CN106967154A - The screening technique of human colon cancer cell specific binding polypeptide and its application - Google Patents
The screening technique of human colon cancer cell specific binding polypeptide and its application Download PDFInfo
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- CN106967154A CN106967154A CN201710183340.6A CN201710183340A CN106967154A CN 106967154 A CN106967154 A CN 106967154A CN 201710183340 A CN201710183340 A CN 201710183340A CN 106967154 A CN106967154 A CN 106967154A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6818—Sequencing of polypeptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The present invention relates to a kind of colon cancer cell surface specific Binding peptide and its application.Comprise the following steps：Clone strain after step (1) expands bacteriophage dodecapeptide storehouse is together incubated with colon cancer cell, discards the clone strain not combined with colon cancer cell, expands the clone strain combined with colon cancer cell after screening；Step (2)：Step (1) is circulated 4 times, phage clone strain constantly enrichment obtains the clone strain for having higher affinity with colon cancer cell, verifies its binding ability repeatedly in the liquid phase；Step (3)：Phage clone strain in (2) is incubated with normal colon epithelial cells, non-specific binding clone strain is removed.Step (4) carries out sequencing to positive clone strain, and step (5) separately verifies the specificity of polypeptide by immunofluorescence in colon cancer cell and tissue.The present invention is exactly that the small peptide specifically bound with colon cancer cell is screened using phage display peptide library technology, as molecular marker or targeting vector etc., for target therapeutic agent.
Description
Technical field
The present invention relates to medical domain, more particularly to a kind of colon cancer cell surface specific Binding peptide screening technique and
It is applied.
Background technology
Colon cancer is one of common malignant tumour of digestive system, and the incidence of disease is only second to stomach cancer and the cancer of the esophagus, has a strong impact on
Human life and health, current China increases colorectal cancer confirmed cases 440,000, annual Died Patients up to 230,000 people newly every year.It is early
It was found that, early excision precancerous lesion and focal canceration can effectively reduce colorectal cancer case fatality rate.But it is due to colon cancer development
Slowly, unobvious more than early symptom, 6% suffers from early stage intestinal cancer, 70%-80% colorectal cancer patients in more than 40 years old Silent cerebral infarction
Middle and advanced stage is come into when making a definite diagnosis, patient's cancer cell more than 25% has been shifted.Metastatic Operation of Colon Carcinoma effects a radical cure effect
Difference, Postoperative recurrent rate is high, it is impossible to which 5 years survival rates of patient of tumor resection are almost 0.Although drug targeting treatment achieves very big
Success, but still have that many not enough antibody molecule amounts as being used to target are big, penetration power is low in tumor tissues, to liver
Toxic action is produced with marrow；Immunogenicity is strong；Preparation process and cycle are longer, expensive etc..Therefore, early diagnosis is explored
Turn into the hot issue of current research work with the effective measures for the treatment of colon cancer.
The different qualities of tumour cell and normal cell, can to find that the molecule of selectively targeted combination tumour cell provides
Can, display technique of bacteriophage can be in the state of tumor cell surface molecule be unknown, and it is swollen that affine screening obtains specific binding
The polypeptide of knurl, helps to find diagnosis and targeted therapy of the new targeted molecular for tumour.
The content of the invention
In order to overcome the above-mentioned deficiencies of the prior art, combined the invention provides a kind of colon cancer cell surface specific many
Peptide and its application in diagnosis of colon cancer and treatment.
The colon cancer cell surface specific Binding peptide that the present invention is provided, it is as follows that it screens step：
Step (1)：Using full cell screening method, phage clone strain in random dodecapeptides storehouse is together incubated with colon cancer cell
Educate, discard the clone strain not combined with described colon cancer cell, the clone strain combined with described colon cancer cell is expanded；
Step (2)：Step (1) is circulated 4 times, the bacteriophage gram combined with the colon cancer cell is enriched with to greatest extent
Grand strain, obtains having higher affinity combination clone strain with colon cancer cell, is expanded；
Step (3)：Clone strain after above-mentioned amplification and normal colon epithelial cells are incubated, negativity screening is carried out, removed
The non-specific clone strain combined with normal cell.
