CN111875673B

CN111875673B - Polypeptide with anti-tumor activity and application thereof

Info

Publication number: CN111875673B
Application number: CN202010762743.8A
Authority: CN
Inventors: 刘晗青; 屠志刚
Original assignee: Jiangsu Laisen Biotechnology Research Institute Co ltd
Current assignee: Jiangsu Laisen Biotechnology Research Institute Co ltd
Priority date: 2020-07-31
Filing date: 2020-07-31
Publication date: 2022-03-11
Anticipated expiration: 2040-07-31
Also published as: CN111875673A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a polypeptide with anti-tumor activity and application thereof. The polypeptide with the anti-tumor activity is finally screened and obtained through the processes of construction of a protein expression vector, expression and purification of target protein, verification of the target protein, phage display panning of a bioactive peptide specifically combined with the target protein, in-vitro detection experiment of the anti-tumor effect of the polypeptide and the like. The polypeptide provided by the invention is obtained by taking Luteinizing Hormone Releasing Hormone Receptor (LHRHR) as a target molecule and carrying out panning by a three-round phage display technology. The provided polypeptide has a short sequence, is easy to synthesize and realize large-scale production, and has important application value in the research and development of antitumor drugs.

Description

Polypeptide with anti-tumor activity and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a polypeptide with anti-tumor activity and application thereof.

Background

Luteinizing Hormone Releasing Hormone (LHRH), also known as gonadotropin releasing hormone (GnRH), is a norpeptide secreted by the pituitary and has important effects on mammalian reproduction. Luteinizing hormone-releasing hormone receptor (LHRHR) is a seven transmembrane G-protein-coupled protein that is activated by GnRH binding and releases Luteinizing Hormone (LH) from the pituitary gland stimulated by tyrosine phosphatase. Recent studies have shown that ovarian cancer is an LHRH-dependent tumor. LHRHR is expressed in ovarian cancer cells higher than normal tissues, and becomes a promising therapeutic target for ovarian cancer.

The phage display technology is a biological technology that inserts the DNA sequence of exogenous protein or polypeptide into the proper position of the structural gene of the coat protein of the phage, so that the exogenous gene is expressed along with the expression of the coat protein, and simultaneously, the exogenous protein is displayed on the surface of the phage along with the reassembly of the phage. The advantage of phage technology is that it is able to recognize the region of interaction of proteins and other molecules without having to have any prior idea of the nature of the interaction. The use of phage display technology has advanced significantly over the last decade. Different screening methods have allowed the isolation and identification of peptides binding to molecules in vitro, in living cells, in animals and in humans. However, the prior art is still lack of reports on research of target drugs on LHRHR by a phage display technology.

Disclosure of Invention

In view of the above, the present invention aims to overcome the defects in the prior art, and the main object of the present invention is to provide a polypeptide having anti-tumor activity and the use thereof. The polypeptide sequence screened by the phage display technology is short, has obvious antitumor activity, has no acute or chronic toxic effect, and has wide clinical application value and prospect.

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

in one aspect, the present invention provides a polypeptide having anti-tumor activity, which has the amino acid sequence: ala Leu Gly Ile Ala His Tyr Lys Trp Gly Ala Val are provided.

In another aspect, the present invention also provides a method for preparing a polypeptide having anti-tumor activity, comprising the following steps:

(1) introducing target genes of extracellular segments of LHRHR into a prokaryotic expression vector pET-30a to construct a target protein expression vector pET-30a/(His)₆-LHRHR：

(2) IPTG induces the expression vector of the target protein to express a large amount, and affinity chromatography purification is carried out to obtain recombinant protein (His)₆-LHRHR；

(3) Recombinant protein (His)₆And fixing LHRHR coating, combining the LHRHR coating with phage in a dodecapeptide phage library, and panning for bioactive peptide specifically combined with target protein to obtain the antitumor active polypeptide.

On the other hand, the invention also provides the application of the polypeptide with the antitumor activity in the preparation of antitumor drugs. In a preferred embodiment, the anti-tumor agent is for the treatment of ovarian cancer.

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

the invention screens out a polypeptide with anti-tumor activity by using a phage display technology. The polypeptide of the invention takes Luteinizing Hormone Releasing Hormone Receptor (LHRHR) as a target molecule, successfully guides a target gene of an extracellular segment of the target LHRHR into a prokaryotic expression vector pET-30a, and after IPTG induction, recombinant protein (His) with a label on the vector is carried out₆LHRHR is abundantly expressed. After two-step affinity chromatography purification, relatively pure recombinant protein (His) is obtained₆-LHRHR. After being coated and fixed, the phage in the random dodecapeptide phage library is combined with the phage, and the phage with high affinity is obtained through 3 rounds of panning and enrichment. After amplification and extraction of the panned monoclonal phage, the panned monoclonal phage is sequenced, and the polypeptide with the highest frequency of occurrence is synthesized. Proved by verification, the polypeptide provided by the invention has obvious antitumor activity and no acute or chronic toxic effect. The polypeptide provided by the invention has a short sequence and is easy to transport in vivo; the whole polypeptide production process is short in time consumption, low in cost, easy to operate and easy to realize large-scale production, and has important application value in the research and development of anti-tumor drugs and wide clinical application value and prospect.

