CN105713070B

CN105713070B - Polypeptide specifically bound with human breast cancer cells and application thereof

Info

Publication number: CN105713070B
Application number: CN201610085381.7A
Authority: CN
Inventors: 侯颖春; 肖丽; 高晓杰; 何慧敏; 薛琴琴; 韩娟娟; 达苗苗; 马乐乐; 马妮; 程思楠
Original assignee: Shaanxi Normal University
Current assignee: Shaanxi Normal University
Priority date: 2016-02-15
Filing date: 2016-02-15
Publication date: 2020-04-07
Anticipated expiration: 2036-02-15
Also published as: CN105713070A

Abstract

The invention relates to a polypeptide specifically combined with human breast cancer cells and application thereof, wherein the amino acid sequence of the related polypeptide is as follows: HPLGPTWRIPDT are provided. The related application is the application of the polypeptide specifically bound with the human breast cancer cell MCF-7 of the invention to specifically bind with human breast cancer. The breast cancer targeted polypeptide obtained by screening and preliminary identification has obvious breast cancer cell binding specificity, and can be used for developing breast cancer early-stage targeted diagnostic reagents and high-efficiency low-toxicity targeted therapeutic drugs.

Description

Polypeptide specifically bound with human breast cancer cells and application thereof

Technical Field

The invention relates to the field of biotechnology, and provides a polypeptide (sequence) specifically bound to human breast cancer cells (MCF-7 cells) and having good binding specificity and sensitivity to the human breast cancer cells by an improved peptide library subtractive screening method.

Technical Field

Breast cancer (breast cancer) is the first-ranked malignancy in women, accounts for 30% of new malignant tumors in women, and seriously harms women's health. According to the international cancer research center, about 46 ten thousand women die of breast cancer each year worldwide, accounting for about 13.7% of all women's malignant tumor deaths. In recent years, the incidence of breast cancer tends to rise year by year in China, particularly in some big cities and areas where coastal economy develops rapidly.

Currently, the main treatment methods for breast cancer include image examination, tumor marker detection, needle biopsy, and the like, but these methods may also cause problems such as cancer cell diffusion and trauma. Most of the existing tumor markers are breast cancer serum tumor markers, but most of the markers have low specificity and sensitivity and unobvious treatment effect. Therefore, it is a hot spot of the current research to find some new breast tumor markers as soon as possible, and the key element is the acquisition of molecular fragments with specific target binding activity to cancer cells and cancer tissues.

Disclosure of Invention

The invention aims to provide a polypeptide sequence which is obtained by screening a bacteriophage 12 peptide library through improved peptide library reduction screening and specifically binds to breast cancer cells, and an experimental result for identifying the specific affinity of the polypeptide and the breast cancer cells, so that the polypeptide has great application value in the aspects of early molecular imaging diagnosis of breast cancer, targeted chemotherapy, breast cancer targeted therapy performed by coupling with other materials and the like.

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

the amino acid sequence of the polypeptide specifically bound by the breast cancer MCF-7 cells is as follows: HPLGPTWRIPDT are provided. The polypeptide can specifically bind to MCF-7 cells, does not recognize human embryonic kidney HEK293 cells (namely normal cells), and has extremely low affinity or no affinity to other tumor cells.

The screening method of the polypeptide specifically bound by the breast cancer MCF-7 cells comprises the steps of taking MCF-7 cell lines cultured in vitro as target cells, taking a human embryonic kidney HEK293 cell line as a non-specific adsorption cell, carrying out 4 rounds of subtractive screening on a phage random 12 peptide library, randomly picking 60 phage clones, and identifying positive clones by ELISA. DNA sequencing is carried out on the positive clones, the amino acid sequence composition and the basic characteristics of the polypeptide are analyzed, finally, 3 strong positive clones with a Consensus sequence (Consensus sequence) are obtained, the specificity and the sensitivity of the strong positive clones are further identified by a cell immunofluorescence method, and the optimal sequence is determined.

The experimental results of various phage clone levels and optimal sequence synthetic peptide probe levels all indicate that the specificity and the sensitivity of the sequence HPLGPTWRIPDT are optimal, so that experimental basis is provided for the polypeptide to be used for early molecular imaging diagnosis, targeted chemotherapy and targeted breast cancer treatment by coupling with other materials in the future.

The invention takes breast cancer MCF-7 cells as target cells, and carries out 4 rounds of subtractive screening on a phage 12 peptide library by using an improved subtractive screening method, and finally obtains 3 common polypeptide sequences which are HPLGPTWRIPDT, NSQARRHSSIDT, ANIDSSHHGNQP respectively. HPLGPTWRIPDT in the test results of a series of identification and analysis experiments has the strongest targeting effect on breast cancer, and the value of the test results in the aspects of breast cancer diagnosis and targeted therapy is suggested.

