CN101033251A - Liver cancer cell specificity internalization short peptide and its in vitro screening and identification - Google Patents
Liver cancer cell specificity internalization short peptide and its in vitro screening and identification Download PDFInfo
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Abstract
This invention relates to the special short peptide of hepatoma cell internalization and its screen and identification in vitro, belonging to the technology of tumor cell peptides screening and identification. It takes hepatoma cell BEL-7404 as the screening target cell, displays the random 12-peptide library for bacteria invader to conduct affinity panning. Through three times of screen, it randomly selects twenty negative colonies for amplification and sequence, to deduct the amino acid sequence of random peptide. Through cell ELISA, immunofluorescence, flow cytometry and other method, it further identifies the combination and internalization of bacteria invader clones and hepatoma cells. From sequencing and sequence analysis of the positive clones, four obtained different sequences have good combination with hepatoma cells, and the screened monoclone can internalize cells.
Description
Technical field
The present invention relates to small peptide and the in-vitro screening and the evaluation of liver cancer cell specificity internalization, belong to the screening of tumor cell specific internalization small peptide and the technical field of evaluation.
Background technology
Liver cancer human beings'health because of its high mortality ratio serious threat.In recent years, along with to the going deep into of oncobiology and cell surface receptor research, targeted therapy demonstrates good therapeutic action, and progressively develops into an important oncotherapy field.The guide molecule that targeted therapy refers to utilize specific performance to act on tumor tissues or organ with the drug targeting tumour, improves the specificity of drug targeting as pharmaceutical carrier effectively, realizes the specific aim and the security of treatment.The guide molecule that generally adopts mainly is monoclonal antibody and fragment thereof at present, and the targeted therapy medicine that is made up by the antibody associated molecule has been obtained great success in recent years.Yet because big, the shortcomings such as immunogenicity is high, tumor-infiltrated ability of molecular weight that the monoclonal antibody molecule had have hindered the development of associated treatment medicine in the clinical treatment field greatly.
Have above-mentioned these shortcomings at the monoclonal antibody molecule, more and more researchers turns one's attention to micromolecular polypeptide class targeted molecular, because it is little that peptide molecule has a molecular weight, fundamentally solved the problem of the high and tumor-infiltrated ability of immunogenicity, become the focus of present development.Display technique of bacteriophage (phage display technique) is to utilize the genetic manipulation means, the exogenous dna fragment of the selected polypeptide of coding and the coat protein gene of filobactivirus are carried out suitable reorganization, make the exogenous dna fragment after the reorganization be transcribed and translate together in company with the coat protein gene of phage, the expressing fusion protein that foreign DNA encoded polypeptides and bacteriophage coat protein are formed is illustrated in the surface of virus.This exogenous peptide still can keep its antigen-specific and biological activity, and amalgamation and expression do not influence the breeding of phage, therefore can carry out in-vitro screening to this allogenic polypeptide.
Traditional triage techniques directly screens at tumor cell specific albumen or tumor cell surface usually, resulting guide molecule also is to combine with tumor cell surface, though solved the effect of local raising medicine concentration, but can not improve the enter problem of medicine effectively, and most of medicine can play a role best after all needing to enter into cell interior to tumour cell.Therefore the screening to specific binding peptides with internalization ability has crucial meaning.
With phage display storehouse screening internalization antibody, be exactly by phage display storehouse and neuron target cell interaction for some time, start the internalization approach, directly filter out by in-vitro screening and can combine the also phage expression small peptide of internalization with cell surface receptor.The present invention is by in-vitro screening technology screening to 4 kind and liver cancer cell BEL-7404 specificity bonded phage small peptide, and the phage small peptide identified by effective means identifying small peptide can internalization.Therefore, in the treatment of liver cancer and diagnosis, the potential using value is arranged.
Summary of the invention
An object of the present invention is to disclose 4 kinds of liver cancer cell specificity internalization small peptides, it is characterized in that the amino acid structure of described small peptide has following sequence:
1.DLNYFTLSSKRE；
2.DLNYLTLSSKRE；
3.DLNYHARSYKRE；
4.NLKLLDSQDWDG。
This type of small peptide not only has the ability of specificity in conjunction with liver cancer cell, can also enter into cell interior effectively by the mediation of associated receptor, can improve the specific killing effect of anti-tumor medicine to liver cancer greatly.
