CN103788181B

CN103788181B - Lung carcinoma cell specific adhesion small peptide and application thereof

Info

Publication number: CN103788181B
Application number: CN201410066647.4A
Authority: CN
Inventors: 孙绪德; 高昌俊; 张莲花; 任鹏程; 吕苗苗; 张玉明; 孙美艳; 杨璐
Original assignee: Fourth Military Medical University FMMU
Current assignee: Fourth Military Medical University FMMU
Priority date: 2014-02-21
Filing date: 2014-02-21
Publication date: 2016-04-20
Anticipated expiration: 2034-02-21
Also published as: CN103788181A

Abstract

The invention provides a kind of can with the small peptide of lung carcinoma cell specific adhesion.Small peptide of the present invention is high to lung carcinoma cell binding specificity, can be used for the judgement of the early clinical diagnosis of lung cancer and the evaluation of result for the treatment of and prognosis.

Description

Lung carcinoma cell specific adhesion small peptide and application thereof

Technical field

The invention belongs to protein and peptide technical field, be specifically related to a kind of can with polypeptide of lung carcinoma cell specific adhesion and its preparation method and application.

Background technology

It is the fastest that lung cancer is that M & M increases, to one of population health and the maximum malignant tumour of life threat.The many countries of immediate and mid-term all report that the M & M of lung cancer all obviously increases, and male lung cancer M & M all accounts for first of all malignant tumours, and women's sickness rate accounts for second, and mortality ratio accounts for second.The cause of disease of lung cancer is still not exclusively clear and definite so far.Therefore, the early diagnosis of lung cancer is significant to adopting rational treatment means, extending the survival time of patient and reducing mortality ratio.

At present following several mode is mainly contained to the detection of lung cancer: x-ray inspection, bronchoscopy, cytolgical examination, exploratory thoracotomy, ECT check and mediastinoscopy.These detection methods otherwise detecting step loaded down with trivial details, or testing cost is high, or Detection accuracy is not high.Just because of this, China is relatively low for the examination rate of lung cancer, and this is also one of major reason causing lung cancer mortality higher.Therefore, this area can be used for the associated protein of pulmonary cancer diagnosis in the urgent need to exploitation.In addition, in order to effectively suppress the development of lung carcinoma cell, this area can be used in the urgent need to exploitation the medicine suppressing lung cancer growth.Wherein, the introducing of targeted drug delivery theory because minimizing the untoward reaction to normal organ while maximizing the anticancer effectiveness of medicine, and the tumor therapeuticing method becoming most valuable and wish.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.

In recent ten years, antibody chip technology becomes the early diagnosis of tumour, the selection systems, pharmacological agent etc. of tumor markers provides new platform, especially uses the most extensive in the screening and identification of tumor markers detects.

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, prostate cancer, mammary cancer, but then little for the relevant report of screening lung cancer target part.

Therefore, the present invention utilize phage display peptide library technology to screen in lung cancer tumor model body to obtain one can be lung cancer-targeted 12 peptides, and in the body of this polypeptide and targeting characteristic identify, to investigate it as target part for transmitting the feasibility of medicine to lung tumors position.

Summary of the invention

The object of the invention is for the deficiencies in the prior art, a polypeptide with lung cancer-targeted characteristic utilizing phage display peptide library technology screening to obtain is provided.

Another object of the present invention is to provide the preparation method of aforementioned polypeptides.

Another object of the present invention is to provide aforementioned polypeptides and is preparing the application in pulmonary cancer diagnosis test 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 lung carcinoma cell as target cell, normal cell is adherent cell, to the Ph.D.-7 of NEWENGLANDBiolabs ^tMphageDisplayPeptideLibraryKit carries out three-wheel in-vitro screening, by strengthening washing dynamics by wheel, obtains the specific phage clone of lung cancer.After above-mentioned screening, take turns respectively at second extraction and the order-checking of carrying out DNA with random picking mono-clonal in third round the selection result.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 the phage clone with lung cancer with stronger avidity, this phage clone is sent to order-checking, and called after LCP3, sequencing result confirms that the aminoacid sequence of LCP3 is as shown in SEQIDNO:2.

