CN105566455A - Oligopeptide PCP13 capable of specifically binding to prostate cancer and application thereof - Google Patents
Oligopeptide PCP13 capable of specifically binding to prostate cancer and application thereof Download PDFInfo
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- CN105566455A CN105566455A CN201610112193.9A CN201610112193A CN105566455A CN 105566455 A CN105566455 A CN 105566455A CN 201610112193 A CN201610112193 A CN 201610112193A CN 105566455 A CN105566455 A CN 105566455A
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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
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57434—Specifically defined cancers of prostate
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- 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
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Abstract
The invention provides a series of polypeptides which are screened and obtained by utilizing a phage display peptide library technology and has target prostate cancer characters. The binding polypeptides provided by the invention are high in specificity and can be used for general survey of prostate cancer high-risk groups, early-stage clinic diagnosis of the prostate cancer and assessment of a therapeutic effect.
Description
Technical field
The invention belongs to protein and peptide technical field, be specifically related to a series of can the small peptide of specific binding prostate cancer.
Background technology
Prostate cancer is one of malignant tumour of serious threat middle-aging male health, and sickness rate rises year by year, and mortality ratio is high, is only second to lung cancer, occupies second.50 for many years, and androgen deprivation therapy is the standard treatment of prostate cancer always, but Most patients finally can develop into Hormone refractory prostate cancer (hormonerefractoryprostatecancer, HRPC).For HRPC, there is no effective therapy at present, and existing treatment means comprises radiotherapy, chemotherapy etc. all can not extend patient vitals and reach more than 1 year.Therefore explore novel effective prostate cancer therapy method, be one of important topic that Urology Surgery is clinical faced always.
Current China is relatively low for the examination rate of prostate cancer, and this is also one of major reason causing prostate cancer mortality ratio higher.Therefore, this area can be used for the associated protein of prostate cancer diagnosis in the urgent need to exploitation.And polypeptide because of molecular weight little, penetration into tissue is good, non-immunogenicity and have higher affinity to cell surface receptor and become the desirable part in targeted delivery research.But known natural receptor-part is to being very limited.
The development of phage display peptide library technology then for finding and characterizing new part and corresponding acceptor provides strong instrument, is not only expected to realize clinical application in the targeted therapy of tumour, also contributes to the theoretical investigation explaining disease mechanism.Display technique of bacteriophage is by inserting in phage DNA by corresponding expression of polypeptides on pIII capsid protein by specific fragment, thus build bridge between the DNA and capsid protein of phage, make the polypeptide ligand of various target molecule be able to Rapid identification by screening in external or body.Be developed so far, apply this technology and successfully screen the target polypeptide obtained for multiple human tumors such as liver cancer, colorectal carcinoma, mammary cancer, but then little for the relevant report of screening prostate cancer targeting part.
Therefore, the present invention utilize phage display peptide library technology to screen in tumor model of prostate cancer body to obtain 16 can the small peptide of target prostate cancer, and the target characteristic of this polypeptide is identified, to investigate it as target part for transmitting the feasibility of medicine to tumor of prostate position.
Summary of the invention
The object of the invention is for the deficiencies in the prior art, a series of polypeptide with target prostate cancer characteristic utilizing phage display peptide library technology screening to obtain is provided.
Another object of the present invention is to provide aforementioned polypeptides and is preparing the application in prostatic cancer diagnostic reagent kit.
Object of the present invention is achieved by following technical proposals:
By the method for phage display peptide library subtractive screening tumor cell specific Binding peptide or target peptide be: usually screen by two clones, by Phage display random peptide library first by not screening containing the normal cell of target antigen, unconjugated supernatant is again by the cell containing target antigen, carry out next round screening after filtering out Phage amplification, finally obtain specific binding peptides.
First, adopt above-mentioned phage display peptide library subtractive triage techniques, using prostate cancer LNCaP and PC3 cell as target cell, normal cell is adherent cell, to the Ph.D.-7 of NEWENGLANDBiolabs
tMphageDisplayPeptideLibraryKit carries out four-wheel in-vitro screening, by strengthening washing dynamics by wheel, obtains the phage clone of prostatic cancer specific.After above-mentioned screening, respectively at second taking turns, in third round and fourth round the selection result, random picking mono-clonal carries out extraction and the order-checking of DNA.The peptide sequence that the Analysis and Screening of applying biological Bioinformatic tool obtains, enzyme-linked immunosorbent assay is for analyzing candidate phage mono-clonal to the binding ability of cell simultaneously, thus identify 16, the phage with prostate cancer cell with stronger avidity and clone, these 16 clones are sent to order-checking, and difference called after PCP1-16, sequencing result confirms that the aminoacid sequence of PCP1-16 is respectively as shown in SEQIDNO:1-16.
