CN106589066A

CN106589066A - Human ovarian carcinoma cell specifically binding polypeptide and application thereof

Info

Publication number: CN106589066A
Application number: CN201610966698.1A
Authority: CN
Inventors: 侯颖春; 何慧敏; 肖丽; 马妮; 程思楠; 黎金媚; 杨倩
Original assignee: Shaanxi Normal University
Current assignee: Shaanxi Normal University
Priority date: 2016-11-03
Filing date: 2016-11-03
Publication date: 2017-04-26
Anticipated expiration: 2036-11-03
Also published as: CN106589066B

Abstract

The invention relates to human ovarian carcinoma cell SKOV3 specifically binding polypeptide and an application thereof, wherein the amino acid sequence of the polypeptide is ASPLAPWSSVGP and the application is specific combination with human ovarian carcinoma cells by means of the human ovarian carcinoma cell SKOV3 specifically binding polypeptide. The ovarian carcinoma cell targeted polypeptide, which is obtained through screening and initial verification, has significant specificity of combination with the human ovarian carcinoma cells and can be used for researching a target diagnosis reagent for ovarian carcinoma in early stage and delivery of high-effect and low-toxic target medicines.

Description

The polypeptide of Proliferation of Human Ovarian Cell specific binding and its application

Technical field

The present invention relates to biological technical field, is screened to Proliferation of Human Ovarian Cell (SKOV3 with the peptide storehouse abatement screening method for improveing Cell) polypeptide (sequence) that specifically binds, there is good binding specificity and sensitivity to Proliferation of Human Ovarian Cell.

Background technology

Ovarian cancer (Ovarian cancer) is one of common gynecological tumor, and its sickness rate is only second to cervical cancer and son Carcinoma of corpus uteri, occupy the 3rd.Ovarian cancer is the most fatal in gynecologic malignant tumor, and feature is latent disease development, develop rapidly, patient Clinical cure rate is low.At present, the Therapeutic Method of ovarian cancer includes operative therapy, chemotherapy, X-ray therapy and immunotherapy Deng, but because of its incidence of occult, early ovarian cancer non-evident sympton and sign, easily cause and fail to pinpoint a disease in diagnosis or mistaken diagnosis, when to be found in Late period, so optimal opportunity of operation is lost, more using conservative treatment.However, radiotherapy, chemotherapeutic toxicity are greatly, be also easy to produce dosage according to Bad property and Drug Resistance etc., effect is undesirable, and the five year survival rate of patient is still very low.Therefore, find a kind of sensitivity high And the early screening diagnostic method of high specificity, so as to the mortality rate for reducing ovarian cancer be also the key issue that faces at present it One.

In recent years, the molecular imaging for developing rapidly brings new wishing with diagnosis, treatment that targeted therapy is ovarian cancer Hope, wherein, filter out that ovarian cancer cell surface specific is strong, the targeted molecular that sensitivity is high becomes the task of top priority.

Phage peptide library is screened for ovarian cancer cell targeted molecular, the early diagnosiss of ovarian cancer and targeted therapy are provided New approach, key therein is the acquisition of the polypeptide fragment for having special target binding activity to cancerous cell, cancerous tissue.

The content of the invention

It is an object of the present invention to obtain the polypeptide sequence to ovarian cancer cell specific binding by 12 peptide library selection of phage Row, there is provided identify the sequence and ovarian cancer cell and the experimental result of the specificity and sensitivity of tissue combination, to prove that this is more Peptide is coupled the application valency for carrying out the aspects such as ovarian cancer targeted therapy in the early molecule imaging diagnosises and other medicines of ovarian cancer Value.

In order to realize above-mentioned task, the present invention takes following technical solution：

The polypeptide that ovarian cancer SKOV3 cell-specific is combined, its aminoacid sequence is：ASPLAPWSSVGP.The polypeptide energy Ovarian cancer cell specifically bound enough, and nonrecognition normal cell/tissue and other tumor cell/tissues.

With ovarian cancer SKOV3 cell as target cell, with 293 cell line of Human Embryonic Kidney HEK as non-specific adsorption cell, to change The good peptide storehouse method of residues carries out 5 wheel screenings to 12 peptide storehouse of phage random, and 60 phage clones of random picking are reflected with ELISA Determine positive colony.Positive colony is sequenced, acquisition possesses the positive colony of consensus sequence (Consensus sequence) Group 9, it is representative that each group takes a clone, identifies its specificity and sensitivity with cellular immunofluorescence method to them, it is determined that most Good consensus sequence.Synthesize the peptide probes of FITC labellings again with this optimal consensus sequence, the probe is carried out to ovarian cancer cell/group Knit, normal cell/tissue, other cancerous cells/tissues are detected, determine its specificity and sensitivity.

