CN105622723A

CN105622723A - Oligopeptide PCP3 specifically combined with prostatic cancer and application thereof

Info

Publication number: CN105622723A
Application number: CN201610107418.1A
Authority: CN
Inventors: 华国光
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-03-24
Filing date: 2014-03-24
Publication date: 2016-06-01
Also published as: CN105601717A; CN105541972A; CN105566455A; CN105601716A; CN105622724A; CN105541973A; CN103897037A; CN105541971A; CN105541974A; CN103897037B

Abstract

The invention relates to an oligopeptide PCP3 specifically combined with prostatic cancer and application thereof and provides a series of polypeptides which have targeting characteristics of prostate cancer and are obtained through screening with a phage display peptide library technology. The combined polypeptides are high in specificity and can be used for general investigation of prostate cancer high-risk groups, early clinical diagnosis of prostatic cancer and evaluation of the treatment effect.

Description

The short peptide PCP3 of specific binding prostate cancer and application thereof

Technical field

The invention belongs to protein and peptide technical field, be specifically related to a series of can the short 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 height, is only second to lung cancer, occupies the 2nd. 50 for many years, and endocrine therapy treatment 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's life and reach more than 1 year. Therefore explore novel effective prostate cancer therapy method, it is 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 of causing prostate cancer mortality ratio higher. Therefore, this area urgently needs to develop the associated protein that can be used for prostate cancer diagnosis. And polypeptide because of molecular weight little, penetration into tissue is good, non-immunogenicity and desirable part cell surface receptor being had higher affinity and becoming in targeted delivery research. But known natural receptor-part is to being very limited.

The development of phage display peptide library technology then provides strong instrument for finding and characterize new part and corresponding acceptor, is not only expected to realize clinical application in the targeted therapy of tumour, also contributes to explaining the theoretical investigation of disease mechanism. Display technique of bacteriophage 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 so that the polypeptide ligand of various target molecule is able to Rapid identification by screening in external or body. It is developed so far, applies this technology and successfully screen the target polypeptide obtained for multiple human tumors such as liver cancer, colorectal carcinoma, mammary cancer, but the relevant report for screening prostate cancer targeting part is then little.

Therefore, the present invention utilize phage display peptide library technology screen in tumor model of prostate cancer body obtain 16 can target to the short peptide of prostate cancer, and the target of this polypeptide has been identified to characteristic, to investigate it as target to part for transmitting the feasibility of medicine to tumor of prostate position.

Summary of the invention

It is an object of the invention to for the deficiencies in the prior art, it is provided that a series of utilize that phage display peptide library technology screening obtains there is the polypeptide of target to prostate cancer characteristic.

It is a further object to provide aforementioned polypeptides in the application preparing in prostatic cancer diagnostic reagent kit.

The object of the present invention is achieved by following technical proposals:

The method screening tumor cell specific Binding peptide or target peptide by phage display peptide library subtractive is: usually screen by two clones, Phage display random peptide library is first screened by not normal cell containing target antigen, in conjunction with supernatant again by containing the cell of target antigen, carry out next after filtering out Phage amplification and take turns screening, finally obtain specific binding peptides.

First, adopting 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 and takes turns in-vitro screening, by strengthening washing dynamics by wheel, obtains the phage clone of prostatic cancer specific. After above-mentioned screening, choose at random respectively at the 2nd taking turns, in three-wheel and fourth round the selection result and get extraction and the order-checking that mono-clonal carries out DNA. The peptide sequence that applying biological Bioinformatic tool Analysis and Screening obtains, enzyme-linked immunosorbent assay is for analyzing candidate's phage mono-clonal to the binding ability of cell simultaneously, thus identify 16, the phage clone with prostate cancer cell with stronger avidity, 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 polypeptide that the present invention's screening obtains 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, show 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 the popular feature of higher affinity, this polypeptide can be used for preparing prostatic cancer diagnostic reagent medicine box, this test kit diagnostic sensitivity height, specificity height, can be used for the evaluation of the early clinical diagnosis of the generaI investigation of prostate cancer high risk population and prostate cancer and result for the treatment of and patient's state of an illness turn return, the judgement of prognosis.

Accompanying drawing explanation

Fig. 1 is the avidity contrast histogram that ELISA detects fourth round screening phage clone and LNCaP and PC3;

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 be former storehouse random phage clone to the avidity of LNCaP, 22 be that former storehouse random phage clones the avidity to PC3.

Fig. 2 is that to the targeting ability qualification of different cell, (phage combines=TU to phage clone relatively_{Phage clone to be measured}/TU_{Blank phage})��

Wherein, Fig. 2 A be phage PCP1-6 respectively to the binding ability result figure of different cell, Fig. 2 B be phage PCP7-11 respectively to the binding ability result figure of different cell, Fig. 2 C is that phage PCP12-16 is respectively to the binding ability result figure of different cell.

Embodiment

The present invention is described further by following examples, but not as the 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 being 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, carries out screening experiment when cell grows to 80%-90% degrees of fusion.

