CN105601716A

CN105601716A - Short peptide PCP11 specifically bound with prostatic cancer and application thereof

Info

Publication number: CN105601716A
Application number: CN201610112187.3A
Authority: CN
Inventors: 华国光
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-03-24
Filing date: 2014-03-24
Publication date: 2016-05-25
Also published as: CN103897037A; CN105601717A; CN105622723A; CN105541973A; CN105541972A; CN103897037B; CN105566455A; CN105622724A; CN105541974A; CN105541971A

Abstract

The invention provides a series of polypeptides with targeted prostatic cancer characteristics. The polypeptides are screened out through the phage display peptide library technology. A binding peptide is high in specificity and can be used for general investigation of prostatic cancer high-risk groups, early clinical diagnosis of the prostatic cancer, and evaluation of the treatment effect.

Description

Small peptide PCP11 and the application thereof of specific binding prostate cancer

Technical field

The invention belongs to protein and peptide technical field, be specifically related to a series of can specific binding prostate cancer shortPeptide.

Background technology

Prostate cancer is one of malignant tumour of serious threat middle-aging male health, and the incidence of disease rises year by year, deadThe rate of dying 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 at present effective therapy, and existing treatment means comprise radiotherapy, chemotherapyReach more than 1 year Deng all not extending patient's life. Therefore explore novel effective prostate cancer therapy method, be uropoiesis alwaysOne of important topic that surgical clinical faces.

China is relatively low for the examination rate of prostate cancer at present, and this is also the weight that causes the prostate cancer death rate higherWant one of reason. Therefore, this area can be used for the GAP-associated protein GAP of prostate cancer diagnosis in the urgent need to exploitation. And polypeptide is because of molecular weightLittle, tissue penetration is good, non-immunogenicity and cell surface receptor is had higher affinity and become in targeted delivery researchDesirable part. But known natural receptor-part is to being very limited.

The development of phage display peptide library technology is for finding and characterizing new part and corresponding acceptor provides strongInstrument, is not only expected to realize clinical practice in the targeted therapy of tumour, also contributes to explain the theoretical research of disease mechanism.Display technique of bacteriophage by specific fragment being inserted in phage DNA and by corresponding expression of polypeptides at pIII capsid proteinUpper, thus between the DNA of bacteriophage and capsid protein, build bridge, make the polypeptide ligand of various target molecules can be by externalOr in body, screening is able to Rapid identification. Be developed so far, apply this technology and successfully screen and obtain for liver cancer, colon cancer, mammary glandThe target polypeptide of the multiple human tumor such as cancer, but little for the relevant report of screening prostate cancer targeting part.

Therefore, utilize phage display peptide library technology to screen in prostate cancer tumor model body to obtain 16 can in the present inventionThe small peptide of target prostate cancer, and the target characteristic of this polypeptide is identified, using investigate its as target part for passingDrug delivery is to the feasibility at tumor of prostate position.

Summary of the invention

The object of the invention is for the deficiencies in the prior art, a series of phage display peptide library technology screenings that utilize are providedThe polypeptide with target prostate cancer characteristic obtaining.

Another object of the present invention is to provide aforementioned polypeptides in the application of preparing in prostatic cancer diagnostic reagent kit.

Object of the present invention is achieved by following technical proposals:

The method of screening tumor cell specific Binding peptide or target peptide by subtracting property of phage display peptide library is: conventionally use twoIndividual clone is screened, and Phage display random peptide library is first by the not normal cell screening containing target antigen, unconjugatedSupernatant, again by the cell containing target antigen, filters out after bacteriophage is increased and carries out next round screening, finally obtains specific bindingPeptide.

