CN113637062A

CN113637062A - Polypeptide for treating glioma and application thereof

Info

Publication number: CN113637062A
Application number: CN202110860180.0A
Authority: CN
Inventors: 金涛; 刘明发; 许海雄; 徐可; 许圳南; 刘衍
Original assignee: Shantou central hospital
Current assignee: Shantou central hospital
Priority date: 2021-07-28
Filing date: 2021-07-28
Publication date: 2021-11-12
Anticipated expiration: 2041-07-28
Also published as: CN113637062B

Abstract

The invention discloses a polypeptide for treating glioma and application thereof. The amino acid sequence of the polypeptide for treating glioma is shown as SEQ ID NO.1, and the amino acid sequence of the secretory polypeptide is shown as SEQ ID NO. 2. The experimental result shows that the polypeptide can be combined with NGAL, block the activation of NGAL on NGALR, and inhibit the invasion and migration capacity of glioma cells, and the polypeptide has a larger application prospect in the preparation of glioma treatment medicines.

Description

Polypeptide for treating glioma and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a polypeptide for treating glioma and application thereof.

Background

Gliomas are the most common refractory tumors of the central nervous system, seriously harming human health. Especially high-grade gliomas, such as glioblastoma multiforme (GBM), have high infiltrates, often penetrate into adjacent normal brain tissue, and are variable in shape without a definite range, and have extremely high lethality; the median survival time of GBM patients is only 12-15 months, the five-year survival rate is less than 5%, and the 1-year survival rate is less than 30%. The invasive growth pattern of gliomas makes the tumor boundaries difficult to define and difficult to clean during surgery, the most important cause of their high recurrence rate and high mortality rate. Chemotherapy dominates in the treatment of malignant glioma for a long time, but traditional chemotherapeutic drugs such as cisplatin, teniposide, carmustine and the like have not ideal treatment effect on glioma, and the clinical actual effective rate is only 20-25%. In recent years, Temozolomide (TMZ) is recommended at home and abroad as a first-line chemotherapeutic drug for treating glioma, the effective rate is less than 40%, and the temozolomide has no inhibition effect on the invasion characteristics of glioma cells. Targeting therapy aiming at specific molecules of tumor cells is more and more emphasized in tumor therapy, so that key molecules for promoting the invasion characteristic of glioma are searched, effective molecular targeted chemotherapeutic drugs capable of inhibiting invasion are developed, and the targeting chemotherapeutic drugs are important subjects in the fields of clinical treatment and basic research of glioma.

Secreted protein NGAL (official name Lipocalin, LCN2) is a neutrophil gelatinase-associated Lipocalin, one of the members of the Lipocalin (Lipocalin) family. NGAL has the characteristics of small molecular weight and difficult degradation, and can be secreted to the outside of cells or discharged out of the body along with urine. The physiological role of NGAL can not only induce the growth of innate immunosuppressive bacteria by capturing iron-containing cells, but can also be involved in maintaining cellular homeostasis by modulating inflammation. In pathological conditions, the biological effects of NGAL are complex. For example, the detection of NGAL in blood and urine has been reported as an early marker of acute kidney injury, and has also been associated with poor prognosis in type II diabetes and myocardial injury. In colon cancer, NGAL is reported to inhibit cell proliferation and epithelial to mesenchymal transition and thereby inhibit tumor progression. However, in cervical cancer, NGAL is ubiquitously expressed and promotes local invasion of tumor cells and lymph node metastasis. In breast cancer, NGAL is knocked out in a mouse body to inhibit breast cancer metastasis, and the NGAL is suggested to have an important tumor metastasis promotion effect. We previously reported that the abnormally elevated expression of NGAL in human glioma tissues, particularly in high-grade gliomas, could serve as an independent factor suggesting a poor prognosis for patients with gliomas, but the biological role and specific mechanism of action of NGAL in pathological conditions, including gliomas, are quite unclear. Currently available studies have shown that NGAL may be involved in regulating ion transport mainly through its receptor protein NGALR (official name of source carrier family 22member 17, SLC22a 17). However, reports on the role of NGALR in diseases, especially in tumors, are relatively rare. The applicant's team pre-reported that NGALR expression was generally elevated in esophageal squamous carcinoma tissues due to low-level methylation of the promoter. Applicants also previously reported that, like their ligand NGAL, NGALR expression in human glioma tissues, and particularly in high grade gliomas, was abnormally elevated and may also serve as an independent factor in suggesting a poor prognosis for patients with gliomas. However, the biological role of NGAL/NGALR in pathological states including gliomas is unclear, and NGALR is especially used as a receptor, and downstream effector proteins and signaling pathways for recruitment after activation by NGAL stimulation have not been reported. It is worth mentioning that NGAL is used as a secretory protein, NGALR is used as a cell receptor, and the molecular characteristics of the NGAL and the NGALR have very important research values for being used as a diagnostic marker and a therapeutic target. Chinese patents CN1874783A, CN1924006A, CN101270158A, etc. all disclose anti-glioma polypeptides or proteins, however, anti-glioma polypeptides against NGAL/NGALR have been reported only rarely.