Get above-mentioned LNCaP and PC3 cell, after cultivating with serum-free DMEM, add bovine serum albumin BSA and close, then add 20 �� l phage peptide libraries, after hatching 1.5h, topple over and remove in conjunction with phage, be inverted bat and get rid of removing residual solution. 4 times are rinsed with washings 0.2% (v/v) TBST damping fluid, add non-specific damping fluid 0.2M glycine-hydrochloric acid (pH2.2) 2ml, sucking-off elutriant, after adding 250 �� l5MTris-HCl (pH9.0) neutralizations, elutriant is gone in RWPE-1 cell, after hatching 3h, collect supernatant, it is the phage that first round screening obtains, being got by the phage obtained utilizes intestinal bacteria to determine the concentration of phage by volumetry on a small quantity, remaining phage-infect intestinal bacteria is increased, for the screening that next is taken turns.

2nd when taking turns screening, and in washings TBST, Tween-20 concentration is increased to 0.5%, is 40min with LNCaP and PC3 incubation time, and washing times is increased to 7 times, and all the other conditions, step are identical with the first round.

When three-wheel screens, 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.

When fourth round is screened, 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.

Measure the phage that fourth round screening obtains drip degree after, LB/IPTG/Xgal flat board is chosen at random and gets blue plaque, preparation 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 �� l dilute after phages and the early stage Escherichia coli bacteria liquid of 200 �� l logarithmic growths mix even, join and it is poured into rapidly the LB solid plate containing IPTG/Xgal after the LB top-agar of 45 DEG C of insulations, 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) droplet degree that namely extension rate obtains every 10 �� l phages.

The enrichment of the phage polypeptide of prostate cancer cell specific combination: as shown in table 1, after the positive phage clones being combined with LNCaP and PC3 takes turns screening through four, the phage rate of recovery improves 1000 times.

The rate of recovery of table 1 positive bacteriophage after four take turns screening

Screening number of times	Add phage number (pfu)	Reclaim phage 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 clone identification that prostatic cancer specific combines: in embodiment 1, phage peptide library is after continuous four take turns 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 �� 10⁴The density in/hole is inoculated in 96 orifice plates, puts CO₂After 20h cultivated by incubator, cell carrying out serum-free process 1h, cleans cell, then fix with paraformaldehyde, 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 1NH₂SO₄Carrying out termination reaction, in enzyme mark hole, reaction solution turns into yellow from blueness, microplate reader 450nm place reading. Choosing at random and get phage former storehouse locus coeruleus in contrast, P/N > 2 is positive.

Result is as shown in Figure 1, Figure 1A, 1B are these 20 phage clones are respectively to the avidity detected result of LNCaP and PC3,21 and 22 as a control group, it is choose the former storehouse phage clone got immediately to the avidity detected result (note: in order to name conveniently, be arranged in order by order from big to small by from the strong and weak different of LNCaP, PC3 avidity by 20 phages) of LNCaP, PC3. Can being 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 the above-mentioned positive colony Phage amplification liquid �� l15%PEG/NaCl (volume ratio of PEG and NaCl is 1: 4) that adds 100, mix even, the room temperature placement centrifugal 15min of 15min, 8000rpm, gets precipitation; With 100 �� lTE (PH=7.0) dissolution precipitations, the saturated phenol of �� lTris that adds 50, turn upside down 2min, then leaves standstill 1min, then the 2min that turns upside down mixes even; The centrifugal 15min of 8000rpm, get upper strata and add 200 �� l2mol/L sodium acetates: dehydrated alcohol (1: 25) precipitation DNA, 20min is left standstill after mixed even, the centrifugal 15min of 8000rpm, removing supernatant, �� l200ml/L ethanol is washed once to add 50, air-dry residual ethanol, dissolving with 30 �� lTE (PH=7.0), agarose gel electrophoresis is identified.

Carrying sequencing primer with peptide storehouse and send to order-checking, PCP1-16 compares the corresponding aminoacid sequence of genetic codon table respectively as shown in SEQIDNO:1-16.

The external qualification of embodiment 5 phage mono-clonal

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, TMB carries out reading by microplate reader after developing the color.

In these 16 phage clones of ELISA detection display, prostate cancer cell LNCaP and PC3 clone are all showed very strong binding ability by PCP1-16, and for other tumour cells (colorectal carcinoma, liver cancer, lung cancer) and the bonding force of normal cell weak (see accompanying drawing 2). The result of this result Binding experiment direct with phage is consistent, illustrate PCP1-16 can identify and target to being 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 comparison 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, and the comparison polypeptide of the stochastic sequence of same concentrations shows unrestraint effect. The Competitive assays effect of SEQIDNO:1 polypeptide shows that to the combination of cell and the behavior entering cell is the effect deriving from SEQIDNO:1 polypeptide to PCP1 phage, instead of the effect of its carrier phage particle. Competitive trials for its corresponding short 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

1. a short peptide, it is characterised in that: can specific binding prostate cancer cell.

2. short peptide as claimed in claim 1, it is characterised in that: it is PCP3, and its aminoacid sequence is respectively as shown in SEQIDNO:3.

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

4. apply as claimed in claim 3, it is characterised in that: described prostatic cell is LNCaP and PC3.

5. short peptide described in the arbitrary item of claim 1-2 is in the application prepared in prostatic cancer diagnostic reagent kit.