First, adopt above-mentioned subtracting property of phage display peptide library triage techniques, using prostate cancer LNCaP and PC3 cell asTarget 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, has obtained prostatic cancer specificPhage clone. After above-mentioned screening, the random picking monoclonal respectively at second taking turns, in third round and fourth round the selection resultCarry out extraction and the order-checking of DNA. Applying biological informatics tool analysis screens the peptide sequence obtaining, simultaneously Enzyme-linked Immunosorbent AssayExperiment, for analyzing the binding ability of candidate bacteriophage monoclonal to cell, has stronger thereby identify with prostate gland cancer cell16 clones of bacteriophage of affinity, these 16 clones are sent to order-checking, and called after PCP1-16 respectively, sequencing result cardThe amino acid sequence of real PCP1-16 is respectively as shown in SEQIDNO:1-16.

The present invention screens the polypeptide obtaining and has following characteristic and advantage through qualification:

Polypeptide can be identified and specific binding prostate cancer cell line LNCaP and PC3 effectively, and to normal prostateClone RWPE-1, and a little less than the binding ability of other tumour cells HepG2, A549, SW480, show for prostate cancer spyDifferent identification and binding ability;

It is little that this polypeptide has molecular weight, and tissue penetration is good, non-immunogenicity and cell surface receptor is had to higher parentWith the popular feature of power, this polypeptide can be used for preparing prostatic cancer diagnostic reagent medicine box, and this kit diagnostic sensitivity is high, specialProperty is high, can be used for the evaluation of prostate cancer people at highest risk's generaI investigation and the early clinical diagnosis of prostate cancer and result for the treatment ofJudgement with patient's Prognosis, prognosis.

Brief description of the drawings

Fig. 1 is the affinity contrast block diagram that ELISA detects fourth round screening phage clone and LNCaP and PC3;

Wherein, Figure 1A is these 20 phage clones of PCP1-20 affinity to LNCaP respectively, and Figure 1B is PCP1-20These 20 bacteriophage monoclonals affinity to PC3 respectively, 21 is that former storehouse random phage is cloned the affinity to LNCaP, 22For former storehouse random phage is cloned the affinity to PC3.

Targeting ability qualification that Fig. 2 is phage clone to different cells (bacteriophage relatively in conjunction with=TU_{Phage clone to be measured}/TU_{Blank bacteriophage})。

Wherein, Fig. 2 A is the bacteriophage PCP1-6 binding ability result figure to different cells respectively, and Fig. 2 B is bacteriophagePCP7-11 is the binding ability result figure to different cells respectively, and Fig. 2 C is the bacteriophage PCP12-16 knot to different cells respectivelyClose capability result figure.

Detailed description of the invention

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 random 12 the subtracting property of peptide storehouse of phage display to screen be combined with prostate gland cancer cell specificity manyPeptide, its concrete steps are as follows:

With after trypsinization Human Prostate Cancer Cells LNCaP and PC3, adjust cell density, be inoculated in coated poly in advanceIn the culture dish of lysine, in the time that growing to 80%-90% degrees of fusion, cell carries out screening experiment.

Get above-mentioned LNCaP and PC3 cell, after cultivating with serum-free DMEM, add bovine serum albumin(BSA) BSA sealing, then addEnter 20 μ l phage peptide libraries, hatch after 1.5h, topple over and remove not in conjunction with bacteriophage, be inverted to clap to get rid of and remove residual solution. With washingLiquid 0.2% (v/v) TBST buffer solution rinses 4 times, adds non-specific buffer solution 0.2M glycine-hydrochloric acid (pH2.2) 2ml, inhalesGo out eluent, add after 250 μ l5MTris-HCl (pH9.0) neutralizations, eluent is gone in RWPE-1 cell, hatch 3hAfter, collect supernatant, be the first round screen bacteriophage obtaining, the bacteriophage obtaining is taken a morsel and utilizes Escherichia coli to pass through dropletThe method of determining is determined the concentration of bacteriophage, and remaining phage-infect Escherichia coli is increased, for the screening of next round.

