CN106589066B

CN106589066B - Polypeptide specifically bound with human ovarian cancer cells and application thereof

Info

Publication number: CN106589066B
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: 2020-06-23
Anticipated expiration: 2036-11-03
Also published as: CN106589066A

Abstract

The invention relates to a polypeptide specifically bound by human ovarian cancer cells SKOV3 and application thereof, wherein the amino acid sequence of the related polypeptide is as follows: ASPLAPWSSVGP are provided. The related application is the application of the polypeptide specifically bound by the human ovarian cancer cell SKOV3 in the invention to specifically bind human ovarian cancer. The ovarian cancer targeted polypeptide obtained by screening and preliminary identification has obvious ovarian cancer cell binding specificity, and can be used for developing ovarian cancer early-stage targeted diagnostic reagents and delivering high-efficiency low-toxicity targeted drugs.

Description

Polypeptide specifically bound with human ovarian cancer cells and application thereof

Technical Field

The invention relates to the field of biotechnology, and provides a polypeptide (sequence) specifically bound to human ovarian cancer cells (SKOV3 cells) by an improved peptide library subtractive screening method, which has good binding specificity and sensitivity to the human ovarian cancer cells.

Background

Ovarian cancer (Ovarian cancer) is one of the common gynecological tumors, with the incidence rate second to cervical cancer and uterine body cancer, and third. Ovarian cancer is the most fatal in gynecological malignant tumors, and is characterized by recessive morbidity, rapid development and low clinical cure rate of patients. At present, the treatment methods of ovarian cancer include surgical therapy, chemotherapy, radiotherapy, immunotherapy and the like, but because the onset of ovarian cancer is hidden, early ovarian cancer has no obvious symptoms and signs and is easy to cause missed diagnosis or misdiagnosis, and the ovarian cancer reaches the middle and late stages when the ovarian cancer is discovered, the best surgical time is lost, and conservative therapy is mostly adopted. However, radiotherapy and chemotherapy have great toxicity, are easy to produce dose dependence, drug resistance and the like, have undesirable effects, and still have low five-year survival rate of patients. Therefore, finding a highly sensitive and specific early screening diagnosis method to reduce the mortality rate of ovarian cancer is also one of the key problems facing today.

In recent years, rapid development of molecular imaging and targeted therapy has brought new hopes for diagnosis and treatment of ovarian cancer, and among them, screening of targeted molecules with high cell surface specificity and high sensitivity for ovarian cancer is an urgent necessity.

The phage peptide library technology provides a new approach for targeted molecular screening of ovarian cancer cells, early diagnosis and targeted therapy of ovarian cancer, wherein the key point is the acquisition of polypeptide fragments with specific targeted binding activity to cancer cells and cancer tissues.

Disclosure of Invention

The invention aims to screen a polypeptide sequence specifically bound to ovarian cancer cells from a phage 12 peptide library, and provide an experimental result for identifying the specificity and sensitivity of the sequence bound to ovarian cancer cells and tissues, so as to prove the application value of the polypeptide in the aspects of early molecular imaging diagnosis of ovarian cancer, targeted therapy of ovarian cancer by coupling with other drugs and the like.

In order to realize the task, the invention adopts the following technical solution:

polypeptide specifically bound by ovarian cancer SKOV3 cells, and the amino acid sequence of the polypeptide is as follows: ASPLAPWSSVGP are provided. The polypeptide is capable of specifically binding to ovarian cancer cells without recognizing normal cells/tissues and other tumor cells/tissues.

Ovarian cancer SKOV3 cells are used as target cells, a human embryonic kidney HEK293 cell line is used as a non-specific adsorption cell, 5 rounds of screening are carried out on a phage random 12 peptide library by an improved peptide library subtraction method, 60 phage clones are randomly picked, and positive clones are identified by ELISA. Sequencing the positive clones to obtain 9 positive clone groups with Consensus sequence (Consensus sequence), taking one clone as a representative in each group, identifying the specificity and sensitivity of each group by using a cell immunofluorescence method, and determining the optimal Consensus sequence. And synthesizing a FITC-labeled peptide probe by using the optimal consensus sequence, detecting ovarian cancer cells/tissues, normal cells/tissues and other cancer cells/tissues by using the probe, and determining the specificity and sensitivity of the probe.

The results of all experiments indicate that the sequence ASPLAPWSSVGP has good specificity and sensitivity to ovarian cancer cells/tissues. Due to the characteristics of small molecular weight, low immunogenicity, high affinity, strong tissue penetrability, easy modification and synthesis and the like, the polypeptide is hopeful to be coupled with a proper fluorescent marker, nano-particles, liposome or antitumor drug and the like, so that the polypeptide is used for early image diagnosis and targeted therapy of tumors and has important value for solving the bottleneck problem of diagnosis and treatment of the current ovarian cancer.

