CN108484734B

CN108484734B - Polypeptide with anti-tumor activity and application thereof

Info

Publication number: CN108484734B
Application number: CN201810242993.1A
Authority: CN
Inventors: 陈龙; 尹会伟
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2018-02-28
Filing date: 2018-03-23
Publication date: 2022-03-25
Anticipated expiration: 2038-03-23
Also published as: CN108484734A

Abstract

The invention relates to a polypeptide with anti-tumor activity, a preparation method thereof and application thereof.

Description

Polypeptide with anti-tumor activity and application thereof

Relating to sequence listing

The present application contains a sequence listing in computer readable form, which is incorporated herein by reference.

Technical Field

The present invention relates to a polypeptide with antitumor activity, polynucleotide for coding it and their application.

Technical Field

Cervical cancer (Cervical cancer) is one of the common gynecological tumors, and the incidence rate of the cancer as a gynecological disease is second to breast cancer and endometrial cancer in developed countries and is the top of the developing countries. For the treatment of cervical cancer, at present, different methods such as surgery, radiotherapy, chemotherapy, and the like can be selected. Such therapies are not ideal for improving the patient's prognosis and increasing survival. Many researchers have shifted their attention to the study of antitumor polypeptides with high activity, less adverse reactions and less susceptibility to drug resistance. However, the stability and selectivity of the antitumor polypeptide are two main factors limiting the application. Due to the effects of filtration of the kidney, degradation of protease and the like, the antitumor polypeptide has poor stability, short half-life, low blood concentration and low bioavailability. Meanwhile, due to poor selectivity of the antitumor polypeptide, the antitumor polypeptide can cause damage to normal cells and tissues of a human body and has certain toxic and side effects on the human body.

Scientists have focused on the study of targeting anti-tumor peptides. It has been found that various tumor-associated proteases are overexpressed in tumor cells or extracellular matrix, and active enzymes such as Matrix Metalloproteinases (MMP) and fibroblast activation protein (FAPot) are found, and the common characteristics of small-molecule antigen substances specifically expressed on the surface of tumor cells or specifically secreted by tumor cells are that the small-molecule antigen substances have both tumor localization and proteolytic enzyme properties. In general, the condensation of cytotoxic parent drugs with polypeptides into hydrolytic substrates for such enzymes allows better targeting of the drug to tumor tissue.

Therefore, the research of an effective method for improving the selectivity and the stability of the polypeptide medicament is an urgent problem to be solved in the research and development of the anti-tumor polypeptide medicament, and has important significance for the clinical application of the anti-tumor polypeptide medicament.

Summary of The Invention

The present invention relates to a polypeptide selected from the group consisting of:

(a) a polypeptide comprising or consisting of the amino acid sequence shown as SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3;

(b) the polypeptide derived from (a) by substituting, deleting or adding one or more amino acids in the amino acid sequence in (a) has antitumor activity.

The invention also relates to polynucleotide for coding the polypeptide, a vector containing the polynucleotide and a host cell.

The invention also relates to a pharmaceutical composition containing the polypeptide and application of the polypeptide in preparing anti-tumor drugs, in particular to drugs for treating diseases related to expression of secretion integrin (integrin) or matrix metalloproteinase 2/9(MMP 2/9).

Drawings

FIG. 1 is an ESI mass spectrum of a LSAP-1 polypeptide prepared by solid phase synthesis of the present invention.

FIG. 2 is an ESI mass spectrum of a LSAP-2 polypeptide prepared by solid phase synthesis of the present invention.

FIG. 3 is an ESI mass spectrum of a LSAP-3 polypeptide prepared by solid phase synthesis of the present invention.

FIG. 4 is a TEM image of LSAP-3 self-assembled form of the polypeptide of the present invention.

FIG. 5 is a graph of LSAP-3 polypeptide of the invention and its activity on Hela cells in the presence of the MMP2/9 inhibitor SB-3 CT.

Detailed Description

In other embodiments of the invention, the polypeptide provided by the invention is a polypeptide comprising an amino acid sequence shown in SEQ ID NO.1, SEQ ID NO. 2 or SEQ ID NO. 3, and modified polypeptides thereof or homologous polypeptides thereof.

In other embodiments of the invention, a polypeptide "comprising SEQ ID NO: 1. the polypeptide "having an amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 3 includes, for example, a polypeptide consisting of SEQ ID NO:1, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. a polypeptide formed by adding a signal peptide sequence in an amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 3, and a polypeptide formed by adding a signal peptide sequence in the amino acid sequence shown in SEQ ID NO: 1. a polypeptide consisting of an amino acid sequence obtained by adding an appropriate tag sequence to the N-terminus and/or C-terminus of the amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 3.

The "modified polypeptide" in the present invention refers to a polypeptide contained in SEQ ID NO: 1. an amino acid sequence obtained by deleting, substituting, inserting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 3, and has anti-tumor activity.