Another object of the present invention is to release a kind of method of in-vitro screening liver cancer cell specificity internalization small peptide.The internalization small peptide that screens with this method can be transported to the medicine orientation in the liver cancer cell, brings into play the pharmacological action of medicine better.
For achieving the above object, the present invention adopts following technical scheme.By the interaction of phage display peptide storehouse and target cell, start the internalization approach, in-vitro screening can with cell surface receptor bonded internalization small peptide.
Now describe technical scheme of the present invention in detail.
A kind of method of in-vitro screening liver cancer cell specificity internalization small peptide is characterized in that:
One, material requested:
Phage display peptide storehouse:
12 peptide phage display library (Ph.D.12 at random
TMPhage Display PeptideLibrary Kit) available from U.S. New England Biolabs company, the titre of its pnagus medius is 1.5 * 10
13Pfu/ml, diversity is 2.7 * 10
9, the host bacterium is E.coli ER2738;
Cell:
Human liver cancer cell BEL-7404 and normal liver cell HL-7702 are available from Shanghai Chinese Academy of Sciences cell bank;
Key reagents:
Subtilisin and chloroquine are available from FLUKA company; PBS:
Contain 130mM NaCl in every liter of solution, 7mM Na
2HPO
4-12H
2O, 3mMNaH
2PO
4, pH 7.5;
TBS: contain 50mM Tris in every liter of solution, 150mM NaCl, pH 7.5;
The sodium deoxycholate of cell pyrolysis liquid: 2%w/v, 10mMTris-HCl, 2mM EDTA, pH 8.0;
Proteinase inhibitor: contain PMSF 100mM, Aprotinin 15uM, Leupeptin 100 μ M, Bestatin 100 μ M in every milliliter, Pepstain A 100 μ M, E-64 80 μ M are dissolved among the DMSO;
Two, the in-vitro screening of internalization small peptide, operation steps:
The first step peptide storehouse that 10 μ l are above-mentioned adds and contains in 1640 substratum of 100 μ M chloroquines and 10%CBS2ml incubated at room 1 hour;
Second step added 1640 substratum that 2ml contains 100 μ M chloroquines and 10%CBS in the culturing bottle of the human liver cancer cell BEL-7404 of vitro culture to 80% polymerization degree, hatched 30 minutes for 37 ℃;
The 3rd step will add in the culturing bottle of handling through second step through the 2ml peptide storehouse that the first step is handled, and hatch 2 hours for 37 ℃;
The 4th step put described culturing bottle 5 minutes on ice, and follow-up operation is all carried out under 4 ℃, contains Mg with 2ml
2+And Ca
2+PBS clean cell 2 times, clean cell 6 times with 2ml PBS again, all be 3 minutes at every turn, clean cell 1～4 time with TBST again, the prescription of TBST is to add 0.05%v/vTween-20 among the TBS;
The 5th step added contains the TBS of subtilisin 3mg/ml and tumour cell that surface bonding has phage was hatched 45 minutes, degraded and cell surface bonded phage, make cell resuspended with the disodium ethylene diamine tetra-acetic acid solution that contains 0.1%, centrifugal 10 minutes of 1500rpm, centrifugal collection obtains single cell suspension, the PBS that contains proteinase inhibitor that adds 5ml was at last hatched 15 minutes, stopped the activity of enzyme;
It is centrifugal that the 6th step was carried out 1500rpm to the solution after handling through the 5th step, and the time is 10 minutes, and collecting cell cleans with PBS, be resuspended in the cell pyrolysis liquid of 200 μ l, and the vibration centrifuge tube, incubated at room 30 minutes adds the MgCl of 50 μ M
2In mixture, in and the EDTA in the lysis buffer and help to infect, the solution after the recovery stays 5 μ l to survey titre, 200 μ l preserve, remaining phage is infected the intestinal bacteria amplification and surveys titre;
The 7th step for remove can with normal liver cell bonded phage, the selective polymerization degree reaches 80% the good HL-7702 of growth conditions, cleans 3 times with PBS, washes not adherent cell off, adds the phage display peptide 10 μ l that screen for the first time, includes phage about 10
11Individual, incubation 1 hour is drawn supernatant, and resulting is not exactly required liver cancer cell specificity internalization phage with normal liver cell bonded phage, puts into 4 ℃ of refrigerators and preserves, and measures the titre of phage, and so far, first round in-vitro screening is finished;
Whole screening process comprises three-wheel altogether, the phage that when second takes turns beginning, adds first round screening back amplification, and third round adopts is second to take turns the phage of screening back amplification, phage after the third round screening contains liver cancer cell specificity internalization small peptide, takes turns to survey with third round screening back from second and has respectively selected 20 locus coeruleus on the flat board of titre and check order and identify.