Disclosure of Invention

The present invention aims to overcome the above-mentioned drawbacks and deficiencies of the prior art and to provide a polypeptide for the treatment of glioma.

The second objective of the invention is to provide a secreted polypeptide for treating glioma.

The third purpose of the invention is to provide the application of the polypeptide in preparing drugs for treating glioma.

The above object of the present invention is achieved by the following technical solutions:

a polypeptide for treating glioma, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1:

PPLHCHYGAFPPNASGWEQPPNASGVSVASAALAASAASRVATSTDPSCSGFAPPDFNHCLKDWDYNGLPVLTTNAIGQWDLVCDLGWQVILEQILFILGFASGYLFLGY。

a secretory polypeptide for treating glioma, the amino acid sequence of said secretory polypeptide is shown in SEQ ID NO. 2:

MHSFPPLLLLLFWGVVSHSPPLHCHYGAFPPNASGWEQPPNASGVSVASAALAASAASRVATSTDPSCSGFAPPDFNHCLKDWDYNGLPVLTTNAIGQWDLVCDLGWQVILEQILFILGFASGYLFLGY, respectively; wherein MHSFPPLLLLLFWGVVSHS is a signal peptide.

The in vitro experiment results show that the polypeptide can be combined with NGAL, the activation of NGAL on NGALR is blocked, the invasion and migration capacity of glioma cells is inhibited, and the polypeptide can be used for preparing glioma treatment medicines.

Therefore, the invention also provides the application of the polypeptide in preparing drugs for treating glioma.

The application of the polypeptide in preparing the medicine for inhibiting glioma migration or invasion.

The invention also provides a preparation method of the secretory polypeptide, which inserts the coding sequence of the secretory polypeptide by selecting a prokaryotic protein expression system to obtain a recombinant plasmid; then introducing the recombinant plasmid into a bacterium for protein expression, screening positive bacteria for amplification culture, inducing expression protein by IPTG, and purifying to obtain the recombinant plasmid.

Preferably, the coding sequence of the secreted polypeptide is as shown in SEQ ID NO. 3.

A drug for treating glioma, which comprises a polypeptide shown in SEQ ID NO.1 or SEQ ID NO.2 or a plasmid or cell line for expressing the polypeptide shown in SEQ ID NO.1 or SEQ ID NO. 2.

Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a polypeptide for treating glioma, wherein the amino acid sequence of the polypeptide for treating glioma is shown as SEQ ID No.1 or SEQ ID No. 2. The research of the invention shows that the polypeptide can be combined with NGAL, block the activation of NGAL to NGALR, inhibit the invasion and migration capacity of glioma cells, and have a wide application prospect in the preparation of glioma treatment medicines.

Drawings

FIG. 1 is the preparation and detection of secreted NGALR peptide fragments. A is a schematic diagram for constructing a secretory NGALR peptide fragment expression plasmid; b, detecting the purification effect of the secretory NGALR peptide fragment; the expression of the secretory NGALR peptide segment linked with the expression tag Flag is shown to be successful.

FIG. 2 shows the effect of secreted NGALR peptide with NGAL and NGALR. A: the secretory NGALR peptide segment can be combined with NGAL; b: the secretory NGALR peptide segment can inhibit the combination of NGAL and NGALR; c: the secretory NGALR peptide segment can inhibit the activation of NF kappa B; the secretory NGALR peptide segment can be combined with NGAL in vitro, block the combination of NGAL and NGALR, and inhibit NF-kB signal channel at the downstream of NGALR.

Figure 3 is a graph of the ability of secreted NGALR peptide fragments to inhibit the invasion of glioma cell lines a172 and U251MG through Matrigel in vitro.

Figure 4 is a graph of the ability of secreted NGALR peptide fragments to inhibit the migration of glioma cell lines a172 and U251MG in vitro.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

EXAMPLE 1 preparation of secreted NGALR peptide fragments

1. Constructing a secretory NGALR peptide fragment expression plasmid: as shown in figure 1A, a pET prokaryotic protein expression system is selected, a secretory NGALR peptide segment DNA sequence is inserted into an AlwN I enzyme cutting site of a pET-31b (+) vector through homologous recombination, the 3' end of the peptide segment DNA sequence is provided with a Flag label, and a terminator adopts TAA. The DNA sequence of the secretory NGALR peptide fragment is shown in SEQ ID NO. 3.

2. Plasmids were obtained by small extraction, introduced into the protein-expressing bacterium Rosetta (DE3) (chloramphenicol resistant bacterium), transformation plated and PCR-identified positive monoclonals.

3. And adding the positive bacteria into 100mL of fresh LB culture medium (added with chloramphenicol and kana antibiotic), and shaking the bacteria for 16h to obtain a small shake bacteria liquid. Further taking 0.2mL of the small shake culture solution, adding the small shake culture solution into 200mL of fresh LB culture medium (added with chloramphenicol and kana antibiotics), and shaking the bacteria for 14h to obtain a large shake culture solution.

