CN105646663A

CN105646663A - Short-chain polypeptides PCP4 capable of being specifically bound to prostate cancer cells and application of short-chain polypeptides PCP4

Info

Publication number: CN105646663A
Application number: CN201610110770.0A
Authority: CN
Inventors: 华国光
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-03-24
Filing date: 2014-03-24
Publication date: 2016-06-08

Abstract

The invention relates to short-chain polypeptides PCP4 capable of being specifically bound to prostate cancer cells and an application of the short-chain polypeptides PCP4 and provides a series of polypeptides obtained through screening with a phage display peptide library technology and having a prostate cancer targeting characteristic. The binding polypeptides have high specificity and can be applied to general survey of high risk groups of the prostate cancer, early clinical diagnosis of the prostate cancer and evaluation of a treatment effect.

Description

The small peptide PCP4 of specific binding carcinoma of prostate and application thereof

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 carcinoma of prostate.

Background technology

Carcinoma of prostate is one of malignant tumor of serious threat middle-aging male health, and sickness rate rises year by year, and mortality rate is high, is only second to pulmonary carcinoma, occupies second. 50 for many years, and androgen deprivation therapy is always up the standard treatment of carcinoma of prostate, but, Most patients eventually develops into Hormone refractory carcinoma of prostate (hormonerefractoryprostatecancer, HRPC). For HRPC, there is no effective therapy at present, and existing treatment means includes radiotherapy, chemotherapy etc. and all can not extend patient vitals and reach more than 1 year. Therefore explore novel effective prostate cancer therapy method, be always up one of clinical encountered important topic of Urology Surgery.

Current China is relatively low for the examination rate of carcinoma of prostate, and this is also one of major reason of causing carcinoma of prostate mortality rate higher. Therefore, this area can be used for the associated protein of prostate cancer diagnosis in the urgent need to exploitation. And polypeptide is little because of molecular weight, 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 amount of.

The development of phage display peptide library technology then provides strong instrument for finding and characterize new part and corresponding receptor, is not only expected to realize clinical practice in the targeted therapy of tumor, it helps explain the theoretical research 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 building bridge between the DNA and capsid protein of phage so that the polypeptide ligand of various target molecules can be able to Rapid identification by external or internal screening. It is developed so far, applies this technology and successfully screen the target polypeptide obtained for multiple human tumors such as hepatocarcinoma, colon cancer, breast carcinoma, 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 the small peptide of targeting carcinoma of prostate, and the targeting characteristic of this polypeptide has been identified, to investigate it as targeting ligand for transmitting the medicine feasibility 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 polypeptide with targeting carcinoma of prostate characteristic utilizing phage display peptide library technology screening to obtain.

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

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

The method screening tumor cell specific Binding peptide or target peptide by subtracting property of phage display peptide library is: screen typically by two cell lines, Phage display random peptide library first passes through the normal cell without target antigen screen, unconjugated supernatant is again through the cell containing target antigen, carry out next round screening after filtering out Phage amplification, finally give specific binding peptides.

First, adopting above-mentioned phage display peptide library subtracting property triage techniques, using carcinoma of prostate LNCaP and PC3 cell as target cell, normal cell is adherent cell, the Ph.D.-7 to NEWENGLANDBiolabs^TMPhageDisplayPeptideLibraryKit carries out four-wheel in-vitro screening, by strengthening washing dynamics by wheel, it is thus achieved that 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 monoclonal carries out extraction and the order-checking of DNA. The peptide sequence that applying biological Bioinformatic tool Analysis and Screening obtains, enzyme-linked immunosorbent assay is for analyzing candidate's phage monoclonal binding ability to cell simultaneously, with prostate gland cancer cell, there is phage 16 clone of stronger affinity thus identifying, these 16 clones are sent to order-checking, and it being respectively designated as PCP1-16, sequencing result confirms that the aminoacid sequence of PCP1-16 is respectively as shown in SEQIDNO:1-16.