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

(1) aminoacid sequence of this polypeptide is: ARLLHWSTKINR, on all four homologous protein structure is not had with the known in US National Biotechnology Information center (NCBI) GenBankDNA sequence library and Swiss-Prot albumen database and albumen, and domestic and foreign literature is also not reported, it is a brand-new polypeptide as can be seen here;

(2) this polypeptide can identify and specific binding lung cancer cell line A549, H1299 effectively, and weak to the binding ability of normal clone HUVEC and other tumour cells HepG2, PC-3, BS517, show for the special identification of lung cancer and binding ability;

(3) to have molecular weight little for this polypeptide, and penetration into tissue is good, non-immunogenicity and cell surface receptor is had to the popular feature of higher affinity, in conjunction with the target ability that this polypeptide is good to lung cancer vivo and vitro, is a kind of targeted delivery systems of lung cancer.

(4) this polypeptide can be used for preparing pulmonary cancer diagnosis reagent kit, this test kit diagnostic sensitivity is high, specificity is high, can be used for the judgement of the evaluation of the generaI investigation of High Risk of Lung Cancer crowd and the early clinical diagnosis of lung cancer and result for the treatment of and patient's Prognosis, prognosis, have broad application prospects.

Accompanying drawing explanation

Fig. 1 is that the avidity of ELISA detection third round screening phage clone and A549 and H1299 contrasts histogram;

Wherein, Figure 1A is that these 10 phage clones of LCP1-10 are respectively to the avidity of A549, Figure 1B is that these 10 phage mono-clonals of LCP1-10 are respectively to the avidity of H1299,11 is the avidity of former storehouse random phage clone to A549, and 12 is the avidity of former storehouse random phage clone to H1299.

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}).

Embodiment

The present invention is described further by following examples, but not as restriction of the present invention.

The screening of embodiment 1 specific combined polypeptide for lung cancer

The present embodiment adopts the polypeptide of the random 12 peptide storehouse subtractive screenings of phage display and lung carcinoma cell specific binding, and its concrete steps are as follows:

After trysinization human lung carcinoma cell (A549) and people's lung normal cell (MRC-5), adjustment cell density, is inoculated in the culture dish wrapped in advance by poly-lysine, when cell grows to 85%-90% degrees of fusion, carries out screening experiment.

Get above-mentioned A549 cell, after cultivating with serum-free DMEM, add bovine serum albumin BSA and close, then add 10 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 0.1% (v/v) TBST wash buffer 3 times, add non-specific damping fluid 0.2M glycine-HCI (pH2.1) 1ml, sucking-off elutriant, after adding 150 μ l1MTris-HCl (pH9.1) neutralizations, elutriant is gone in MRC-5 cell, after hatching 1h, collect supernatant, being the first round screens the phage obtained, being taken a morsel by the phage obtained utilizes intestinal bacteria ER2738 to determine the concentration of phage by volumetry, remaining phage-infect intestinal bacteria ER2738 increases, for the screening of next round.

Second when taking turns screening, and in washings TBST, Tween-20 concentration is increased to 0.3%, is reduced to 45min with A549 incubation time, and washing times is increased to 6 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 0.5%, is reduced to 30min with A549 incubation time, and washing times is increased to 8 times, and all the other conditions, step are identical with the first round.

After measuring the titre of the phage that third round screening obtains, the blue plaque of random picking on LB/IPTG/Xgal flat board, prepare phage mono-clonal for the identification of.

The phagocytosis scale of construction of embodiment 2 after screening and amplification

By in embodiment 1, after the phage that often wheel screening obtains carries out 100 doubling dilutions with LB substratum, get 10 μ l dilute after phage and the early stage intestinal bacteria ER2738 bacterium 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, spend the night, 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 lung carcinoma cell specific combination: as shown in table 1, the positive phage clones be combined with A549 phage rate of recovery after three-wheel screens improves 100 times.

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

Embodiment 3ELISA identifies phage polypeptide

The phage polypeptide positive clone identification of lung cancer specific binding: in embodiment 1, phage peptide library is after continuous three-wheel subtractive screening, random choose 12 phage clones, utilize the ordinary method ELISA method preliminary evaluation phage clone of this area to the avidity of A549, H1299.