Step (4)：Rear clone strain will finally be expanded and carry out sequencing, peptide sequence is analyzed.
It is preferred that using being screened and being verified in the liquid phase in step (2), experiment, which is repeated several times, after 4 wheel screenings compares
Each monoclonal is combined front and rear average accumulation rate with colon cancer cell, detects the cell binding ability of each monoclonal, with average
Accumulation rate determines positive phage clones strain higher than standard more than an order of magnitude of negative control group.
It is preferred that in described step (4), described peptide sequence is glimmering by being immunized in colon cancer cell and tissue
Light is verified.
Compared with prior art, beneficial effects of the present invention are as follows：
Of the invention initiative employs display technique of bacteriophage, using the means of genetic engineering by external source peptide or protein base
Because of insertion bacteriophage specific protein gene, many peptide or proteins of foreign gene coding are presented on bacteriophage in the form of fusion protein
Surface, many peptide or proteins being demonstrated can keep relative space structure and bioactivity.Phage library is washed in a pan by biological
Choosing, i.e. phage library are incubated with target molecule, wash away the clone strain not combined with target molecule, are collected, and amplification is enriched with the clone combined
Strain, process iterative cycles 3-5 times result in the phage clone strain specifically bound with target molecule.Pass through gene sequencing
Obtain corresponding polypeptide sequence.This method convenience and high-efficiency, the polypeptide screened, molecular weight is smaller, effectively overcomes conventional monoclonal
The immune allergic reaction of antibody, the present invention filters out 1 and colon cancer cell surface specific knot by display technique of bacteriophage
The polypeptide of conjunction.The polypeptid specificity combine in cell surface, is not combined with normal cell, can effectively distinguish colon cancer cell with
Normal cell, is expected to turn into targeted molecular, diagnosis and targeted therapy for tumour.
Brief description of the drawings：
Figure 1A-C are diabetic mice life cycle result figure after not transplanting and transplant in the embodiment of the present invention；
Fig. 2 be the embodiment of the present invention in transplant before with transplanting after impaired glucose tolerance mouse IPGTT result figures；
Fig. 3 is the result figure of Flow cytometry urine Stem cell surface marker thing in the embodiment of the present invention
Embodiment
The present invention is further described with reference to specific embodiment, to more fully understand the present invention.
Embodiment one：
First, screening technique：
The present embodiment colon cancer cell surface specific Binding peptide screening technique, comprises the following steps：
Step (1)：Using full cell screening method, phage clone strain in random dodecapeptides storehouse is together incubated with colon cancer cell
Educate, discard the clone strain not combined with described colon cancer cell, the clone strain combined with described colon cancer cell is expanded；
Step (2)：Step (1) is circulated 4 times, the bacteriophage gram combined with the colon cancer cell is enriched with to greatest extent
Grand strain, obtains having higher affinity combination clone strain with colon cancer cell, is expanded；
Step (3)：Clone strain after above-mentioned amplification and normal colon epithelial cells are incubated, negativity screening is carried out, removed
The non-specific clone strain combined with normal cell.
Step (4)：Rear clone strain will finally be expanded and carry out sequencing, peptide sequence is analyzed.
Described peptide sequence is also verified by immunofluorescence.
2nd, phage titre determines (Titration methods)
Antibiotic-free LB fluid nutrient mediums after sterilizing carry out 100 times to the phage supernatants after amplification and wait dilution, so
100ul is respectively taken from each titre diluted afterwards in centrifuge tube, is mixed with the XL1Blue bacterium solutions of 500ul exponential phases,
The LB top agars that 3ml is incubated in 57 DEG C of thermostat water baths are added, the rapid LB solids for being poured over tetracyclin resistance after mixing
On flat board, after slightly solidifying, incubated overnight in 37 DEG C of incubators, the quantity for counting plaque in second day, the drop of bacteriophage are placed on
Spend the number of the bacteriophage included in (pfu/100ul)=bacteriophage number * extension rates, the i.e. phage supernatants per 100ul
Amount, determines the titre of the bacteriophage supernatant amplified according to the method, then is adjusted to fix titre by the dilution of its titre, uses
In inspection cell binding ability.