Drawings

FIG. 1 shows the protein expression vector pET-30a/(His)₆-construction of LHRHR;

FIG. 2 is SDS-PAGE validating recombinant protein (His)₆-expression and purification profile of LHRHR;

FIG. 3 is a graph showing the results of the distribution of phage panning sequences.

Detailed Description

The invention discloses a polypeptide with anti-tumor activity and application thereof, and can be realized by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention. The methods, devices and materials used in the examples which follow, if not specifically indicated, are all conventional and commercially available methods, devices and materials used in the art.

Reagent: BamHI, Hind III, T4 DNA ligase, DH 5. alpha. competent cells, BL21 (DE 3) strain, isopropyl-. beta. -D-thiogalactoside (IPTG) were all from Shanghai Biotech, vector pET-30a from King-rui Biotech, and Ni-NTA from Li Ji Biotech, Inc.

Example 1: pET-30a/(His)₆Construction of-LHRHR protein expression vector

Construction of pET-30a/(His) as shown in FIG. 1₆LHRHR, the gene sequence (1-125 AA, NM-000233.4) of the LHRHR is searched from a Genbank database, a PCR primer is designed, and the sequence of an upstream primer is as follows: 5' -CGGGATCCATGAAGCAGCGGTTCTCGGC-3' (BamH I), the sequence of the downstream primer is: 5' -CCAAGCTTGCTCCGGGCTCAATGTATCT-3' (Hind III), the sequence of the cleavage site is underlined. The PCR product and the vector pET-30a were digested with BamHI and Hind III at 37 ℃ for 3 hours and ligated with T4 DNA ligase at 16 ℃ for 12 hours. Transforming the ligation product into DH5 alpha competent cells, then coating the transformation product on a kanamycin-resistant (50 mu g/ml) LB plate for culture until a single colony grows out, selecting the single colony, extracting plasmids for enzyme digestion verification, sequencing the recombinant plasmids to obtain the recombinant plasmids pET-30a/(His)₆- LHRHR。

Example 2: (His)₆Expression, purification and validation of LHRHR

The recombinant plasmid pET-30a/(His) prepared in example 1 was introduced into a cell₆LHRHR was transformed into BL21 (DE 3) strain, recombinant plasmids were selected using kanamycin-resistant LB plate and cultured to OD in 10 ml of LB liquid medium (containing 50. mu.M kanamycin)₆₀₀After about 0.5, the culture is inoculated into a plurality of bottles of LB liquid culture medium according to the volume ratio of 1: 10, and is cultured to OD by violent shaking at 37 DEG C₆₀₀About 0.5, adding isopropyl-beta-D-thiogalactoside (IPTG) with final concentration of 1 mM, and inducing at 37 deg.C for 10 hr to obtain bacteriaAnd (4) liquid.

The bacterial suspension is centrifuged, the supernatant is removed, the bacterial pellet is resuspended in the lysate (50 mM Tris-HCl, 20 mM imidazole, 100 mM NaCl, 10% glycerol, 1% Triton, 1 mM protease inhibitor PMSF, 1 mg/ml lysozyme, pH 8.0) at a ratio of bacterial suspension to lysate = 20:1 by volume, the supernatant is placed on ice for 30 min, sonicated, centrifuged at 12000 g for 30 min, and the total protein is collected. Performing BCA quantification on the total protein, adding Ni-NTA into the protein solution according to the ratio of the protein to the; loading the column, equilibrating with lysate, washing the column with 5 times the volume of lysate, and finally collecting the eluted target protein with 10 times the volume of eluent (250 mM imidazole, other components are the same as the lysate).

Expression and purification of the target protein were verified by SDS-PAGE, and the collected target protein sample was quantified by BCA method, and loaded with a protein amount of 30 to 50. mu.g (2 to 5. mu.g of purified protein) per well. The 12% polyacrylamide gel (SDS-PAGE) was run at 100V for 100 min to isolate the protein, and the result is shown in FIG. 2, where the target band is more evident at 27 kD. .

Example 3: bioactive peptide specifically combined with LHRHR (LHRHR) by phage display panning

(1) Immobilization of the target protein: 600. mu.l of a target protein solution (0.1M NaHCO) at a concentration of 17. mu.g/ml₃pH 8.6) were added to a six-well plate, placed on a shaker with gentle shaking and incubated overnight at 4 ℃. Using TBST (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% [ v/v ]]Tween-20) 6 times, and blocking solution (0.1M NaHCO)₃ pH 8.6，5 mg/ml BSA，0.02% NaN₃) And sealing for 1 h.

