore plate, removing culture solution, washing with PBS for 5min for 3 times.

And (4): cell slide was fixed with ice methanol at room temperature for 10 min.

And (5): absorbing and removing the methanol, and airing at room temperature until the hair turns white;

and (6): washing with PBS for 5min for 3 times;

and (7): adding 10ug/ml polypeptide 200 ul/well, the control group is random peptide group CBP-R, the experimental group is polypeptide CBP-1 obtained by screening: LPKTVSSDMSLN, placing into 37 deg.C incubator for 15 min;

and (8): PBS wash 3 times, 5min each time, and protect from light.

And (9): adding 10ug/ml DAPI, incubating at room temperature for 10-30min, washing with PBS for 3 times, 5min each time, and protecting from light.

Step (10): the glycerol seals were observed under a confocal microscope and recorded by photography (FIG. 2).

2. Verification of polypeptide specificity on tissue

Step (1): carrying out OCT embedding on fresh colon cancer and tissues beside the cancer, and cutting into sections with the thickness of 4 um;

step (2): washing with immunostaining washing solution for 5min, adding frozen slice rapid antigen repairing solution for repairing, and maintaining at room temperature for 5 min;

and (3): washing with immunostaining solution for 5 times, each time for 10 min;

and (4): blocking with 3% BSA + 0.05% TitonX-100 at room temperature for 60 min;

and (5): washing with 0.1% TitonX-100 for 3 times, each for 5 min;

and (6): carefully delineating the contour of an immune group painting pen, adding 10ug/ml of polypeptide control group into each section to obtain a random peptide group CBP-R, and screening an experimental group to obtain a polypeptide CBP-1: LPKTVSSDMSLN, 200ul, incubating at 4 deg.C for 30 min;

and (7): PBS wash 3 times, each time for 5 min;

and (8): adding 10ug/ml DAPI into each slice, 200ul, and standing at room temperature for 10min

And (9): PBS wash 3 times, each time for 5 min;

step (10): protected from light, observed under a fluorescent microscope and photographed (fig. 3).

the invention creatively adopts the phage display technology, utilizes the means of genetic engineering to insert the exogenous peptide or protein gene into the specific protein gene of the phage, the polypeptide or protein coded by the exogenous gene is presented on the surface of the phage in the form of fusion protein, and the displayed polypeptide or protein can keep relative spatial structure and biological activity. The phage library is subjected to biopanning (biopanning), namely the phage library is incubated with colon cancer whole cells, clone strains which are not combined with the cancer cells are washed away, the combined clone strains are collected, amplified and enriched, the process is repeatedly circulated for 3-5 times, and phage clone strains which are specifically combined with the colon cancer can be obtained. Screening and verifying in a liquid phase, completely collecting colon cancer cells in a whole dish each time, and fully combining the colon cancer cells with phage in a liquid phase state for a certain time, so that the quantity of the combined cells each time is consistent, the quantity of phage binding sites on the cell surface is consistent, and the cells in each experiment are in a fresh growth state; the combined screening experiment is carried out in the liquid phase, each washing process is carried out by more than 10 times of blowing and beating processes, and 5 minutes of low-speed centrifugation is carried out, the washing capacity of PBST relative to PBS is greatly enhanced, so the washing intensity in the liquid phase environment is actually greater than that in the solid phase surface screening process. The influence of nonspecific binding action can be better reduced. And (3) in the processes of washing, blowing and centrifuging, attention should be paid to ensure the integrity of the cell structure as much as possible, otherwise, cell membrane rupture can cause loss of target phage polypeptides, the binding capacity of each monoclonal cell is detected by a method of average enrichment rate before and after cell binding, and the positive phage clone strain is determined by a standard that the average enrichment rate is more than one order of magnitude higher than that of a negative control group. Finally, non-specifically bound clones bound to normal colonic epithelium were removed by incubation with normal colonic epithelial cells. The utilization of normal cells as subtractive cells is still a major controversial at present, and scholars believe that the negative selection can better remove bacteriophage polypeptides not specifically bound, but also believe that due to the very complex molecular structure and distribution on the surface of human cells, it is possible to make the specific bacteriophage polypeptide clone lose function in the negative selection process. Finally, the corresponding polypeptide sequence is obtained by gene sequencing.

The method is convenient and efficient, the screened polypeptide has small molecular weight, and the immune anaphylactic reaction of the traditional monoclonal antibody is effectively overcome. The polypeptide is specifically bound on the cell surface, is not bound with normal cells, is expected to become a targeting molecule, and is used for diagnosis and targeted treatment of colon cancer.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. The human colon cancer cell line COLO320HSR specific binding polypeptide is characterized in that the amino acid sequence of the polypeptide is as follows: LPKTVSSDMSLN are provided.

2. Use of a polypeptide according to claim 1 for the preparation of a medicament for the treatment of human colon cancer.