The present invention screens the polypeptide obtained and has following characteristic and advantage through qualification:
Polypeptide can identify and specific binding prostate cancer cell line LNCaP and PC3 effectively, and to normal prostate cell line RWPE-1, and the binding ability of other tumour cells HepG2, A549, SW480 is weak, shows the identification for prostate cancer specific and binding ability;
It is little that this polypeptide has molecular weight, penetration into tissue is good, non-immunogenicity and cell surface receptor is had to the popular feature of higher affinity, this polypeptide can be used for preparing prostatic cancer diagnostic reagent medicine box, this test kit diagnostic sensitivity is high, specificity is high, can be used for the judgement of the evaluation of the generaI investigation of prostate cancer high risk population and the early clinical diagnosis of prostate cancer and result for the treatment of and patient's Prognosis, prognosis.
Accompanying drawing explanation
Fig. 1 is that the avidity of ELISA detection fourth round screening phage clone and LNCaP and PC3 contrasts histogram;
Wherein, Figure 1A is that these 20 phage clones of PCP1-20 are respectively to the avidity of LNCaP, Figure 1B be these 20 phage mono-clonals of PCP1-20 respectively to the avidity of PC3,21 is the avidity of random phage clone in former storehouse to LNCaP, and 22 be the avidity that former storehouse random phage clones to PC3.
Fig. 2 is to the targeting ability of different cell, phage clone identifies that (phage combines=TU relatively
treat survey phage clone/ TU
blank phage).
Wherein, Fig. 2 A is phage PCP1-6 be phage PCP7-11 to the binding ability result figure of different cell, Fig. 2 B is respectively that phage PCP12-16 is respectively to the binding ability result figure of different cell to the binding ability result figure of different cell, Fig. 2 C respectively.
Embodiment
The present invention is described further by following examples, but not as restriction of the present invention.
The screening of embodiment 1 prostatic cancer specific Binding peptide
The present embodiment adopts the random 12 peptide storehouse subtractive of phage display to screen the polypeptide be combined with prostate gland cancer cell specificity, and its concrete steps are as follows:
After trysinization Human Prostate Cancer Cells LNCaP and PC3, adjustment cell density, is inoculated in the culture dish wrapped in advance by poly-lysine, when cell grows to 80%-90% degrees of fusion, carries out screening experiment.
Get above-mentioned LNCaP and PC3 cell, after cultivating with serum-free DMEM, add bovine serum albumin BSA and close, then add 20 μ l phage peptide libraries, after hatching 1.5h, topple over removing in conjunction with phage, be inverted bat and get rid of removing residual solution.With washings 0.2% (v/v) TBST wash buffer 4 times, add non-specific damping fluid 0.2M glycine-HCI (pH2.2) 2ml, sucking-off elutriant, after adding 250 μ l5MTris-HCl (pH9.0) neutralizations, elutriant is gone in RWPE-1 cell, after hatching 3h, collect supernatant, being the first round screens the phage obtained, being taken a morsel by the phage obtained utilizes intestinal bacteria to determine the concentration of phage by volumetry, remaining phage-infect intestinal bacteria is increased, for the screening of next round.
Second when taking turns screening, and in washings TBST, Tween-20 concentration is increased to 0.5%, and be 40min with LNCaP and PC3 incubation time, washing times is increased to 7 times, and all the other conditions, step are identical with the first round.
During third round screening, in washings TBST, Tween-20 concentration is increased to 0.8%, is reduced to 25min with LNCaP and PC3 incubation time, and washing times is increased to 10 times, and all the other conditions, step are identical with the first round.
During fourth round screening, in washings TBST, Tween-20 concentration is increased to 1.0%, is reduced to 20min with LNCaP and PC3 incubation time, and washing times is increased to 15 times, and all the other conditions, step are identical with the first round.
After measuring the titre of the phage that fourth round screening obtains, the blue plaque of random picking on LB/IPTG/Xgal flat board, prepare phage mono-clonal for the identification of.
The amplification of embodiment 2 phage
By in embodiment 1, after the phage that often wheel screening obtains carries out 100 doubling dilutions with LB substratum, get 20 μ l dilute after phage and the early stage Escherichia coli bacteria liquid of 200 μ l logarithmic growths mix, the LB solid plate containing IPTG/Xgal is poured into rapidly after joining the LB top-agar be incubated in 45 DEG C, process 24 hours, the spot number that the dull and stereotyped upper phage of counting can grow, is then multiplied by with this number plaque forming unit (pfu) titre that namely extension rate obtains every 10 μ l phages.
The enrichment of the phage polypeptide of prostate cancer cell specific combination: as shown in table 1, the positive phage clones be combined with LNCaP and PC3 phage rate of recovery after four-wheel screens improves 1000 times.