Second takes turns while screening, and in cleaning solution TBST, Tween-20 concentration is increased to 0.5%, with LNCaP and PC3 incubation timeFor 40min, washing times is increased to 7 times, and all the other conditions, step are identical with the first round.

When third round screening, in cleaning solution TBST, Tween-20 concentration is increased to 0.8%, with LNCaP and PC3 incubation timeBe reduced to 25min, and washing times is increased to 10 times, all the other conditions, step are identical with the first round.

When fourth round screening, in cleaning solution TBST, Tween-20 concentration is increased to 1.0%, with LNCaP and PC3 incubation timeBe reduced to 20min, and washing times is increased to 15 times, all the other conditions, step are identical with the first round.

Measure after the titre of bacteriophage of fourth round screening acquisition, on LB/IPTG/Xgal flat board, random picking blueness is bittenBacterial plaque, preparation bacteriophage monoclonal for the identification of.

The amplification of embodiment 2 bacteriophages

By in embodiment 1, every bacteriophage LB culture medium of taking turns screening acquisition carries out after 100 doubling dilutions, gets 20 μ l rareRelease the early stage Escherichia coli bacteria liquid of rear bacteriophage and 200 μ l logarithmic growths and mix, join the top agar in the LB of 45 DEG C of insulationsBe poured into rapidly afterwards the LB solid plate that contains IPTG/Xgal, process 24 hours, the spot that the dull and stereotyped upper bacteriophage of counting can growNumber, is then multiplied by extension rate and obtains plaque forming unit (pfu) titre of every 10 μ l bacteriophages with this number.

The enrichment of the phage polypeptide of prostate gland cancer cell specific bond: as shown in table 1, the sun of being combined with LNCaP and PC3Property phage clone bacteriophage rate of recovery after four-wheel screening improve 1000 times.

The rate of recovery of table 1 positive bacteriophage after four-wheel screening

Screening number of times	Add bacteriophage number (pfu)	Reclaim bacteriophage number (pfu)	The rate of recovery
				1	10¹¹	10²	10^-9
2	10¹¹	10²	10^-9
				3	10¹¹	10⁴	10^-7
4	10¹¹	10⁵	10^-6

Embodiment 3ELISA identifies phage polypeptide

The phage polypeptide positive colony qualification of prostatic cancer specific combination: in embodiment 1, phage peptide library is through connectingAfter continuous subtracting property of four-wheel screening, 20 phage clones of random choose, utilize the conventional method ELISA method Preliminary Identification of this area to biteThalline is cloned the affinity to LNCaP and PC3.

LNCaP and PC3 are pressed to 1 × 10⁴The density in/hole is inoculated in 96 orifice plates, puts CO₂Incubator is cultivated after 20h, to cellCarry out serum-free and process 1h, clean cell, then fix with paraformaldehyde, PBS washes once, PBS-after TritonX-100 processesBSA sealing, adds bacteriophage monoclonal, hatches 2h; Add HRP-antiM13 antibody, hatch 1.5h for 37 DEG C; With TMB colour developing, addEnter isopyknic 2NHCl or 1NH₂SO₄Carry out cessation reaction, in enzyme mark hole, reactant liquor becomes yellow from blueness, ELIASA 450nmPlace's reading. In contrast, P/N > 2 is positive for random picking prophage storehouse locus coeruleus.

Result as shown in Figure 1, is that these 20 phage clones detect the affinity of LNCaP and PC3 respectively in Figure 1A, 1BAs a result, 21 and 22 as a control group, is the former storehouse phage clone of the picking immediately affinity testing result to LNCaP, PC3(note: in order to name conveniently, 20 bacteriophages are complied with by order from big to small by different from the power of LNCaP, PC3 affinityInferior arrangement). From Fig. 1, can be clear that very much the affinity of these 20 bacteriophage monoclonals of PCP1-20 to LNCaP and PC3All, higher than the bacteriophage monoclonal of random choose, wherein No. 1-16 clone is the strongest to the affinity of LNCaP and PC3, respectively lifePCP1-16 by name.