IPTG stimulation of bacterial expression of proteins: the temperature of the shake-out bacteria liquid is reduced to 16 ℃, 100mM IPTG (final concentration is 1mM) and MgCl are directly added into 200mL of the bacteria liquid₂、ZnCl₂(final concentration is 50 mu M), shaking the bacteria for 12h, collecting the bacteria block, and carrying out ultrasonic lysis on the bacteria until the bacteria liquid is uniform and has good fluidity.

5. Protein purification: filtering with 0.45mm filter head to obtain protein liquid, and purifying with AKTApurifier 10 high pressure column chromatography system (replacing protein by dialysis method).

6. The purification effect of the secretory NGALR peptide fragment is determined by identifying the expression of the Flag tag through an immunoblotting experiment, and the result is shown in figure 1B, which indicates that the secretory NGALR peptide fragment linked with the expression tag Flag is successfully expressed.

Example 2 secreted NGALR peptide fragments can bind to NGAL and block NGAL from activating NGALR

1.293FT cells were transfected with secretory NGALR peptide fragment expression plasmid and NGAL-HA expression plasmid, and the secretory NGALR peptide fragment was enriched by Flag magnetic beads using co-immunoprecipitation, and found to be capable of binding to NGAL (FIG. 2A).

2.293FT cells were transfected with NGAL-HA plasmid, which was found to bind to NGALR by co-immunoprecipitation, and further the expression plasmid of high expression secretory NGALR peptide fragment could inhibit the binding of NGAL to NGALR (FIG. 2B).

3. Luciferase report experiments are used for detecting the activity of the NF kappa B signal channel at the downstream of NGALR in glioma cell lines A172 and U251MG, the activity of NF kappa B luciferase is obviously improved after NGAL is highly expressed, and the activation of NF kappa B can be inhibited by further treating with secretory NGALR peptide (figure 2C).

Example 3 secreted NGALR peptide fragments can inhibit invasion and migration of glioma cells

1. Invasion test: 50uL matrigel was previously spread evenly in a transwell chamber, and after digestion counting the differently treated glioma cells A172 and U251MG were resuspended in basal medium and added to the upper chamber layer and the lower chamber layer in complete medium containing 10% FBS. After 36h of cell incubator culture, the cells were fixed and stained with crystal violet for 15min, and the cells were observed under a microscope to cross the chamber. It was found that after treatment with the secreted NGALR peptide fragment, the invasive capacity of glioma cells a172 and U251MG was significantly reduced (fig. 3).

2. Migration experiment: the transwell chamber was filled with complete medium containing 10% FBS in the upper layer without matrigel, and cell suspensions of different treatments of glioma cells A172 and U251MG were prepared in basal medium. After 36h of cell incubator culture, the cells were fixed and stained with crystal violet for 15min, and the cells were observed under a microscope to cross the chamber. It was found experimentally that the migratory capacity of glioma cells a172 and U251MG was significantly reduced after treatment with the secreted NGALR peptide fragment (fig. 4).

The results show that the secretory NGALR peptide segment can inhibit the invasion and migration capacity of glioma cells, and has a wide application prospect in preparing glioma treatment medicines.

Sequence listing

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<120> polypeptide for treating glioma and application thereof

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atgcactcat tcccaccact actactacta ctattctggg gagtagtatc acactcacca 60

cctctgcact gccactacgg tgcattcccg ccgaacgcat caggctggga acaaccnccg 120

aacgcttcag gcgtgtccgt cgcatcagca gccctcgcag cttctgctgc atcacgcgtc 180

gctacctcaa ccgacccttc atgctcaggc ttcgctccac ccgacttcaa ccactgcctg 240

aaagactggg actacaacgg actgccagtc ctcacgacga acgcaatagg ccaatgggac 300

ctcgtctgcg acctcggatg gcaagtcata ctggaacaaa tactcttcat actcggattc 360

gcatctggat acctcttcct cggatac 387

Claims

1. A polypeptide for treating glioma, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

2. A secretory polypeptide for treating glioma, wherein the amino acid sequence of the secretory polypeptide is shown as SEQ ID NO. 2.

3. Use of the polypeptide of claim 1 or 2 for the manufacture of a medicament for the treatment of glioma.

4. Use of a polypeptide according to claim 1 or 2 for the manufacture of a medicament for inhibiting glioma migration or invasion.

5. The method for preparing the secretory polypeptide of claim 2, wherein a prokaryotic protein expression system is selected and inserted into the coding sequence of the secretory polypeptide to obtain a recombinant plasmid; then introducing the recombinant plasmid into a bacterium for protein expression, screening positive bacteria for amplification culture, inducing expression protein by IPTG, and purifying to obtain the recombinant plasmid.

6. The method of claim 5, wherein the coding sequence of the secreted polypeptide is as shown in SEQ ID No. 3.

7. A drug for treating glioma is characterized in that the drug contains polypeptide shown in SEQ ID NO.1 or SEQ ID NO.2 or plasmid or cell line for expressing the polypeptide shown in SEQ ID NO.1 or SEQ ID NO. 2.

8. The therapeutic agent of claim 7, further comprising a pharmaceutically acceptable excipient.