The rate of recovery of table 1 positive bacteriophage after four-wheel screening
Screening number of times | Add phage number (pfu) | Reclaim phage number (pfu) | The rate of recovery |
1 | 10 11 | 10 2 | 10 -9 |
2 | 10 11 | 10 2 | 10 -9 |
3 | 10 11 | 10 4 | 10 -7 |
4 | 10 11 | 10 5 | 10 -6 |
Embodiment 3ELISA identifies phage polypeptide
The phage polypeptide positive clone identification that prostatic cancer specific combines: in embodiment 1, phage peptide library is after continuous four-wheel subtractive screening, random choose 20 phage clones, utilize the ordinary method ELISA method preliminary evaluation phage clone of this area to the avidity of LNCaP and PC3.
By LNCaP and PC3 by 1 × 10
4the density in/hole is inoculated in 96 orifice plates, puts CO
2incubator carries out serum-free process 1h to cell after cultivating 20h, cleaning cell, then fixes with paraformaldehyde, and PBS washes once, and after TritonX-100 process, PBS-BSA closes, and adds phage mono-clonal, hatches 2h; Add HRP-antiM13 antibody, hatch 1.5h for 37 DEG C; With TMB colour developing, add isopyknic 2NHCl or 1NH
2sO
4carry out termination reaction, in enzyme mark hole, reaction solution becomes yellow from blueness, microplate reader 450nm place reading.In contrast, P/N > 2 is positive to random picking prophage storehouse locus coeruleus.
Result as shown in Figure 1, be that these 20 phage clones are respectively to the avidity detected result of LNCaP and PC3 in Figure 1A, 1B, 21 and 22 as a control group, the avidity detected result (note: in order to name conveniently, 20 phages by from the power of LNCaP, PC3 avidity different by order are from big to small arranged in order) of former storehouse phage clone to LNCaP, PC3 of picking immediately.Can be clear that very much from Fig. 1, these 20 phage mono-clonals of PCP1-20 are to the avidity of LNCaP and PC3 all higher than the phage mono-clonal of random choose, and wherein No. 1-16 clone is the strongest to the avidity of LNCaP and PC3, respectively called after PCP1-16.
Embodiment 4 phage DNA sequencing
Amplification embodiment 3 positives clone PCP1-16, and get above-mentioned positive colony Phage amplification liquid and add 100 μ l15%PEG/NaCl (volume ratio of PEG and NaCl is 1: 4), mix, the centrifugal 15min of room temperature placement 15min, 8000rpm, gets precipitation; With 100 μ lTE (PH=7.0) dissolution precipitations, add the saturated phenol of 50 μ lTris, turn upside down 2min, then leave standstill 1min, then 2min mixing of turning upside down; The centrifugal 15min of 8000rpm, get upper strata and add 200 μ l2mol/L sodium acetates: dehydrated alcohol (1: 25) precipitation DNA, 20min is left standstill after mixing, the centrifugal 15min of 8000rpm, remove supernatant, add 50 μ l200ml/L ethanol and wash once, air-dry residual ethanol, dissolve with 30 μ lTE (PH=7.0), agarose gel electrophoresis is identified.
Carry sequencing primer with peptide storehouse and send to order-checking, PCP1-16 contrasts the corresponding aminoacid sequence of genetic codon table respectively as shown in SEQIDNO:1-16.
The monoclonal external qualification of embodiment 5 phage
Cell ELISA method is for the identification of 16 phage clones (PCP1-16) of embodiment 3 to the specific binding capacity of prostate cancer cell, and the blank phage of M13KE is with comparing.By phage clone with fixing, close after cell (LNCaP/PC3/HepG2/A549/SW480/RWPE-1) educate 2 hours altogether after, with the anti-M13 phage antibody of HRP/, the phage combined is detected, after TMB colour developing, carries out reading by microplate reader.
In these 16 phage clones of ELISA detection display, PCP1-16 all shows very strong binding ability to prostate cancer cell LNCaP and PC3 clone, and for other tumour cells (colorectal carcinoma, liver cancer, lung cancer) and Normocellular bonding force weak (see accompanying drawing 2).This result is consistent with the result of the direct Binding experiment of phage, PCP1-16 identifiable design is described and target is incorporated into prostate cancer cell, simultaneously to normal cell and other tumour cells in conjunction with few.