According to the invention, ovarian cancer SKOV3 cells are used as target cells, 5 rounds of subtractive screening are carried out on a phage 12 peptide library by using an improved subtractive screening method, and finally 9 common polypeptide sequences are obtained, wherein the common polypeptide sequences are AWPASFLTQKAL, ASPLAPWSSVGP, QPTTDNRDIRSK, KLHDWADSFSLI, YDHHGQSVMPRA, KSGNIWCPCPNF, WSNALTVTSTYG, ELIPSGSQTYLS, NIAFTTTQHGNR respectively. ASPLAPWSSVGP in the ovarian cancer gene is found to have the strongest targeting effect on ovarian cancer through a series of identification and analysis experiments, and the value of the gene in the aspects of diagnosis and targeted treatment of ovarian cancer is suggested.

In the aspect of ovarian cancer detection, the polypeptide marked by the isotope or fluorescence can be specifically combined with ovarian cancer cells/tissues, is suitable for tumor imaging and molecular image diagnosis, and has important significance for early detection of ovarian cancer, cancer focus positioning and curative effect evaluation; in the aspect of targeted therapy of ovarian cancer, the characteristics of high specificity, small molecular weight, strong penetrating power and high affinity are expected to be utilized to couple with the chemotherapeutic drug, so that the purpose of targeted delivery and drug delivery is achieved, and the non-specificity and toxic and side effects of the chemotherapeutic drug can be greatly reduced.

Drawings

FIG. 1 is a scheme for the preparation of the polypeptides of the invention;

FIG. 2 is the N-terminal sequence of a random dodecapeptide pIII fusion protein;

FIG. 3 is a private cryptography sub-table;

FIG. 4 shows the result of ELISA identification of the affinity of phage clones to ovarian cancer cells SKOV 3;

FIG. 5 is an immunofluorescence identification of the affinity of a representative positive phage clone for cells of 9 consensus sequences, wherein A: r5+ SKOV 3; b: r6+ SKOV 3; c: r10+ SKOV 3; d: r12+ SKOV 3; e: r18+ SKOV 3; f: r20+ SKOV 3; g: r33+ SKOV 3; h: r46+ SKOV 3; i: r47+ SKOV 3; j: URP + SKOV 3; k: r5+ HEK 293; l: r6+ HEK 293; m: r10+ HEK 293; n: r12+ HEK 293; o is R18+ HEK 293; p is R20+ HEK 293; q is R33+ HEK 293; r is R46+ HEK 293; s is R47+ HEK 293; t: URP + HEK293.DAPI stain nuclei; ELISA identification results of the affinity of the phage clones and ovarian cancer cells SKOV 3;

FIG. 6 is an identification of the affinity of clone S7(ASPLAPWSSVGP) for target cells, wherein A: SKOV3+ S7; b: hek293+ S7; c: SKOV3+ URP; d: hek293+ URP; e: SKOV3+ PBS; f: hek293+ PBS; DAPI staining of nuclei;

fig. 7 is an identification of the binding specificity of the synthetic polypeptide (ASPLAPWSSVGP) to target cells, where a: SKOV3+ positive polypeptide; b: SKOV3+ negative polypeptide; c: a Hek293+ positive polypeptide; d: a Hek293+ negative polypeptide; DAPI staining of nuclei;

FIG. 8 shows the identification of the binding specificity of the synthetic polypeptide (ASPLAPWSSVGP) to different tissues of ovarian cancer, wherein the tissue arrangement of the human ovarian cancer tissue chip is shown in FIG. 8-A; the binding specificity of the synthetic polypeptide probes to different tissues of ovarian cancer is shown in FIG. 8-B; the binding specificity of the control polypeptide probe to different tissues of ovarian cancer is shown in FIG. 8-C.

The invention is further described below with reference to the accompanying drawings.

Detailed Description

The invention utilizes an improved peptide library subtractive screening method to screen a polypeptide sequence which can be specifically and sensitively combined with human ovarian cancer cells/tissues, and verifies the high specificity and sensitivity of the sequence to the combination of the human ovarian cancer cells/tissues by correlation experiments.

According to the invention, human ovarian cancer SKOV3 cells are used as target cells, human embryonic kidney HEK293 cells are used as non-specific adsorption cells, 5 rounds of subtractive screening are carried out on a phage random 12 peptide library, and a phage peptide sequence with specific binding force with ovarian cancer cells/tissues is searched, so that a foundation is laid for further research and development of ovarian cancer early-stage targeted diagnostic reagents and high-efficiency low-toxicity targeted therapeutic drugs, and a specific technical route is shown in figure 1.