By A549, H1299 by 1 × 10 ⁵the density in/hole is inoculated in 96 orifice plates, puts CO ₂incubator carries out serum-free process 1h to cell after cultivating 24h, cleaning cell, then fixes with paraformaldehyde, and PBS slightly washes, TritonX-100 process, and PBS-BSA closes, and adds phage mono-clonal, hatches 2h; Add HRP-antiM13 antibody, hatch Ih for 37 DEG C; With TMB colour developing (every hole 50 μ lTMB, colour developing 10-15 minute), add isopyknic 1NHCl or 2NH 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, in Figure 1A, 1B be these 10 phage clones of LCP1-10 respectively to the avidity detected result of A549, H1299,11 and 12 as a control group, is that the former storehouse phage clone of picking is immediately to the avidity detected result of A549, H1299.Can be clear that very much from Fig. 1, No. 3 clone is the strongest to the avidity of A549, H1299, called after LCP3.

Embodiment 4 phage DNA sequencing

Amplification embodiment 3 positives clone LCP3, and get above-mentioned positive colony Phage amplification liquid and add 200 μ l20%PEG/NaCl (volume ratio of PEG and NaCl is 1: 4), mix, the centrifugal 10min of room temperature placement 15min, 10000rpm, gets precipitation; With 100 μ lTE (PH=8.0) dissolution precipitations, add the saturated phenol of 100 μ lTris, turn upside down 1min, then leave standstill 1min, the more about 1min that turns upside down mixes; The centrifugal 5min of 10000rpm, get upper strata and add 300 μ l3mol/L sodium acetates: dehydrated alcohol (1: 25) precipitation DNA, about 30min is left standstill after mixing, the centrifugal 10min of 10000rpm, remove supernatant, add 100 μ l700ml/L ethanol and wash once, air-dry residual ethanol, dissolve with 40 μ lTE (PH=8.0), agarose gel electrophoresis is identified.

Carry sequencing primer with peptide storehouse and send to order-checking, sequencing primer is: 5 '-CCCTCATAGTTAGCGTAACG-3 ', insert base sequence apart from object fragment as shown in SEQIDNO:1 at a distance of 96bp, LCP3, the corresponding aminoacid sequence of contrast genetic codon table is respectively as shown in SEQIDNO:2.

The bioinformatic analysis of specific combined polypeptide for lung cancer sequence: sequencing result finds homologous sequence at NCBIGenBankDNA sequence library and Swiss-Prot albumen database by BLAST.Find that known in above-mentioned 12 peptide sequences and NCBIGeneBankDNA sequence library and Swiss-Prot Protein Data Bank and albumen do not have on all four homologous protein structure, and domestic and foreign literature is not reported, prove that the present invention screens the part of new lung cancer surface associated antigen thus.

The monoclonal targeting qualification of embodiment 5 phage

Cell ELISA method is for the identification of the phage clone LCP3 of embodiment 3 to the specific binding capacity of lung carcinoma cell, and the blank phage of M13KE is with comparing.By phage clone with fixing, close after cell (A549/H1299/MRC-5/HepG2/PC-3/BS517/HUVEC) educate 2 hours altogether after, with the anti-M13 phage antibody of HRP/, the phage combined is detected, after TMB colour developing, carry out reading by microplate reader.

ELISA detection display is in phage clone, LCP3 all shows stronger binding ability to lung cell A549 and H1299 clone, and for other tumour cells (liver cancer, prostate cancer, mammary cancer) and Normocellular bonding force weak (see accompanying drawing 2).This result is consistent with the result of the direct Binding experiment of phage, LCP3 identifiable design is described and target is incorporated into lung carcinoma cell, simultaneously very weak to the bonding force of normal cell and other tumour cells.Simultaneously as can be seen from the result of accompanying drawing 2, relative to lung cancer H1299 clone, the binding ability of LCP3 to lung cancer cell line A549 is stronger.

In addition, in competitive assay, the introducing of lower concentration ARLLHWSTKINR polypeptide can suppress the LCP3 of 71% to combine, and contrast polypeptide does not then show this kind of restraining effect.On the other hand, ARLLHWSTKINR polypeptide can suppress the LCP3 of 95% by A549 internalization, the control peptide performance unrestraint effect of same concentrations.The Competitive assays effect of ARLLHWSTKINR polypeptide shows that LCP3 is to the combination of cell and the behavior entering cell is the effect deriving from ARLLHWSTKINR polypeptide, instead of the effect of its carrier phage particle.

Claims

1. a lung carcinoma cell specific adhesion small peptide, is characterized in that: its aminoacid sequence is as shown in SEQIDNO:2.

2. polypeptide according to claim 1 is in the purposes of the preparation of preparation detection of lung cancer cell, and described lung carcinoma cell is A549, H1299.