Another object of the present invention provides a kind of method of identifying liver cancer cell specificity internalization small peptide.
For achieving the above object, the present invention adopts following technical scheme.
A kind of method of identifying liver cancer cell specificity internalization small peptide is characterized in that:
One, material requested:
The phage mono-clonal that above-mentioned in-vitro screening obtains, host bacterium are E.coli ER2738;
Cell:
Human liver cancer cell BEL-7404, BEL-7402, normal liver cell HL-7702, normal people's embryonic kidney cells 293 are available from Shanghai Chinese Academy of Sciences cell bank;
Key reagents:
Rabbit phage-resistance M13 antiserum(antisera) is so kind as to give by the Wang Linfa researcher of Australian CSIRO animal health institute (Australia Animal Health Laboratory);
The anti-M13 antibody of HRP-rabbit is available from Sigma company;
The goat anti-rabbit igg of FITC mark is available from the magnificent company in Shanghai;
Subtilisin and chloroquine are available from FLUKA company;
Add 0.05%v/vTween-20 among the PBST05:PBS;
PBS-S: the PBS that contains 0.1% Saponin/TSM;
PBS-SB: the PBS-S that contains the BSA of 1%w/v;
Cell pyrolysis liquid: 50mMTris-HCl, 150mMNaCl, 0.2g/LNaN
3, 1g/L SDS, 5g/L Sodium desoxycholate;
D-Hank ' s:NaCl 8.00g/L, KCl 0.40g/L, Na
2HPO
4-12H
2O 0.12g/L, KH
2PO4 0.06g/L, D-glucose (D-Glucose) 1.00g/L, NaHCO3 0.35g/L;
Proteinase inhibitor: contain PMSF 100mM, Aprotinin 15 μ M, Leupeptin 100 μ M, Bestatin 100 μ M in every milliliter, Pepstain A 100 μ M, E-64 80 μ M are dissolved among the DMSO;
Two, the authentication method of liver cancer cell specificity internalization small peptide has following four steps, the concrete operations step:
The first step cell ELISA is identified the bonding force of phage mono-clonal and cell: select the good human liver cancer cell BEL-7404 of growth conditions to digest, with cell suspension, concentration transfers to 5 * 10 after the numeration
5Cells/ml adds 96 porocyte culture plates respectively, and the every hole of cell plate adds 100 μ l cell suspensions, spend the night in 37 ℃ of cell culture incubators, make cell attachment, D-Hanks buffer solution for cleaning cell, serum free medium was cultivated 1 hour, blocking solution 100 μ l are sealed up in every hole, the prescription of liquid blockaded is to add 4% skim-milk among the PBST05, blockades 1 hour for 37 ℃, and plate is washed with PBST05 in every hole, 3 times repeatedly, every hole adds phage mono-clonal 10
10Individual, the wild type phage of same quantity is made negative control, do not add phage clone and do blank, 4 ℃ of incubations 2 hours, the same plate of washing, every hole adds rabbit phage-resistance antibody 100 μ l, rabbit phage-resistance M13 antibody with the described liquid of blockading with the dilution of 1: 1000 weight ratio, 37 ℃ of incubations 1.5 hours; Wash plate, every hole adds the goat anti-rabbit igg antibody 100 μ l of HRP mark again, and goat-anti rabbit enzyme labelled antibody dilutes with the weight ratio with 1: 2500 with the described liquid of blockading, 37 ℃ of incubations 1 hour are washed plate, and every hole adds OPD substrate colour developing liquid, 37 ℃ were developed the color every hole enzyme-added stop buffer 2M H 10～15 minutes
2SO
450 μ l, color development stopping, microplate reader 490nm wavelength readings;