The present invention polypeptide that obtains of screening is identified has following characteristic and advantage:

Polypeptide can efficiently identify and specific binding prostate cancer cell line LNCaP and PC3, and to normal prostate cell line RWPE-1, and the binding ability of other tumor 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 is high, specificity is high, can be used for the judgement of the evaluation of the early clinical diagnosis of the generaI investigation of carcinoma of prostate high-risk group and carcinoma of prostate and therapeutic effect and patient's Prognosis, prognosis.

Accompanying drawing explanation

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

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

Fig. 2 is that the phage clone targeting ability to different cells identifies that (phage combines=TU relatively_Treat _{Survey phage clone}/TU_{Blank phage})��

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

Detailed description of the invention

The present invention is described further by following example, but not as the 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 the polypeptide being combined with prostate gland cancer cell specificity, and it specifically comprises the following steps that

After trypsinization Human Prostate Cancer Cells LNCaP and PC3, adjust cell density, be inoculated in and be coated in the culture dish of poly-D-lysine in advance, when cell length to 80%-90% degrees of fusion, carry out screening experiment.

Take above-mentioned LNCaP and PC3 cell, after cultivating with serum-free DMEM, add bovine serum albumin BSA and close, add 20 �� l phage peptide libraries, after hatching 1.5h, topple over removing and be not associated with phage, be inverted bat and get rid of removing residual solution. With cleaning mixture 0.2% (v/v) TBST wash buffer 4 times, add non-specific buffer 0.2M glycine-HCI (pH2.2) 2ml, sucking-off eluent, after adding 250 �� l5MTris-HCl (pH9.0) neutralizations, eluent is gone in RWPE-1 cell, after hatching 3h, collect supernatant, it is the phage that first round screening obtains, the phage obtained being taken utilizes escherichia coli to determine the concentration of phage by titrimetry on a small quantity, remaining phage-infect escherichia coli expands, for the screening of next round.

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

During third round screening, in cleaning mixture TBST, Tween-20 concentration is increased to 0.8%, reduces to 25min with LNCaP and PC3 incubation time, and washing times increases to 10 times, and all the other conditions, step are identical with the first round.

During fourth round screening, in cleaning mixture TBST, Tween-20 concentration is increased to 1.0%, reduces to 20min with LNCaP and PC3 incubation time, and washing times increases 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, random picking blueness plaque on LB/IPTG/Xgal flat board, preparation phage monoclonal is used for identifying.

The amplification of embodiment 2 phage

By in embodiment 1, after the phage LB culture medium that often wheel screening obtains carries out 100 doubling dilutions, after taking 20 �� l dilutions, phage mixes with the Escherichia coli bacteria liquid of 200 �� l logarithmic growth early stages, join and be 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 speckle number that on counting flat board, phage can grow, is then multiplied by extension rate with this number and namely obtains plaque forming unit (pfu) titre of every 10 �� l phagies.

The enrichment of the phage polypeptide of prostate gland cancer cell specific bond: as shown in table 1, the positive phage clones being combined with LNCaP and PC3 phage response rate after four-wheel screens improves 1000 times.

Table 1 is the response rate of positive bacteriophage after four-wheel screens

Screening number of times	Add phage number (pfu)	Reclaim phage number (pfu)	The response rate
				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 subtracting property of four-wheel is screened, 20 phage clones of random choose, utilize the conventional method ELISA method Preliminary Identification phage clone of this area affinity to LNCaP and PC3.

By LNCaP and PC3 by 1 �� 10⁴The density in/hole is inoculated in 96 orifice plates, puts CO₂After incubator cultivates 20h, cell carrying out serum-free and processes 1h, clean cell, then fix with paraformaldehyde, PBS washes once, and after TritonX-100 processes, PBS-BSA closes, and adds phage monoclonal, hatches 2h; Add HRP-antiM13 antibody, hatch 1.5h for 37 DEG C; Develop the color with TMB, add isopyknic 2NHCl or 1NH₂SO₄Terminating reaction, in enzyme mark hole, reactant liquor becomes yellow, microplate reader 450nm place reading from blueness.Random picking prophage storehouse locus coeruleus is as comparison, and P/N > 2 is positive.