Further, in competitive trials, the introducing of the SEQIDNO:1 polypeptide of lower concentration can suppress the PCP1 phage of 69% and the combination of LNCaP, and the contrast polypeptide of stochastic sequence does not then show this kind of restraining effect.On the other hand, SEQIDNO:1 polypeptide can suppress the PCP1 phage of 93% by LNCaP internalization, the contrast polypeptides exhibit unrestraint effect of the stochastic sequence of same concentrations.The Competitive assays effect of SEQIDNO:1 polypeptide shows that PCP1 phage is to the combination of cell and the behavior entering cell is the effect deriving from SEQIDNO:1 polypeptide, instead of the effect of its carrier phage particle.Competitive trials for its corresponding small peptide of PCP2-16 phage all has above-mentioned similar test-results.
The immunohistochemical methods qualification of the present embodiment is this area universal method adopted, and reagent involved in test and reaction conditions are also that those skilled in the art are in common knowledge.
Claims (5)
1. a small peptide, is characterized in that: can specific binding prostate cancer cell.
2. small peptide as claimed in claim 1, is characterized in that: it is PCP13, and its aminoacid sequence is respectively as shown in SEQIDNO:13.
3. the small peptide as shown in claim 1-2 detects the application in the preparation of prostate cancer cell in preparation.
4. apply as claimed in claim 3, it is characterized in that: described prostatic cell is LNCaP and PC3.
5. the small peptide described in any one of claim 1-2 is preparing the application in prostatic cancer diagnostic reagent kit.
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CN201410115439.9A CN103897037B (en) | 2014-03-24 | 2014-03-24 | The small peptide of specific binding prostate cancer and application thereof |
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CN201610113149.XA Pending CN105541974A (en) | 2014-03-24 | 2014-03-24 | Short peptides PCP10 specifically binding to prostatic cancer cells and application of short peptides PCP10 |
CN201610107546.6A Pending CN105622724A (en) | 2014-03-24 | 2014-03-24 | Oligopeptide PCP8 specifically combined with prostatic cancer and application thereof |
CN201410115439.9A Active CN103897037B (en) | 2014-03-24 | 2014-03-24 | The small peptide of specific binding prostate cancer and application thereof |
CN201610112187.3A Pending CN105601716A (en) | 2014-03-24 | 2014-03-24 | Short peptide PCP11 specifically bound with prostatic cancer and application thereof |
CN201610112231.0A Pending CN105601717A (en) | 2014-03-24 | 2014-03-24 | Short peptide PCP15 specifically bound with prostatic cancer and application thereof |
CN201610112193.9A Pending CN105566455A (en) | 2014-03-24 | 2014-03-24 | Oligopeptide PCP13 capable of specifically binding to prostate cancer and application thereof |
CN201610110725.5A Pending CN105541973A (en) | 2014-03-24 | 2014-03-24 | Short peptides PCP16 specifically binding to prostatic cancer cells and application of short peptides PCP16 |
CN201610107661.3A Pending CN105541971A (en) | 2014-03-24 | 2014-03-24 | Short peptide PCP5 for specifically binding prostatic cancer and application thereof |
CN201610107418.1A Pending CN105622723A (en) | 2014-03-24 | 2014-03-24 | Oligopeptide PCP3 specifically combined with prostatic cancer and application thereof |
CN201610109511.6A Pending CN105541972A (en) | 2014-03-24 | 2014-03-24 | Short peptides PCP7 specifically binding to prostatic cancer cells and application of short peptides PCP7 |
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CN201610113149.XA Pending CN105541974A (en) | 2014-03-24 | 2014-03-24 | Short peptides PCP10 specifically binding to prostatic cancer cells and application of short peptides PCP10 |
CN201610107546.6A Pending CN105622724A (en) | 2014-03-24 | 2014-03-24 | Oligopeptide PCP8 specifically combined with prostatic cancer and application thereof |
CN201410115439.9A Active CN103897037B (en) | 2014-03-24 | 2014-03-24 | The small peptide of specific binding prostate cancer and application thereof |
CN201610112187.3A Pending CN105601716A (en) | 2014-03-24 | 2014-03-24 | Short peptide PCP11 specifically bound with prostatic cancer and application thereof |
CN201610112231.0A Pending CN105601717A (en) | 2014-03-24 | 2014-03-24 | Short peptide PCP15 specifically bound with prostatic cancer and application thereof |
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CN201610107661.3A Pending CN105541971A (en) | 2014-03-24 | 2014-03-24 | Short peptide PCP5 for specifically binding prostatic cancer and application thereof |
CN201610107418.1A Pending CN105622723A (en) | 2014-03-24 | 2014-03-24 | Oligopeptide PCP3 specifically combined with prostatic cancer and application thereof |
CN201610109511.6A Pending CN105541972A (en) | 2014-03-24 | 2014-03-24 | Short peptides PCP7 specifically binding to prostatic cancer cells and application of short peptides PCP7 |
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CN105541972A (en) | 2016-05-04 |
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CN105601716A (en) | 2016-05-25 |
CN105622723A (en) | 2016-06-01 |
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