The second step competitive ELISA is identified the bonding force of phage mono-clonal and cell: get one bottle of BEL-7404 tumour cell that growth conditions is good, get off with trysinization, clean several times with PBS, put into the ice chest effect 1.5 hours after adding an amount of proteinase inhibitor, make the abundant cracking of cell, the albumen that lysing cell obtains directly carried out the 12000rpm high speed centrifugation 1.5 hours in 4 ℃, the supernatant that obtains is the tumor cell membrane protein solution, with extractive membranin with different extent of dilution: 1: 2,1: 4,1: 20,1: 50,1: 100,1: 200,4 ℃ of wrapper sheets that spend the night were got the growth conditions good cell and are forwarded in 96 orifice plates, add the phage mono-clonal in the hole of membranin is arranged, the add-on in every hole is about 10
10Individual and membranin were hatched 1 hour for 37 ℃, got in 96 orifice plates that supernatant is added to cell, and the operation of the method and the first step is identical;
The 3rd step immunofluorescence detects the internalization of phage display peptide: every hole adds 10 in being placed with 24 orifice plates of cover glass in advance
5Individual cell, incubated overnight, use D-Hank ' s to wash not adherent cell, cell and mono-clonal were all hatched 30 minutes with 37 ℃ of the substratum that contains the 100uM chloroquine in advance in advance, the phage mono-clonal is added in the hand-hole, 37 ℃ act on 2 hours, and it is inferior to give a baby a bath on the third day after its birth with cold PBST05, each 5 minutes, wash 2 times with the 0.1 μ M glycine-HCL that contains 1%BSA and pH2.2, each 5 minutes, it was inferior to give a baby a bath on the third day after its birth with cold PBST05 again, each 5 minutes, cell is fixed 15 minutes for 37 ℃ with 4% Paraformaldehyde 96, hatched 15 minutes with PBS-S, increase the perviousness of cell, blockaded 15 minutes with PBS-SB, hatched 1.5 hours for 37 ℃ with the PBS-SB that contains 0.1% rabbit phage-resistance M13 IgG, PBST cleans 4 times, with the PBS-B that contains goat anti-rabbit antibody IgG of FITC conjugated to dilute 37 ℃ of effects 1 hour, the weight ratio of goat anti-rabbit antibody IgG and PBS-B is 1: 30, PBST cleans 4 times, with buffering glycerine mounting, slide is tipped upside down on the sheet glass, fluorescent microscope detects;
The internalization of the 4th step flow cytometer detection by quantitative phage display peptide: treat that the bottle cell covers with, add 10
9Individual mono-clonal is in culturing bottle, 37 ℃ act on 2 hours, establish contrast simultaneously: add M13, blank, former storehouse, normal cell, for stoping further endocytosis, culturing bottle was put 5 minutes on ice, outwelled supernatant, PBST cleans 3 times, TBST cleans 3 times, adds the TBS 1ml that contains subtilisin 1.5mg/ml, hatches 15 minutes with cell, the phage of degradation of cell surface bonding, with dropper cell is blown and beaten, single cell suspension, forward in the doffer manages, centrifugal 7 minutes of 1000rpm, remove supernatant, PBST cleans 2 times, adds 4% Paraformaldehyde 96,100 μ l, fix 15 minutes for 37 ℃, PBST cleans 2 times, adds the PBS that contains 3%BSA and 0.1% saponin, fixes 30 minutes for 37 ℃, PBST cleans 2 times, add rabbit phage-resistance M13 IgG antibody, 37 ℃ act on 1.5 hours, and rabbit phage-resistance M13 antiserum(antisera) dilutes with 1: 1000 weight ratio with the described liquid of blockading, PBST cleans 2 times, the goat anti-rabbit igg antibody that adds the FITC mark, 37 ℃ act on 1 hour, and the goat anti-rabbit igg antibody of FITC mark and the weight ratio of PBS-B are 1: 30, PBST cleans 2 times, upflowing cell instrument fluorescence intensity.
Compare with background technology, the present invention has the following advantages: the specificity height, and good effectiveness, the internalization ability that especially polypeptide had is for tumour medicine is had crucial meaning in effective carrying of cancer location in the future.
Description of drawings
Fig. 1: BEL-7404 is to the screening and the enrichment in phage random 12 peptide storehouses.