3rd, the method for filtering out the clone strain combined with described colon cancer cell is as follows：
Using the thin COLO320HSR cells of colon cancer as target cell, people's normal colonic mucosa epithelial cell NCM460 is thin for absorption
Born of the same parents, carry out 4 and take turns Cycle Screening, keep the bacteriophage input amount of each round.Specific screening sequence is as follows：
Step (1)：1 ware COLO320HSR cells are taken, the cell 3000rpm 5min wherein suspended are collected, are used
1ml PBS are resuspended, and adherent cell is blown and beaten with 2ml PBS, is placed in two EP pipes, the suspension cell of resuspension is also divided into two
Part is mixed in the cell of two EP pipes, and 6000rpm 3min are washed 1 time, abandoning supernatant, notes removing supernatant totally, so
The paraformaldehydes of 500ul 4% are often added in pipe afterwards, 20min is fixed at room temperature.Same method processing normal colon epithelial cells
NCM460。
Step (2)：Washed twice with PBS 10000rpm 3min, supernatant is removed totally, often 0.5ml5% is added in pipe
Skim milk, closes 1h, 10000rpm 3min, and centrifugation removes milk, then cell is resuspended with 0.5ml PBS, mixes one
In individual EP pipes.
Step (3)：Titre is 10 after being diluted in above-mentioned Titration titer determinations method12/ 100ul bacteriophage takes
50ul keeps sample in case titer determination, takes the bacteriophage of the identical titres of 1ml to add in the COLO320HSR cells fixed, gently blow
Beat and mix cell and bacteriophage, then low speed shakes 1h at room temperature by suspension, treats that bacteriophage and COLO320HSR cells fill
Divide after combining, 8000rp, centrifugation 3min discards supernatant, then add after 1ml 1%PBST (containing 1%Tween-20) resuspension,
8000rpm, centrifuges 3min, same method precipitation repeated washing 4 times, wash away non-specific binding in precipitation or not with carefully
The bacteriophage that born of the same parents combine.
Step (4)：It will precipitate to be resuspended with 0.5ml XL1 Blue bacterium solutions and mix, be placed on 37 DEG C of shaking tables and cultivate 1h,
1000rpm, centrifuges 5min, collects supernatant, the normal colon epithelial cells NCM460 after transferring them to above-mentioned fixation and closing
In, it is resuspended after mixing and shakes 1h, 10000rpm, 5min at room temperature, the common 500ul of supernatant is collected by centrifugation.
Step (5)：Taking out 50ul is used for titer determination, and remaining part adds the XL1Blue bacterium of 1.5ml exponential phases
Liquid, 37 DEG C of incubator overnights after mixing.
Step (6)：Bacteriophage-bacterium solution mixed liquor of amplification overnight is taken out, 8000rpm centrifugations 20min removes bacterial precipitation,
Supernatant is collected, then PEG8000/Nacl is added according to 1/4 volume of supernatant, stands 3 hours on ice, treat in liquid
13000rpm, 30min, 4 DEG C of constant temperature centrifugations, remove supernatant after bacteriophage is fully precipitated, and are resuspended and precipitated with 0.5ml PBS.
Step (7)：According to Titration titer determination methods, under identical experiment condition, determine respectively before often wheel screening
The phage supernatants added in cell, the phage supernatants not overnight after the screening kept sample, the screening that PEG8000 is collected
The titre of the phage supernatants of amplification is stayed overnight afterwards, the bacteriophage accumulation rate after calculating often wheel screening=(do not stayed overnight after screening
The volume of phage supernatants after the titre * screenings of phage supernatants)/(the phage titre * in cell is added to before screening
The volume of addition).