Result is as shown in Figure 1, Figure 1A, 1B are these 20 phage clones affinity testing results to LNCaP and PC3 respectively, 21 and 22 as a control group, it it is the former storehouse phage clone affinity testing result (note: in order to name conveniently, 20 phagies are arranged in order by order from big to small by different from the power of LNCaP, PC3 affinity) to LNCaP, PC3 of picking immediately. It is clear that the affinity of LNCaP and PC3 is above the phage monoclonal of random choose by these 20 phage monoclonals of PCP1-20 from Fig. 1, wherein No. 1-16 clone is the strongest to the affinity of LNCaP and PC3, is respectively designated as PCP1-16.

Embodiment 4 phage DNA sequencing

Amplification embodiment 3 positives clone PCP1-16, and take above-mentioned positive colony Phage amplification liquid and add 100 �� l15%PEG/NaCl (volume ratio of PEG and NaCl is 1: 4), mixing, room temperature is placed 15min, 8000rpm and is centrifuged 15min, takes precipitation; With 100 �� lTE (PH=7.0) dissolution precipitations, adding the 50 saturated phenol of �� lTris, turn upside down 2min, then stands 1min, then 2min mixing of turning upside down; 8000rpm is centrifuged 15min, take upper strata and add 200 �� l2mol/L sodium acetates: dehydrated alcohol (1: 25) precipitation DNA, 20min is stood after mixing, 8000rpm is centrifuged 15min, remove supernatant, add 50 �� l200ml/L ethanol and wash once, 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 monoclonal external qualification of embodiment 5 phage

Cell ELISA method is used for 16 phage clones (PCP1-16) identifying embodiment 3 specific binding capacity to prostate gland cancer cell, and M13KE blank phage 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.

ELISA detection shows in these 16 phage clones, prostate gland cancer cell LNCaP and PC3 cell line are all presented with very strong binding ability by PCP1-16, and for other tumor cells (colon cancer, hepatocarcinoma, pulmonary carcinoma) and Normocellular adhesion weak (see accompanying drawing 2). This result and phage are consistent directly in conjunction with the result of experiment, illustrate that PCP1-16 can recognize that and targeting is incorporated into prostate gland cancer cell, simultaneously to normal cell and other tumor cells in conjunction with few.

Further, in competitive trials, the introducing of the SEQIDNO:1 polypeptide of low concentration can suppress the PCP1 phage of 69% and the combination of LNCaP, and the comparison polypeptide of random sequence does not then show this kind of inhibitory action. On the other hand, SEQIDNO:1 polypeptide can suppress the PCP1 phage of 93% by LNCaP internalization, the comparison polypeptides exhibit unrestraint effect of the random sequence of same concentrations. The Competitive assays effect of SEQIDNO:1 polypeptide shows that the knot of cell is combined into the behavior of cell and is derived from the effect of SEQIDNO:1 polypeptide rather than the effect of its carrier phage granule by PCP1 phage. Competitive trials for its corresponding small peptide of PCP2-16 phage is respectively provided with above-mentioned similar result of the test.

The SABC of the present embodiment identifies the method generally in the art being to adopt, and reagent involved in test and reaction condition are also that those skilled in the art are in common knowledge.

Claims

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

2. small peptide as claimed in claim 1, it is characterised in that: it is PCP4, and its aminoacid sequence is respectively as shown in SEQIDNO:4.

3. the application in the preparation of preparation detection prostate gland cancer cell of the small 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. the application in preparing prostatic cancer diagnostic reagent kit of the small peptide described in any one of claim 1-2.