In a preferred embodiment of the invention, the modification of an amino acid in the modified polypeptide or the polypeptide homologous thereto is a "conservative modification". For example, "conservative substitutions" refer to the substitution of one or more amino acid residues with other amino acids that are chemically similar, without substantially altering the activity of the protein. Examples thereof include a case where a certain hydrophobic residue is substituted with another hydrophobic residue, and a case where a certain polar residue is substituted with another polar residue having the same charge.

Functionally similar amino acids that can be conservatively substituted are well known in the art for the correspondence of amino acids. Specifically, the nonpolar (hydrophobic) amino acid includes alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, and the like. Examples of the polar (neutral) amino acid include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of the positively charged (basic) amino acid include arginine, histidine, and lysine. Examples of the negatively charged (acidic) amino acid include aspartic acid and glutamic acid.

By "homologous polypeptide" in the context of the present invention is meant a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1. an amino acid sequence represented by SEQ ID NO. 2 or SEQ ID NO. 3 having at least 85%, at least preferably 90%, at least preferably 92%, at least preferably 93%, at least preferably 94%, at least preferably 95%, at least preferably 96%, at least preferably 97%, at least preferably 98%, at least preferably 99%, more preferably 100% homology (sequence identity).

For The purposes of The present invention, The degree of sequence identity between two amino acid sequences is determined using The Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J.Mol.biol.48: 443-. The optional parameters used are gap open penalty (gap open penalty)10, gap extension penalty (gap extension penalty)0.5 and EBLOSUM62 (EMBOSS version of BLOSUM 62) substitution matrices. The output of the Needle labeled "highest identity" (obtained using the-nobrief option) is used as the percent identity and is calculated as follows:

(same residue X100)/(alignment Length-Total number of gaps in alignment)

The polypeptides of the invention may be natural, synthetic, semi-synthetic, or recombinantly produced. The polypeptide of the present invention can be produced by genetic engineering, by known peptide synthesis, or by digesting the polypeptide of the present invention with an appropriate peptidase.

In a preferred embodiment of the invention, the polypeptide of the invention is encoded by a recombinant DNA sequence in a host cell according to conventional bioengineering procedures to produce a polypeptide product, and may be synthesized by Solid Phase Synthesis or liquid Phase Synthesis, for example by the Solid Phase Biosystem synthesizer or the Pioneer Peptide synthesizer according to the procedures described by Steward and Young (Steward, J.M. and Young, J.D.), Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company, Rockford, I11., (1984)). In a preferred embodiment of the invention, the polypeptide of the invention can be prepared by Fmoc chemical synthesis starting from a solid-phase synthesis resin.

The present invention further provides polynucleotides encoding the polypeptides described above. The polynucleotides of the present invention can be used for in vivo or in vitro production of the polypeptides of the present invention as described above, or can be used for gene therapy of diseases attributable to genetic abnormalities in the gene encoding the polypeptides of the present invention. Any form of the polynucleotide of the present invention may be used so long as it encodes the polypeptide of the present invention, including mRNA, RNA, cDNA, genomic DNA, and chemically synthesized polynucleotides. The polynucleotide of the present invention includes a DNA comprising the specified nucleotide sequence and a barrel-fused sequence thereof, so long as the resulting DNA encodes the polypeptide of the present invention.

Polynucleotides of the invention can be prepared by methods known to those skilled in the art. For example, a polynucleotide of the invention can be prepared as follows: a cDNA library is prepared from cells expressing the polypeptide of the present invention, and hybridization is carried out using a partial sequence of the DNA thereof as a probe. cDNA libraries can be prepared, for example, by methods described in molecular cloning, written by Sambrook et al, cold spring harbor laboratory Press (1989); alternatively, a commercially available cDNA library can be used.

In addition, by sequencing the nucleotides of the resulting cDNA, the translated region encoded by the cDNA can be determined routinely, and the amino acid sequence of the polypeptide of the present invention can be easily obtained. Furthermore, genomic DNA can be isolated by screening a genomic DNA library using the obtained cDNA or a part thereof as a probe.

The present invention also provides a genetic engineering vector containing the polynucleotide encoding the polypeptide of the present invention. The genetic engineering vector can be a common vector or an expression vector. Specifically, commercially available expression vectors suitable for use in prokaryotic cells generally carry selectable markers and origins of cell replication, bacterial promoters such as lacI, T7, lambda PL and trp, and other genetic elements of the known cloning vector pBR322(ATCC 37017). Such commercially available vectors include pGEM (Promega) and pKK223-3 (Pharmacia). Suitable vectors derived from pBR322 can be selected depending on the appropriate promoter selected and the structural gene sequence to be expressed. GST prokaryotic expression systems may also be used in the present invention. Vectors suitable for eukaryotic cells carry eukaryotic promoters such as CMV, SV40, etc., and such vectors include pMT-hIL-3 (Madamong, Dichunhui, Ponzeny et al (1991) high tech letters 11: 26-29), pQE-9(Qiagen), pD10, pNH18A (Stratagene), pKK233-3, pDR540, pRIT5(Pharmacia), and pcDNA3, pCI, pWLNEO, pSG (Stratagene), pSVL Pharmacia).