Step (8)：1-7 processes 3-5 times repeatedly, the positive clone strain combined with colon cancer cell is constantly enriched with (Figure 1A),
21 clone strains are selected, carry out that its combination of checking and ability, wherein 2#, 5#, 8# is repeated several times in liquid phase with COLO320HSR,
The average accumulation rate of 19# clone strains is apparently higher than more than an order of magnitude of negative control group, it is thus determined that positive bacteriophage
Clone strain (Figure 1B).
4th, the determined dna sequence of positive phage clones and analysis
The fast purifying of sequencing template
This method is very quick, without phenol or chromatography, can produce sufficiently pure template and carry out manually or automatically double deoxidation
Sequencing.
Step (1)：After plaque amplification centrifugation, 500 μ l are transferred to a fresh centrifuge tube containing bacteriophage supernatant.
Step (2)：Plus 200 μ l PEG/NaCl, overturn and mix, room temperature places 10min.
Step (3)：10min is centrifuged, supernatant is abandoned.
Step (4)：Of short duration centrifugation, carefully sucks remaining supernatant.
Step (5)：Sediment is resuspended in 100 μ l iodide buffer solutions, adds 250 μ l ethanol.It is incubated at room temperature 10min.
The incubation at room temperature of short time precipitates single stranded phage DNA (ssDNA) and most of phage proteins are kept in the solution.
Step (6)：10min is centrifuged, supernatant is abandoned.With 70% ethanol precipitation, of short duration vacuum drying.
Step (7)：Precipitation is resuspended in 30 μ lTE [10mM Tris-HCl (pH8.0), 1mM EDTA].
Step (8)：5 μ l above-mentioned template solution is carried out35S or33The manual dideoxy sequencing of P marks, or dye marker
Automatic cycle dideoxy sequencing.According to the difference of sequence measurement, suitably increase and decrease template consumption.
Phage single-chain DNA sequencing：The corresponding template liquid 5ul of above-mentioned positive colony, sequencing analysis polypeptide are taken respectively
Sequence (Fig. 1 C).
5th, specificity of the immunofluorescence in checking synthesis polypeptide
1st, polypeptid specificity is verified on cell, step is as follows
Step (1)：Pre-wash, autoclaved cover glass are put into 24 well culture plates.
Step (2)：Take colon cancer cell COLO320HSR and people's normal colonic mucosa epithelial cell that growth conditions are good
NCM460, cell suspension is made with the RPMI-1640 culture mediums containing 10% hyclone, after cell count, is added by individual 8000 hole
Enter to the culture of 24 holes and pull.
Step (3)：It is placed on incubator to continue to cultivate, treats cell fusion up to 80%, take out orifice plate, nutrient solution is abandoned in suction, is used
PBS 3 times, each 5min.
Step (4)：Cell climbing sheet, room temperature, 10min are fixed with ice methanol.
Step (5)：Methanol is abandoned in suction, is dried at room temperature to turning white；
Step (6)：Washed with PBS 3 times, each 5min；
Step (7)：10ug/ml polypeptide 200ul/ holes are added, control group is random peptide group CBP-R, and experimental group is screening
The peptide C BP-1 of acquisition：LPKTVSSDMSLN, inserts 37 DEG C of incubator 15min；
Step (8)：PBS is washed 3 times, each 5min, lucifuge.
Step (9)：10ug/ml DAPI are added, 10-30min is incubated at room temperature, PBS is washed 3 times, each 5min, lucifuge.
Step (10)：Glycerine mounting, is placed in and observes and photograph to record under Laser Scanning Confocal Microscope (Fig. 2).