The equal prompting sequence ASPLAPWSSVGP of each experimental result has good specificity, quick to ovarian cancer cell/tissue Perception.As polypeptide molecular weight is little, immunogenicity is low, affinity is high, penetration into tissue is strong, easily modification and the features such as synthesize, have Prestige makes which be coupled with appropriate fluorescent marker, nano-particle, liposome or antitumor drug etc., so as to be used for the early stage of tumor Diagnostic imaging, targeted therapy, have important value for the current Diagnosis of Ovarian Cancer of solution faces bottleneck problem.

The present invention with ovarian cancer SKOV3 cell as target cell, the abatement screening method improved with us by 12 peptide storehouse of phage Carried out 5 wheel abatement screening, finally obtain 9 common polypeptide sequences, respectively AWPASFLTQKAL, ASPLAPWSSVGP, QPTTDNRDIRSK、KLHDWADSFSLI、YDHHGQSVMPRA、KSGNIWCPCPNF、WSNALTVTSTYG、 ELIPSGSQTYLS、NIAFTTTQHGNR.Find ASPLAPWSSVGP therein for ovum by a series of experiment of identification and analysis The targeting of nest cancer is most strong, has pointed out its value in terms of being used for ovarian cancer diagnosis, targeted therapy.

In ovarian cancer context of detection, ovarian cancer can be specifically bound using isotope or fluorescently-labeled polypeptide thin Born of the same parents/tissue, it is adaptable to tumor imaging and molecular image diagnosis, for ovarian cancer early detection, cancer lesion localization and therapeutic evaluation It is all significant；In terms of ovarian cancer targeted therapy, be expected to using its specificity it is high, molecular weight is little, penetration power is strong, affine The high characteristic of power to be coupled with chemotherapeutics, reaches the purpose of targeted delivery administration, can greatly reduce the non-specific of chemotherapeutics Property and toxic and side effects.

Description of the drawings

Fig. 1 is the syntheti c route figure of polypeptide of the present invention；

N-terminal sequences of the Fig. 2 for random dodecapeptides pIII fusion protein；

Fig. 3 is special purpose system sublist；

Fig. 4 is the ELISA qualification results of phage clone and ovarian cancer cell SKOV3 affinity；

Fig. 5 is the identified by immunofluorescence of the representative positive phage clones with cellular affinity of 9 consensus sequences, wherein A：R5+SKOV3；B：R6+SKOV3；C：R10+SKOV3；D：R12+SKOV3；E：R18+SKOV3；F：R20+SKOV3；G：R33+ SKOV3；H：R46+SKOV3；I：R47+SKOV3；J：URP+SKOV3；K：R5+HEK293；L：R6+HEK293；M：R10+ HEK293；N：R12+HEK293；O:R18+HEK293；P:R20+HEK293；Q:R33+HEK293；R:R46+HEK293；S:R47 +HEK293；T：URP+HEK293.DAPI contaminates nucleus；Phage clone is reflected with the ELISA of ovarian cancer cell SKOV3 affinity Determine result；

Fig. 6 is clone S7 (ASPLAPWSSVGP) and the identification of target cell affinity, wherein A：SKOV3+S7；B：Hek293+ S7；C：SKOV3+URP；D：Hek293+URP；E：SKOV3+PBS；F：Hek293+PBS；DAPI contaminates nucleus；

Binding specificity identifications of the Fig. 7 for synthesis polypeptide (ASPLAPWSSVGP) and target cell, wherein A：SKOV3+ is positive Polypeptide；B：SKOV3+ feminine gender polypeptides；C：Hek293+ positive polypeptides；D：Hek293+ feminine gender polypeptides；DAPI contaminates nucleus；

Fig. 8 is the binding specificity identification of synthesis polypeptide (ASPLAPWSSVGP) and ovarian cancer different tissues, wherein, people's ovum The tissue line of nest cancerous tissue chip is as shown in Fig. 8-A；The binding specificity of synthesis polypeptide probe and ovarian cancer different tissues is such as Shown in Fig. 8-B；The binding specificity of control polypeptide probe and ovarian cancer different tissues is as shown in Fig. 8-C.

Below in conjunction with accompanying drawing, the invention will be further described.

Specific embodiment

The present invention using improvement peptide storehouse abatement screening method screening can specifically sensitivity be incorporated into Proliferation of Human Ovarian Cell/group The peptide sequence knitted, and high degree of specificity that the sequence pair Proliferation of Human Ovarian Cell/tissue combines and quick is demonstrated with related experiment Perception.

The present invention is with human ovarian cancer SKOV3 cell as target cell, thin as non-specific adsorption with 293 cell of Human Embryonic Kidney HEK Born of the same parents, carry out the abatement screening of 5 wheels, find and have specific binding affinity with ovarian cancer cell/tissue to 12 peptide storehouse of phage random Phage display peptide sequence, is that further research and development ovarian cancer early stage targeting diagnosis reagent and high-efficiency low-toxicity target therapeutic agent lay the foundation, Particular technique route is as shown in Figure 1.