Embodiment 4 phage DNA sequencings

Positive colony PCP1-16 in amplification embodiment 3, and get above-mentioned positive colony bacteriophage amplification liquid and add 100 μ l15%PEG/NaCl (volume ratio of PEG and NaCl is 1: 4), mixes, and room temperature is placed 15min, and the centrifugal 15min of 8000rpm, gets precipitation;With 100 μ lTE (PH=7.0) dissolution precipitations, add the saturated phenol of 50 μ lTris, the 2min that turns upside down, then leave standstill 1min, more upper and lowerPutting upside down 2min mixes; The centrifugal 15min of 8000rpm, gets upper strata and adds 200 μ l2mol/L sodium acetates: absolute ethyl alcohol (1: 25) precipitationDNA, mixes rear standing 20min, and the centrifugal 15min of 8000rpm, removes supernatant, adds 50 μ l200ml/L ethanol and washes once, air-dry remnantsEthanol, with 30 μ lTE (PH=7.0) dissolvings, agarose gel electrophoresis qualification.

Carry sequencing primer with peptide storehouse and send to order-checking, the corresponding amino acid sequence of PCP1-16 contrast genetic codon table respectively asShown in SEQIDNO:1-16.

The monoclonal external qualification of embodiment 5 bacteriophage

Cell enzyme linked immunological adsorption method for the identification of 16 phage clones (PCP1-16) of embodiment 3 to prostateThe specific binding capacity of cancer cell, the blank bacteriophage of M13KE is with comparing. Thin by after phage clone and fixing, sealingBorn of the same parents (LNCaP/PC3/HepG2/A549/SW480/RWPE-1) educate after 2 hours altogether, with the anti-M13 phage antibody of HRP/ to combinationBacteriophage detect, after TMB colour developing, carry out reading with ELIASA.

In these 16 phage clones of ELISA detection display, PCP1-16 is to prostate gland cancer cell LNCaP and PC3 cloneAll performance has very strong binding ability, and for other tumour cells (colon cancer, liver cancer, lung cancer) and Normocellular adhesionWeak (seeing accompanying drawing 2). This result is directly consistent in conjunction with the result of experiment with bacteriophage, illustrates that PCP1-16 can identify and target knotClose in prostate gland cancer cell, simultaneously few to the combination of normal cell and other tumour cells.

Further, in competitive trials, the introducing of the SEQIDNO:1 polypeptide of low concentration can suppress 69%The combination of PCP1 bacteriophage and LNCaP, the contrast polypeptide of random sequence does not show this kind of inhibitory action. On the other hand, SEQIDNO:1 polypeptide can suppress 93% PCP1 bacteriophage by LNCaP internalization, the contrast polypeptide performance of the random sequence of same concentrationsUnrestraint effect. The competition inhibition of SEQIDNO:1 polypeptide shows the combination of PCP1 bacteriophage to cell and enters cellBehavior be the effect that derives from SEQIDNO:1 polypeptide, instead of the effect of its carrier phage particle. For PCP2-16The competitive trials of its corresponding small peptide of bacteriophage all has above-mentioned similar result of the test.

The SABC qualification of the present embodiment is this area universal method adopting, and reagent involved in test is with anti-The condition of answering is also that those skilled in the art are in common knowledge.

Claims

1. a small peptide, is characterized in that: can specific binding prostate gland cancer cell.

2. small peptide as claimed in claim 1, is characterized in that: it is PCP11, and its amino acid sequence is respectively as SEQIDNO:Shown in 11.

3. the application of the small peptide as shown in claim 1-2 in the preparation of preparation detection prostate gland cancer cell.

4. application as claimed in claim 3, is characterized in that: described prostatic cell is LNCaP and PC3.

5. the small peptide described in claim 1-2 any one is in the application of preparing in prostatic cancer diagnostic reagent kit.