Fig. 2: in order to identify the bonding force power of phage mono-clonal and liver cancer cell BEL-7404, phage mono-clonal and liver cancer cell are hatched, detect by the OD value.Experimental result such as Fig. 2 illustrate that 4 mono-clonals compare M13 and all with liver cancer cell very strong bonding force is arranged, and No. 1 clone to compare other the clone and the binding ability of liver cancer cell stronger.
Fig. 3: cell ELISA is identified the monoclonal result of phage, in order to identify the bonding force power of phage mono-clonal and liver cancer cell BEL-7404, phage mono-clonal and liver cancer cell is hatched, and detects by the OD value.Experimental result such as Fig. 2 illustrate that 4 mono-clonals compare M13 and all with liver cancer cell very strong bonding force is arranged, and No. 1 clone to compare other the clone and the binding ability of liver cancer cell stronger.
Fig. 4: figure A, D, G, J are respectively the result that 4 kinds of clones and liver cancer cell BEL-7404 are hatched, and the result that B, E, H, K to be respectively C as a result, F that 4 kinds of clones and normal liver cell HL-7702 are hatched be former storehouse and M13 wild type phage and liver cancer cell BEL-7404 are hatched; K, the L result that to be No. 1 clone hatch with liver cancer cell BEL-7402 and normal people's embryonic kidney cells 293.
Fig. 5: totally 4 go, three pictures of every row are respectively that 4 clones and liver cancer cell BEL-7404 are hatched, and add internalization inhibitor colchicine, sucrose fluorescence picture afterwards.
Fig. 6: A-F six width of cloth pictures result that to be respectively phage hatch at different incubation times (5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours) with liver cancer cell BEL-7404.
Fig. 7: different cells and No. 1 clone hatch, by the fluidic cell fluorescence intensity relatively.
Fig. 8: different clones hatch the result with liver cancer cell BEL-7404.
Embodiment
In above summary of the invention, the concrete operations step of the method for in-vitro screening and evaluation liver cancer cell specificity internalization small peptide is disclosed in detail, the content of this part is exactly embodiment.For avoiding style of writing to repeat, just repeat no more herein.
Claims (3)
1,4 kinds of liver cancer cell specificity internalization small peptides is characterized in that the amino acid structure of described small peptide has following sequence:
1.DLNYFTLSSKRE；
2.DLNYLTLSSKRE；
3.DLNYHARSYKRE；
4.NLKLLDSQDWDG。
2, a kind of method of in-vitro screening liver cancer cell specificity internalization small peptide, described liver cancer cell specificity internalization small peptide is exactly the described liver cancer cell specificity internalization of claim 1 small peptide, it is characterized in that:
One, material requested:
Phage display peptide storehouse:
12 peptide phage display library (Ph.D.12 at random
TMPhage Display PeptideLibrary Kit) available from U.S. New England Biolabs company, the titre of its pnagus medius is 1.5 * 10
13Pfu/ml, diversity is 2.7 * 10
9, the host bacterium is E.coli ER2738;
Cell:
Human liver cancer cell BEL-7404 and normal liver cell HL-7702 are available from Shanghai Chinese Academy of Sciences cell bank;
Key reagents:
Subtilisin and chloroquine are available from FLUKA company; PBS:
Contain 130mM NaCl in every liter of solution, 7mM Na
2HPO
4-12H
2O, 3mMNaH
2PO
4, pH 7.5;
TBS: contain 50mM Tris in every liter of solution, 150mM NaCl, pH 7.5;
The sodium deoxycholate of cell pyrolysis liquid: 2%w/v, 10mMTris-HCl, 2mM EDTA, pH 8.0;
Proteinase inhibitor: contain PMSF 100mM, Aprotinin 15uM, Leupeptin 100 μ M, Bestatin 100 μ M in every milliliter, Pepstain A 100 μ M, E-64 80 μ M are dissolved among the DMSO;
Two, the in-vitro screening of internalization small peptide, operation steps:
The first step peptide storehouse that 10 μ l are above-mentioned adds and contains in 1640 substratum of 100 μ M chloroquines and 10%CBS2ml incubated at room 1 hour;