2nd, tissue verifies polypeptid specificity
Step (1)：Take fresh colon cancer and cancer beside organism OCT to embed, be cut into 4um slabs,；
Step (2)：Immunostaining cleaning solution washs 5min, adds frozen section rapid antigen reparation liquid and is repaired, room
The lower 5min of temperature；
Step (3)：Immunostaining cleaning solution is washed 5 times, each 10min；
Step (4)：Closed with 3%BSA+0.05%TitonX-100, room temperature 60min；
Step (5)：0.1%TitonX-100 is washed 3 times, each 5min；
Step (6)：Immune group paintbrush carefully irises out profile, and the polypeptide control group that every section adds 10ug/ml is random
Peptide group CBP-R, experimental group screening obtains peptide C BP-1：LPKTVSSDMSLN, 200ul, 4 DEG C of incubation 30min；
Step (7)：PBS is washed 3 times, each 5min；
Step (8)：Every section adds 10ug/mlDAPI, 200ul, at room temperature 10min
Step (9)：PBS is washed 3 times, each 5min；
Step (10)：Lucifuge, is placed in fluorescence microscopy Microscopic observation and takes pictures (Fig. 3).
Compared with prior art, beneficial effects of the present invention are as follows：
Of the invention initiative employs display technique of bacteriophage, using the means of genetic engineering by external source peptide or protein base
Because of insertion bacteriophage specific protein gene, many peptide or proteins of foreign gene coding are presented on bacteriophage in the form of fusion protein
Surface, many peptide or proteins being demonstrated can keep relative space structure and bioactivity.Phage library is washed in a pan by biological
(biopanning), i.e. phage library and the full cell incubation of colon cancer are selected, the clone strain not combined with cancer cell is washed away, collected,
Amplification, is enriched with the clone strain combined, and process iterative cycles 3-5 times result in the bacteriophage specifically bound with colon cancer
Clone strain.Screened and verified in the liquid phase, the colon cancer cell of whole ware collected completely be placed on liquid phase state every time
Lower and bacteriophage carries out the abundant combination of certain time, on the one hand ensure that the cell concentration combined every time is consistent, so as to ensure thin
The bacteriophage binding site quantity of cellular surface is consistent, and the state of cell is all the cell of fresh Growth in experiment every time；In liquid phase
In the screening experiment that is combined, its each washing process will pass through the piping and druming process of more than 10 times, and will be by 5 minutes
Low-speed centrifugal, washabilities of the PBST with respect to PBS greatly enhance, so the washing intensity in liquid phase environment is actual than solid phase table
Face screening process is big.The influence of non-specific binding effect can preferably be mitigated.Need to wash piping and druming and centrifugal process again
In should note ensureing cyto-architectural integrality as far as possible, otherwise cause membranolysis to cause some target phage polypeptides
Loss, the method for combining front and rear average accumulation rate by cell detects the binding ability of each monoclonal cell, with average
Accumulation rate determines positive phage clones strain higher than standard more than an order of magnitude of negative control group.Finally, by with just
Normal colon epithelial cell is incubated the non-specific binding clone for removing and being combined with normal colonic epithelia.By the use of normal cell as disappearing
Subtract cell and still suffer from larger dispute at present, there is scholar to think that this negativity screening can preferably remove biting for non-specific binding
Thalline polypeptide, but also it is believed that, due to human cell surface's molecular structure and be distributed it is extremely complex, it is possible to negativity screening
During make specific bacteriophage polypeptide clone loss of function, the present invention in order to avoid the latter, not in four-wheel in-vitro screening
During design negativity screening, but the screening of negativity is carried out after four-wheel is screened, it is intended to do not influence target phage many
The influence of Non-specific phage polypeptide is reduced on the premise of peptide function as far as possible.Finally, corresponding polypeptide is obtained by gene sequencing
Sequence.
This method convenience and high-efficiency, the polypeptide screened, molecular weight is smaller, effectively overcomes the immune of conventional monoclonal antibody
Allergic reaction, the present invention filters out the polypeptide combined with colon cancer cell surface specific by display technique of bacteriophage.This is more
Peptide specific is combined in cell surface, is not combined with normal cell, is expected to turn into targeted molecular, diagnosis and target for colon cancer
To treatment.