Materials and methods

1.1 Main test materials

1.1.1 cell line human ovarian carcinoma cell line SKOV3, purchased from ATCC (Maryland, USA) in the United states. Human embryonic kidney cell line HEK293, which is preserved in the laboratory.

1.1.2 phage random dodecapeptide library 1.5 × 10¹³pfu/ml. Purchased from NEB (Boston, USA); the library kit comprises host bacterium e.

1.2 Experimental methods

1.2.1 cell culture

Complete culture in RPMI 1640 medium at 37 deg.C and saturated humidity and 5% CO₂An incubator.

1.2.2 preparation of host bacterium E.coli ER2738

Coli ER2738 using LB-Tet medium.

1.2.3 phage random dodecapeptide library subtractive selection

The human embryonic kidney cell HEK293 is taken as a negative adsorption cell for pre-adsorption to eliminate normal cell targeted clone, then the human ovarian cancer cell SKOV3 is taken as a target cell for elutriation by an improved subtractive screening method, and 5 rounds are repeated. The subduction screening method greatly simplifies the experimental difficulty and improves the screening efficiency.

After amplification of the selected phage clones, the affinity to the target cell SKOV3 was identified by ELISA.

1.2.4 determination of Positive phage clone polypeptide sequences

Phage positive clones, irrelevant clones were sequenced, polypeptide sequences were translated into amino acid sequences and Consensus sequences were obtained according to the genetic codon table (see fig. 2, fig. 3).

1.2.5 identification of binding specificity of Consensus sequences

The binding specificity and sensitivity were identified by conventional cellular immunofluorescence using representative clones of the Consensus sequence.

1.2.6 statistical analysis

Data analysis and data results Using SPSS 16.0 data processing software

Is represented by P<0.01 indicates that the difference is extremely significant, P<0.05 denotes significant difference, P>0.05 indicated no significant difference, no statistical significance, and multiple comparisons between groups were processed using the Duncan test.

Second, experimental results

2.1 ELISA identification and sequencing of Positive clones

38 positive phage clones were obtained, of which clone R20 had the highest binding specificity to SKOV3 cells, and the results are shown in FIG. 4. The positive phage clones had 9 consensus sequences, designated OSP1-OSP 9:

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

2.2 binding specificity of Positive phage clones

The results are shown in fig. 5-6, the positive phage clones all bound SKOV3 cells but not HEK293 cells, and clone R20(ASPLAPWSSVGP) was the best, indicating that the positive polypeptide has good targeting property.

2.3 specificity and sensitivity identification of synthetic FITC-labeled polypeptide ASPLAPWSSVGP

The results are shown in FIGS. 7 to 8. Polypeptide ASPLAPWSSVGP specifically binds to the cell surface of SKOV3 and not to HEK293.

Nucleotide sequence list electronic file

<110> university of Shanxi university

<120> polypeptide specifically bound to human ovarian cancer cells and application thereof

<141>

<160>

<210>1

<211>12

<212> amino acid

<213> OSP1 sequence

<220>

<223>

<400>1

AWPASFLTQKAL

<210>2

<211>12

<212> amino acid sequence

<213> OSP2 sequence

<220>

<223>

<400>2

ASPLAPWSSVGP

<210>3

<211>12

<212> amino acid sequence

<213> OSP3 sequence

<220>

<223>

<400>3

QPTTDNRDIRSK

<210>4

<211>12

<212> amino acid sequence

<213> OSP4 sequence

<220>

<223>

<400>4

KLHDWADSFSLI

<210>5

<211>12

<212> amino acid sequence

<213> OSP5 sequence

<220>

<223>

<400>5

YDHHGQSVMPRA

<210>6

<211>12

<212> amino acid sequence

<213> OSP6 sequence

<220>

<223>

<400>6

KSGNIWCPCPNF

<210>7

<211>12

<212> amino acid sequence

<213> OSP7 sequence

<220>

<223>

<400>7

WSNALTVTSTYG

<210>8

<211>12

<212> amino acid sequence

<213> OSP8 sequence

<220>

<223>

<400>8

ELIPSGSQTYLS

<210>9

<211>12

<212> amino acid sequence

<213> OSP9 sequence

<220>

<223>

<400>9

NIAFTTTQHGNR

Claims

1. A polypeptide specifically bound by a human ovarian cancer cell, wherein the amino acid sequence of the polypeptide is: ASPLAPWSSVGP are provided.

2. Use of the polypeptide of claim 1 for the preparation of a preparation that specifically binds to human ovarian cancer cells.