(2) Screening for specifically bound phage: the six well plates were washed 10 times with TBST. The amplified phage were diluted with TBST to a copy number of 10⁹~10¹¹In between, the diluted phage was added to a six-well plate to bind to the target protein and incubated at room temperature for about 60 min. TBST was washed 10 times, and patted dry after each wash. Adding eluent, and collecting phage specifically binding target protein.

(3) Extraction of monoclonal phage information: phage amplification and repeat the panning 2 rounds above. After 3 rounds of panning, the phage eluted in the last round were plated on LB/IPTG/Xgal plates after infecting host bacteria ER2738 with the phage. After 12 h, the phage blue spots grew, and the blue spots were picked and subjected to monoclonal phage amplification. The monoclonal phage is used as a template, PCR primers 5'-TTATTCGCAATTCCTTTAG-3' and 5'-CCCTCATAGTTAGCGTAACG-3' are designed to amplify random polypeptide sequences, amplified products are sequenced, and the proportion of various random polypeptides is analyzed.

After three rounds of phage panning, 23 phage blue spots are picked up, amplified respectively, then phage are collected, primers are designed to carry out PCR amplification on random polypeptide insertion sequences, and sequencing is carried out on the obtained phage random polypeptide sequences. FIG. 3 is a graph showing the distribution of phage panning sequences, as shown in FIG. 3, after 3 rounds of panning, a total of 7 polypeptide sequences are obtained, which account for 72%, 9%, 7%, 5%, 4%, 2%, 1%, and the highest percentage of the finally selected polypeptide sequences are: ala Leu Gly Ile Ala His Tyr Lys Trp Gly Ala Val are provided.

Example 4: detection of anti-tumor action of polypeptide

In this example, MTT method was used to detect the anti-tumor effect of the polypeptides selected in example 3: healthy SKOV3, OVCAR3, OVCAR8 and a2780 cells (all purchased from the central academy) were cultured separately, trypsinized, centrifuged, and cell counted using 96-well plates containing 2500 cells per 90 μ L RPMI1640 medium. Diluting with PBS to obtain polypeptide with different concentrations, adding 10 μ L polypeptide solution with different concentrations into each well, and repeating each group for 3 wells containing 5% CO₂Culturing in a cell culture box at 37 ℃ for 72 h, and then testing. And adding 10 mu L of 5 mg/mL MTT solution into each well to be detected, incubating the wells in an incubator for 1.5 h, observing blue-purple formazan crystals under a microscope, pouring out supernatant, adding 100 mu L DMSO into each well to dissolve the crystals, shaking up, and finally measuring the light absorption value of each well at the wavelength of 550 nm by using a microplate reader to calculate the relative survival rate of the cells.

TABLE 1 comparison table of cell survival rates of ovarian cancer cell lines.

	SKOV3	OVCAR3	OVCAR8	A2780
					IC₅₀（μM)	4.79	12.11	9.31	5.37

When IC₅₀The proliferation of tumor cells can be well inhibited when the concentration of the polypeptide (the inhibition rate of which reaches 50%) is in a lower concentration range, and as can be seen from table 1, the polypeptide screened by the invention can kill ovarian cancer tumor cells, particularly has a good inhibition effect on OVCAR3 cell lines, and can be applied to the development of targeted drugs in ovarian cancer treatment.

Example 5: observation of toxic Effect of polypeptide on mice in vivo

Dividing Kunming mice of 6-8 weeks into 4 groups at random, wherein three groups are high, medium and low dose test groups, the fourth group is control group (normal saline), and each group has 6 mice; the polypeptide obtained in example 3 was diluted with physiological saline to 200 (low dose group), 1000 (medium dose group) and 5000. mu.g/ml (high dose group), respectively, and then injected into mice via tail vein. Various changes in the behavioral indicators of the mice were observed and recorded.

TABLE 2 Observation of the toxic Effect of the Polypeptides in mice

As shown in Table 2, after the polypeptide provided by the invention is injected into a mouse body respectively by tail vein with low, medium and high doses, no obvious influence is caused on factors such as normal diet, excretion and exercise of the mouse, all recorded ethological indications are normal, and no death phenomenon of the mouse is found one day and one week after the injection, which indicates that the polypeptide provided by the invention has no obvious acute or chronic toxic effect on the polypeptide entering the mouse body.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims

1. A polypeptide having anti-tumor activity, wherein the amino acid sequence of the polypeptide is: ala Leu Gly Ile Ala His Tyr Lys Trp Gly Ala Val are provided.

2. Use of the polypeptide having an antitumor activity according to claim 1 for the preparation of an antitumor agent, wherein the tumor is ovarian cancer.