In the aspect of breast cancer detection, the polypeptide labeled by an isotope or fluorescence can be specifically combined with breast cancer cells/tissues, is suitable for tumor imaging and molecular image diagnosis, and has important significance for early detection of breast cancer, location of cancer focus and curative effect evaluation; in the aspect of targeted therapy of breast cancer, the breast cancer is expected to be coupled with traditional chemotherapeutic drugs (such as cisplatin, adriamycin and the like) by utilizing the characteristics of high specificity, small molecular weight, strong penetrating power and high affinity, so that the aim of targeted delivery administration is achieved, and the non-specificity and the toxic and side effects of the chemotherapeutic drugs 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 results of two ELISA identifications of affinity of 60 phage clones to MCF-7 cells;

FIG. 5 is an immunofluorescence identification of cell affinity of representative positive phage clones for three consensus sequences, wherein: A. c, E are: the affinity of the phage clones Q35, Q3, Q41 for cellular MCF-7; B. d, F are: the affinity of the phage clones Q35, Q3, Q41 to the cell HEK 293; G. h is respectively as follows: affinity of PBS, URPs to cellular MCF-7;

FIG. 6 is an affinity identification of clone Q35(HPLGPTWRIPDT) with other tumor cells, wherein: a: MCF-7; b: SiHa (cervical cancer cells); c: caco2 (colorectal cancer cells); d: SGC-7901 (gastric cancer cells); e: SMMC-7721 (hepatoma cells); f: eca-109 (esophageal cancer cells);

fig. 7 is a dose effect of polypeptide (HPLGPTWRIPDT) binding specifically to cells, wherein: A. b, C, D, E is polypeptide combined with breast cancer cell MCF-7; F. g, H, I, J polypeptide is combined with HEK293 human embryonic kidney cell, and the two groups of concentrations are respectively 10 μ M, 15 μ M, 20 μ M, 25 μ M and 30 μ M;

FIG. 8 is a time effect of polypeptide (HPLGPTWRIPDT) binding specifically to cells, wherein: A. b, C, D, E is polypeptide combined with breast cancer cell MCF-7; F. g, H, I, J the polypeptide is combined with human embryonic kidney HEK293, and the incubation time of the two groups is 10min, 20min, 40min, 60min and 80min respectively;

fig. 9 is a binding site of the polypeptide (HPLGPTWRIPDT) to a breast cancer cell, wherein: A. c: FITC-positive polypeptide and MCF-7, HEK293 cells; B. d: FITC-negative polypeptide and MCF-7, HEK293 cells;

FIG. 10 is the binding specificity of polypeptide (HPLGPTWRIPDT) to other tumor cells, where: a: SiHa (cervical cancer cells); b: SKV3 (ovarian cancer cells); c: caco2 (colorectal cancer cells); d: SGC-7901 (gastric cancer cells); e: SMMC-7721 (hepatoma cells); f: eca-109 (esophageal cancer cells);

fig. 11 is a flow cytometer for detecting the binding specificity of a polypeptide (HPLGPTWRIPDT), wherein: a: breast cancer MCF-7 cells; b: HEK293 cells. Green: FITC-Brca Probe; red: FITC-Ctrl Probe; black: PBS;

fig. 12 shows competitive inhibition of polypeptide (HPLGPTWRIPDT): after the target cells are incubated by the polypeptide (HPLGPTWRIPDT), the target cells are incubated by phage clone Q35, and after washing, the specific binding force of the polypeptide is analyzed according to the strength of fluorescence emitted by the phage. The competitive inhibition is positively correlated with the polypeptide concentration, which indicates that the polypeptide specificity is good.

The invention is further illustrated by the following figures and specific examples.

Detailed Description

Phage display technology was created in 1985 by g.p. smith et al, university of Missouri, usa, as a technology capable of specifically screening polypeptides or proteins. The technology displays the polypeptide coded by the target gene on the surface of the phage in the form of fusion protein, and the polypeptide or the protein is expressed by fusion with the capsid protein of the phage, and the displayed polypeptide or protein can keep relatively independent spatial structure and biological activity. At present, the phage display technology has been widely applied to the research in the aspects of tumor marker screening, tumor drug targeted transportation, polypeptide drug development and the like.