First, materials and methods

1.1 major experimental materials

1.1.1 cell strain human oophoroma cell line SKOV3, is purchased from U.S. ATCC (Maryland, USA).HEKC Strain HEK293, is that this laboratory is preserved.

1.1.2 phage random dodecapeptide storehouse 1.5 × 10¹³pfu/ml.It is purchased from U.S. NEB (Boston, USA)；Storehouse reagent Box includes Host Strains E.coli ER2738.

1.2 experimental technique

1.2.1 cell culture

With 1640 complete mediums of RPMI, 37 DEG C, saturated humidity, 5%CO₂Incubator.

1.2.2 the preparation of Host Strains E.coli ER2738

E.coli ER2738 special strain therefores, use LB-Tet culture medium.

1.2.3 phage random dodecapeptide storehouse abatement screening

Cloned with excluding normal cell targeting using HEKC HEK293 as negative adsorption cell preadsorption, then with people Ovarian cancer cell SKOV3 carries out washing in a pan sieve with the abatement screening method for improveing as target cell, repeats 5 and takes turns.The abatement screening method is significantly Simplify experiment difficulty and improve screening efficiency.

With ELISA method identification and the affinity of target cell SKOV3 after selected phage clone amplification.

1.2.4 the measure of positive phage clones peptide sequence

Phage positive colony, unrelated clone are sequenced, according to genetic code sub-table (referring to Fig. 2, Fig. 3), will Peptide sequence is translated into aminoacid sequence and obtains Consensus sequences.

1.2.5Consensus the binding specificity identification of sequence

Specificity and sensitivity are combined by regular growth immunofluorescence with the representative clone of Consensus sequences The identification of property.

1.2.6 statistical analysis

Data analysiss are carried out using 16.0 data processing softwares of SPSS, data result is usedRepresent, P<0.01 represents Difference is extremely notable, P<0.05 represents significant difference, P>0.05 explanation difference is not notable, not statistically significant, multiple comparisons between group Processed using Duncan inspections.

2nd, experimental result

The ELISA identifications of 2.1 positive colonies and sequencing

Positive phage clones 38 are obtained, wherein clone R20 and SKOV3 Cell binding specificity highests, as a result such as Fig. 4 It is shown.Positive phage clones have 9 consensus sequences, are respectively designated as OSP1-OSP9：

(1) OSP1, peptide sequence be AWPASFLTQKAL, comprising clone have R1, R10, R17, R26, R27, R32, R40, R57；(2) OSP2, peptide sequence be ASPLAPWSSVGP, comprising clone have R3, R13, R20, R25, R45；(3) OSP3, peptide sequence is QPTTDNRDIRSK, comprising clone have R4, R6, R12, R15, R37；(4) OSP4, peptide sequence are KLHDWADSFSLI, comprising Clone has R5；(5) OSP5, peptide sequence be YDHHGQSVMPRA, comprising clone have R8, R16, R18, R36, R39, R48；(6) OSP6, peptide sequence be KSGNIWCPCPNF, comprising clone have R12；(7) OSP7, peptide sequence be WSNALTVTSTYG, comprising clone There are R28, R30, R31, R33, R49, R60；(8) OSP8, peptide sequence be ELIPSGSQTYLS, comprising clone have R34, R35, R44, R47；(9) OSP9, peptide sequence be NIAFTTTQHGNR, comprising clone have R46, R58.The peptide sequence of two negative clones is： HMNTYTQTRSPP and NWHNSSAQVYQG.The peptide sequence of unrelated clone is：LTSDYMASSSRS.

The binding specificity of 2.2 positive phage clones

As a result, as shown in Fig. 5-Fig. 6, positive phage clones are combined SKOV3 cells and are not combined with HEK293 cells, R20 (ASPLAPWSSVGP) is cloned as optimal, illustrate that the positive polypeptides have good targeting.

The specificity of 2.3 synthesis FITC labeling polypeptide ASPLAPWSSVGP and sensitivity identification

As a result as shown in Figs. 7-8.Polypeptide A SPLAPWSSVGP specific bond in SKOV3 cell surfaces not with HEK293 With reference to.

Nucleotides sequence list e-file

<110>Shaanxi Normal University

<120>The polypeptide of Proliferation of Human Ovarian Cell specific binding and its application

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KLHDWADSFSLI

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YDHHGQSVMPRA

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KSGNIWCPCPNF

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WSNALTVTSTYG

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Claims

1. the polypeptide of Proliferation of Human Ovarian Cell specific binding, it is characterised in that the aminoacid sequence of the polypeptide is： ASPLAPWSSVGP。

2. polypeptide described in claim 1 combines the application of Proliferation of Human Ovarian Cell.

3. polypeptide described in claim 1 is used for the application for preparing treatment human ovarian cancer medicine.