Second step added 1640 substratum that 2ml contains 100 μ M chloroquines and 10%CBS in the culturing bottle of the human liver cancer cell BEL-7404 of vitro culture to 80% polymerization degree, hatched 30 minutes for 37 ℃;
The 3rd step will add in the culturing bottle of handling through second step through the 2ml peptide storehouse that the first step is handled, and hatch 2 hours for 37 ℃;
The 4th step put described culturing bottle 5 minutes on ice, and follow-up operation is all carried out under 4 ℃, contains Mg with 2ml
2+And Ca
2+PBS clean cell 2 times, clean cell 6 times with 2ml PBS again, all be 3 minutes at every turn, clean cell 1～4 time with TBST again, the prescription of TBST is to add 0.05%v/vTween-20 among the TBS;
The 5th step added contains the TBS of subtilisin 3mg/ml and tumour cell that surface bonding has phage was hatched 45 minutes, degraded and cell surface bonded phage, make cell resuspended with the disodium ethylene diamine tetra-acetic acid solution that contains 0.1%, centrifugal 10 minutes of 1500rpm, centrifugal collection obtains single cell suspension, the PBS that contains proteinase inhibitor that adds 5ml was at last hatched 15 minutes, stopped the activity of enzyme;
It is centrifugal that the 6th step was carried out 1500rpm to the solution after handling through the 5th step, and the time is 10 minutes, and collecting cell cleans with PBS, be resuspended in the cell pyrolysis liquid of 200 μ l, and the vibration centrifuge tube, incubated at room 30 minutes adds the MgCl of 50 μ M
2In mixture, in and the EDTA in the lysis buffer and help to infect, the solution after the recovery stays 5 μ l to survey titre, 200 μ l preserve, remaining phage is infected the intestinal bacteria amplification and surveys titre;
The 7th step for remove can with normal liver cell bonded phage, the selective polymerization degree reaches 80% the good HL-7702 of growth conditions, cleans 3 times with PBS, washes not adherent cell off, adds the phage display peptide 10 μ l that screen for the first time, includes phage about 10
11Individual, incubation 1 hour is drawn supernatant, and resulting is not exactly required liver cancer cell specificity internalization phage with normal liver cell bonded phage, puts into 4 ℃ of refrigerators and preserves, and measures the titre of phage, and so far, first round in-vitro screening is finished;
Whole screening process comprises three-wheel altogether, the phage that when second takes turns beginning, adds first round screening back amplification, and third round adopts is second to take turns the phage of screening back amplification, phage after the third round screening contains liver cancer cell specificity internalization small peptide, takes turns to survey with third round screening back from second and has respectively selected 20 locus coeruleus on the flat board of titre and check order and identify.
3, a kind of method of identifying liver cancer cell specificity internalization small peptide is characterized in that:
One, material requested:
The phage mono-clonal that above-mentioned in-vitro screening obtains, host bacterium are E.coli ER2738;
Cell:
Human liver cancer cell BEL-7404, BEL-7402, normal liver cell HL-7702, normal people's embryonic kidney cells 293 are available from Shanghai Chinese Academy of Sciences cell bank;
Key reagents:
Rabbit phage-resistance M13 antiserum(antisera) is so kind as to give by the Wang Linfa researcher of Australian CSIRO animal health institute (Australia Animal Health Laboratory);
The anti-M13 antibody of HRP-rabbit is available from Sigma company;
The goat anti-rabbit igg of FITC mark is available from the magnificent company in Shanghai;
Subtilisin and chloroquine are available from FLUKA company;
Add 0.05%v/vTween-20 among the PBST05:PBS;
PBS-S: the PBS that contains 0.1% Saponin/TSM;
PBS-SB: the PBS-S that contains the BSA of 1%w/v;
Cell pyrolysis liquid: 50mMTris-HCl, 150mMNaCl, 0.2g/LNaN
3, 1g/LSDS, 5g/L Sodium desoxycholate;
D-Hank ' s:NaCl 8.00g/L, KCl 0.40g/L, Na
2HPO
4-12H
2O 0.12g/L, KH
2PO40.06g/L, D-glucose (D-Glucose) 1.00g/L, NaHCO30.35g/L;