The invention screens a polypeptide sequence which can be specifically and sensitively combined with human breast cancer cells by using the peptide library and an improved peptide library subtractive screening method, and verifies the high specificity and sensitivity of the sequence to the combination of the human breast cancer cells by using correlation experiments. The polypeptide has the characteristics of high affinity, small molecular weight, strong tissue penetrability, low immunogenicity, easy synthesis and the like, can be connected with other effector molecules, can be used for early molecular imaging diagnosis of breast cancer, forms tumor three-dimensional imaging, greatly improves the early diagnosis rate of the breast cancer, and can evaluate the treatment effect; the targeting chemotherapeutic component for breast cancer can greatly reduce the toxicity of the drug and improve the curative effect when being used as the guiding component of the chemotherapeutic drug for breast cancer, so that the chemotherapy becomes one of the treatment methods which are really beneficial to patients; the polypeptide is coupled with the nano liposome or nano drug, so that a better targeted treatment effect can be achieved; the method is used for research of searching the related ligand on the surface of the tumor cell and provides a new target for tumor treatment.

The invention takes human breast cancer MCF-7 cells as target cells, carries out 4 rounds of subtractive screening on a phage peptide library by an improved peptide library subtractive screening method, searches for a phage peptide sequence with specific binding force with the breast cancer MCF-7 cells, and lays a foundation for further research and development of early-stage targeted diagnostic reagents and high-efficiency low-toxicity targeted therapeutic drugs for breast cancer, and the specific technical route is shown in figure 1.

Materials and methods

1.1 Main test materials

1.1.1 cell lines human breast cancer cell line MCF-7, purchased from American ATCC (Rockville, USA), human embryonic kidney cell line HEK293, and stored in the laboratory.

1.1.2 phage random dodecapeptide library storage: with glycerol 1:1 in TBS solution, 1.5X 10¹³pfu/ml. Complexity: 2.7 × 10⁹And (4) a transformant. Coat gene-96 gIII sequencing primer for vector M13 KE: 5' -^HOCCC TCA TAGTTA GCG TAA CG-3’。

1.1.3 host bacterium E.coli ER2738, and glycerol 1:1, storing in an ultra-low temperature refrigerator at-80 ℃.

1.2 Experimental methods

1.2.1 cell culture

Complete culture in DMEM at 37 deg.C, saturated humidity and 5% CO₂An incubator.

1.2.2 preparation of host bacterium E.coli ER2738

Coli ER2738 is a proprietary strain of the NEB company's ph.d series phage peptide library. The mixture was shaken overnight at 300rpm in LB-Tet medium on a 37 ℃ constant temperature shaker.

1.2.3 phage random dodecapeptide library in vitro fast subtractive screening

The human embryonic kidney cell HEK293 was pre-adsorbed as a negative adsorbing cell to exclude the abnormal cell targeted clone, and then the targeted breast cancer clone was screened by an improved subtractive screening method (fractionation Biopanning) using the human breast cancer cell MCF-7 and as a target cell, and 4 rounds were repeated. Based on the conventional classical Biopanning method (see kit instructions) provided by New England BioLabs inc, NEB, the phage peptide library kit manufacturer, the modified subtractive screening method was characterized as follows for each round of Biopanning: (1) each round of the first step increases the pre-adsorption of the thrown phage peptide library by normal cells to remove the non-cancer cell targeted phage clones as much as possible; (2) the volume of 25ml of the bacterial liquid in the amplification step required in the conventional step is changed to 3 ml. The change greatly simplifies the experimental difficulty and improves the screening efficiency; (3) due to the change of item (2), at least 60 or more phage clones can be randomly selected in the last round (generally 4 rounds of Biopanning).

After the final round of randomly selecting 60 clones, the affinity of the clones to the target cell MCF-7 is identified by ELISA method.

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 means 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

36 positive phage clones were obtained, of which clone Q35 had the highest binding specificity to MCF-7 cells, and the results are shown in FIG. 4. Positive phage clones had 3 consensus sequences: HPLGPTWRIPDT, NSQARRHSSIDT, ANIDSSHHGNQP are provided.

2.2 binding specificity of Positive phage clones

The results are shown in fig. 5 and 6, and the positive phage clones all bound MCF-7 cells but not HEK293 cells, as best clone Q35(HPLGPTWRIPDT), indicating good targeting of the positive polypeptide.

2.3 specificity and sensitivity identification of polypeptide HPLGPTWRIPDT

The results are shown in FIGS. 7 to 12. The polypeptide HPLGPTWRIPDT specifically binds to MCF-7 cells and not to HEK293 and other cells.

Claims

1. A polypeptide that specifically binds to a human breast cancer cell, wherein the amino acid sequence of the polypeptide is: HPLGPTWRIPDT are provided.

2. Use of a polypeptide according to claim 1 for the preparation of a preparation which specifically binds to human breast cancer cells.