The specific embodiment of the present invention is described in detail above, but it is intended only as example, and the present invention is not limited
It is formed on particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and
Substitute also all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and
Modification, all should be contained within the scope of the invention.
Claims (5)
1. screening technique and its application of a kind of colon cancer cell surface specific Binding peptide, it is characterised in that including following
Step：
Step (1)：Using full cell screening method, phage clone strain in random dodecapeptides storehouse is together incubated with colon cancer cell,
The clone strain not combined with described colon cancer cell is discarded, the clone strain combined with described colon cancer cell is expanded；
Step (2)：Step (1) is circulated 4 times, the clone strain combined with the colon cancer cell is enriched with to greatest extent, is obtained
There is higher affinity combination clone strain with colon cancer cell, expanded, the clone strain after being expanded；
Step (3)：Clone strain after described amplification and normal colon epithelial cells are incubated, negativity screening is carried out, remove with
The non-specific clone strain that normal cell is combined, the clone strain after finally being expanded.
Step (4)：Rear clone strain will finally be expanded and carry out sequencing, peptide sequence is analyzed.
2. colon cancer cell surface specific Binding peptide screening technique according to claim 1, it is characterised in that step
(2) using being screened and being verified in the liquid phase in, more each monoclonal of experiment and colon cancer cell is repeated several times after 4 wheel screenings
With reference to front and rear average accumulation rate, the cell binding ability of each monoclonal is detected, negative control group is higher than with average accumulation rate
An order of magnitude more than standard determine positive phage clones strain.
3. colon cancer cell surface specific Binding peptide screening technique according to claim 1, it is characterised in that described
The step of (4) described in peptide sequence also by immunofluorescence verify.
4. human colon carcinoma specific binding polypeptide, it is characterised in that the amino acid sequence of the polypeptide is：LPKTVSSDMSLN.
5. polypeptide described in claim 4 is used for the application for preparing treatment human colon carcinoma medicine.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108610396A (en) * | 2018-04-26 | 2018-10-02 | 中国医科大学 | A kind of specific polypeptide of targeting human colon cancer cell |
| CN108640976A (en) * | 2018-04-26 | 2018-10-12 | 中国医科大学 | A kind of polypeptide with human colon cancer cell specific binding |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101492505A (en) * | 2008-12-24 | 2009-07-29 | 广东药学院 | Specific combined polypeptide for lung cancer, preparation and uses thereof |
| CN102127153A (en) * | 2010-12-16 | 2011-07-20 | 陕西师范大学 | Caco-2 cell surface specific binding polypeptide and screening method thereof |
| CN103145803A (en) * | 2012-12-13 | 2013-06-12 | 东南大学 | Polypeptide in specific binding with breast cancer brain metastases cells |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101492505A (en) * | 2008-12-24 | 2009-07-29 | 广东药学院 | Specific combined polypeptide for lung cancer, preparation and uses thereof |
| CN102127153A (en) * | 2010-12-16 | 2011-07-20 | 陕西师范大学 | Caco-2 cell surface specific binding polypeptide and screening method thereof |
| CN103145803A (en) * | 2012-12-13 | 2013-06-12 | 东南大学 | Polypeptide in specific binding with breast cancer brain metastases cells |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108610396A (en) * | 2018-04-26 | 2018-10-02 | 中国医科大学 | A kind of specific polypeptide of targeting human colon cancer cell |
| CN108640976A (en) * | 2018-04-26 | 2018-10-12 | 中国医科大学 | A kind of polypeptide with human colon cancer cell specific binding |
| WO2019205866A1 (en) * | 2018-04-26 | 2019-10-31 | 中国医科大学 | Specific polypeptide targeting human colon cancer cells |
| WO2019205867A1 (en) * | 2018-04-26 | 2019-10-31 | 中国医科大学 | Polypeptide specifically binding to human colon cancer cells |
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