Proteinase inhibitor: contain PMSF 100mM, Aprotinin 15 μ M, Leupeptin 100 μ M, Bestatin 100 μ M in every milliliter, Pepstain A 100 μ M, E-64 80 μ M are dissolved among the DMSO;
Two, the authentication method of liver cancer cell specificity internalization small peptide has following four steps, the concrete operations step:
The first step cell ELISA is identified the bonding force of phage mono-clonal and cell: select the good human liver cancer cell BEL-7404 of growth conditions to digest, with cell suspension, concentration transfers to 5 * 10 after the numeration
5Cells/ml adds 96 porocyte culture plates respectively, and the every hole of cell plate adds 100 μ l cell suspensions, spend the night in 37 ℃ of cell culture incubators, make cell attachment, D-Hanks buffer solution for cleaning cell, serum free medium was cultivated 1 hour, blocking solution 100 μ l are sealed up in every hole, the prescription of liquid blockaded is to add 4% skim-milk among the PB ST05, blockades 1 hour for 37 ℃, and plate is washed with PBST05 in every hole, 3 times repeatedly, every hole adds phage mono-clonal 10
10Individual, the wild type phage of same quantity is made negative control, do not add phage clone and do blank, 4 ℃ of incubations 2 hours, the same plate of washing, every hole adds rabbit phage-resistance antibody 100 μ l, rabbit phage-resistance M13 antibody with the described liquid of blockading with the dilution of 1: 1000 weight ratio, 37 ℃ of incubations 1.5 hours; Wash plate, every hole adds the goat anti-rabbit igg antibody 100 μ l of HRP mark again, and goat-anti rabbit enzyme labelled antibody dilutes with the weight ratio with 1: 2500 with the described liquid of blockading, 37 ℃ of incubations 1 hour are washed plate, and every hole adds OPD substrate colour developing liquid, 37 ℃ were developed the color every hole enzyme-added stop buffer 2M H 10～15 minutes
2SO
450 μ l, color development stopping, microplate reader 490nm wavelength readings;
The second step competitive ELISA is identified the bonding force of phage mono-clonal and cell: get one bottle of BEL-7404 tumour cell that growth conditions is good, get off with trysinization, clean several times with PBS, put into the ice chest effect 1.5 hours after adding an amount of proteinase inhibitor, make the abundant cracking of cell, the albumen that lysing cell obtains directly carried out the 12000rpm high speed centrifugation 1.5 hours in 4 ℃, the supernatant that obtains is the tumor cell membrane protein solution, with extractive membranin with different extent of dilution: 1: 2,1: 4,1: 20,1: 50,1: 100,1: 200,4 ℃ of wrapper sheets that spend the night were got the growth conditions good cell and are forwarded in 96 orifice plates, add the phage mono-clonal in the hole of membranin is arranged, the add-on in every hole is about 10
10Individual and membranin were hatched 1 hour for 37 ℃, got in 96 orifice plates that supernatant is added to cell, and the operation of the method and the first step is identical;
The 3rd step immunofluorescence detects the internalization of phage display peptide: every hole adds 10 in being placed with 24 orifice plates of cover glass in advance
5Individual cell, incubated overnight, use D-Hank ' s to wash not adherent cell, cell and mono-clonal were all hatched 30 minutes with 37 ℃ of the substratum that contains the 100uM chloroquine in advance in advance, the phage mono-clonal is added in the hand-hole, 37 ℃ act on 2 hours, and it is inferior to give a baby a bath on the third day after its birth with cold PBST05, each 5 minutes, wash 2 times with the 0.1 μ M glycine-HCL that contains 1%BSA and pH2.2, each 5 minutes, it was inferior to give a baby a bath on the third day after its birth with cold PBST05 again, each 5 minutes, cell is fixed 15 minutes for 37 ℃ with 4% Paraformaldehyde 96, hatched 15 minutes with PBS-S, increase the perviousness of cell, blockaded 15 minutes with PBS-SB, hatched 1.5 hours for 37 ℃ with the PBS-SB that contains 0.1% rabbit phage-resistance M13 IgG, PBST cleans 4 times, with the PBS-B that contains goat anti-rabbit antibody IgG of FITC conjugated to dilute 37 ℃ of effects 1 hour, the weight ratio of goat anti-rabbit antibody IgG and PBS-B is 1: 30, PBST cleans 4 times, with buffering glycerine mounting, slide is tipped upside down on the sheet glass, fluorescent microscope detects;
The internalization of the 4th step flow cytometer detection by quantitative phage display peptide: treat that the bottle cell covers with, add 10
9Individual mono-clonal is in culturing bottle, 37 ℃ act on 2 hours, establish contrast simultaneously: add M13, blank, former storehouse, normal cell, for stoping further endocytosis, culturing bottle was put 5 minutes on ice, outwelled supernatant, PBST cleans 3 times, TBST cleans 3 times, adds the TBS 1ml that contains subtilisin 1.5mg/ml, hatches 15 minutes with cell, the phage of degradation of cell surface bonding, with dropper cell is blown and beaten, single cell suspension, forward in the doffer manages, centrifugal 7 minutes of 1000rpm, remove supernatant, PBST cleans 2 times, adds 4% Paraformaldehyde 96,100 μ l, fix 15 minutes for 37 ℃, PBST cleans 2 times, adds the PBS that contains 3%BSA and 0.1% saponin, fixes 30 minutes for 37 ℃, PBST cleans 2 times, add rabbit phage-resistance M 13 IgG antibody, 37 ℃ act on 1.5 hours, and rabbit phage-resistance M 13 antiserum(antisera)s dilute with 1: 1000 weight ratio with the described liquid of blockading, PBST cleans 2 times, the goat anti-rabbit igg antibody that adds the FITC mark, 37 ℃ act on 1 hour, and the goat anti-rabbit igg antibody of FITC mark and the weight ratio of PBS-B are 1: 30, PBST cleans 2 times, upflowing cell instrument fluorescence intensity.
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|---|---|---|---|
| CN 200610148050 CN101033251A (en) | 2006-12-27 | 2006-12-27 | Liver cancer cell specificity internalization short peptide and its in vitro screening and identification |
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| CN101492505B (en) * | 2008-12-24 | 2011-11-23 | 广东药学院 | Specific combined polypeptide for lung cancer, preparation and uses thereof |
| CN101768210B (en) * | 2007-10-29 | 2012-02-29 | 昆明医学院第一附属医院 | Tumor targeting polypeptide and preparation method thereof |
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| CN101412750B (en) * | 2008-10-08 | 2011-06-15 | 南京农业大学 | Bursin specific binding peptide and screening method thereof |
| CN101492505B (en) * | 2008-12-24 | 2011-11-23 | 广东药学院 | Specific combined polypeptide for lung cancer, preparation and uses thereof |
| CN101870723B (en) * | 2009-04-24 | 2012-07-04 | 上海交通大学医学院附属瑞金医院 | Short peptide and immunosuppressant containing the same and application thereof |
| CN102060909B (en) * | 2009-11-11 | 2013-01-02 | 中国医学科学院放射医学研究所 | Tumor specific target polypeptide and application thereof |
| CN102033056B (en) * | 2010-10-14 | 2012-05-30 | 中国科学院化学研究所 | Method for evaluating anti-cancer drug inhibiting aggregation of membrane protein receptors |
| CN102033056A (en) * | 2010-10-14 | 2011-04-27 | 中国科学院化学研究所 | Method for evaluating anti-cancer drug inhibiting aggregation of membrane protein receptors |
| CN102558300A (en) * | 2010-12-29 | 2012-07-11 | 中国人民解放军军事医学科学院毒物药物研究所 | Polypeptides capable of antagonizing corticotropin-releasing factor receptors 1 and application of polypeptides |
| CN102558300B (en) * | 2010-12-29 | 2015-02-11 | 中国人民解放军军事医学科学院毒物药物研究所 | Polypeptides capable of antagonizing corticotropin-releasing factor receptors 1 and application of polypeptides |
| CN105659088A (en) * | 2013-06-26 | 2016-06-08 | 菲洛吉卡有限公司 | Method of monitoring cellular trafficking of peptides |
| CN105601716A (en) * | 2014-03-24 | 2016-05-25 | 朱育盼 | Short peptide PCP11 specifically bound with prostatic cancer and application thereof |
| CN111481657A (en) * | 2020-06-18 | 2020-08-04 | 北京欣颂生物科技有限公司 | JZY-17 and use of compounds for the preparation of a medicament for the treatment of psoriasis |
| CN116003523A (en) * | 2022-07-27 | 2023-04-25 | 大连理工大学 | Neuron-specific enolase-specific binding short peptide and application thereof |
| CN116003523B (en) * | 2022-07-27 | 2024-04-05 | 大连理工大学 | Neuron-specific enolase-specific binding short peptide and application thereof |
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