The present invention also provides a host cell containing a vector of the present invention, which may be used to express a polypeptide of the present invention, including but not limited to: prokaryotic hosts such as E.coli, Bacillus, Streptomyces, and the like; eukaryotic hosts such as: saccharomyces, Aspergillus, insect cells such as Drosophila S2 and Spodoptera frugiperda Sf 9, animal cells such as CHO, COS (monkey kidney fibroblast Cell line, Gluzman (Cell 23: 175, 1981) human Cell lines such as the PC-3 Cell line, DU145 Cell line and other Cell lines capable of expressing compatible vectors.

The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide, polynucleotide, vector, host cell of the invention; and a pharmaceutically acceptable carrier or excipient, if necessary. Pharmaceutically acceptable carriers or excipients refer to non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or other formulation excipients.

The invention also provides an application of the polypeptide in preparing antitumor drugs, in particular an application in treating diseases related to expression of secretion integrin (integrin) or matrix metalloproteinase 2/9(MMP 2/9). Diseases associated with the expression of secretory integrin (integrin) or matrix metalloproteinase 2/9(MMP2/9) are, for example, cervical diseases, more preferably, the diseases are cervical cancers.

In preferred embodiments of the present invention, for example, the medicament may be administered orally, e.g., as sugar-coated tablets, capsules, microcapsules, etc., as desired; or non-orally, e.g., in the form of a sterile solvent or suspension in water for injection or any other pharmaceutically acceptable liquid. For example, the compounds may be combined with a pharmaceutically acceptable carrier or vehicle, such as sterile water, saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders and the like, in a unit dosage form for acceptable pharmaceutical administration. The amount of active ingredient in these formulations is within the appropriate dosage range indicated to be desired.

Examples of additives which can be used in the form of tablets or capsules are binding agents such as gelatin, corn starch and acacia, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin and alginic acid; lubricants such as magnesium stearate; sweetening agents such as sucrose, lactose or saccharin; fragrances such as peppermint. When the unit dosage form is a microcapsule, the above ingredients may also include a liquid carrier, such as an oil. Sterile compositions for injection may be formulated following conventional vehicle for pharmaceutical administration such as distilled water for injection. Physiological saline, dextrose and other isotonic liquids, including adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride, can be used as aqueous solutions for injection. These may be used in combination with suitable solubilizers, such as alcohols, in particular ethanol, polyols such as propylene glycol and polyethylene glycol, nonionic surfactants such as polysorbate 80. Sesame oil or soybean oil may be used as the oily liquid, and may be used in combination with methyl benzoate, benzyl alcohol as a solubilizer, and may be used in combination with a buffer such as a phosphate buffer, an acetic acid buffering liquid; analgesics such as procaine hydrochloride; stabilizers, such as benzyl alcohol; formulated with an antioxidant. The prepared injection can be filled into a suitable ampoule.

The pharmaceutical compounds of the invention may be administered to a patient using methods well known to those skilled in the art, such as intra-arterial, intravenous, transdermal injection, and intranasal, transbronchial, intramuscular, or oral administration. The dosage and method of administration vary according to the weight and age of the patient and the method of administration; and the skilled person can select it as usual. If the compound is encoded by DNA, the DNA may be inserted into a vector for gene therapy and the vector administered for treatment. The dose and administration method vary depending on the body weight, age, and symptoms of the patient, but can be appropriately selected by those skilled in the art.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, which are intended as illustrations of several aspects of the invention.

Example 1: preparation of LSAP-1 Polypeptides

The synthesis of the LSAP-1 polypeptide adopts a solid-phase synthesis method, and comprises the following steps for synthesizing 0.25 millimole polypeptide:

(1) activating resin: wang resin (purchased from Gill Biochemical Shanghai Co., Ltd.) to which Fmoc-protected glycine was attached was weighed out, poured into a clean and anhydrous solid phase reactor, and 5ml of DCM (dichloromethane) was added for dissolution and activation overnight;

(2) resin cleaning: pumping out the liquid in the reactor, adding 4ml DMF (N, N-dimethylformamide), shaking for 1min, pumping out, and repeating the operation for 8 times; collecting a small amount of activated resin for Kaiser detection;

(3) removing Fmoc protection: after the solvent is drained, 4ml of DMF solution containing 20% piperidine is added, the mixture is placed in a shaking table, and after shaking for 5min, the solvent is drained; adding 4ml of DMF solution containing 20% piperidine, placing in a shaking table, and shaking for 20 min;

(4) resin washing and piperidine removal: after the solvent is drained, 4ml of DMF solution is added, the mixture is placed in a shaking table and shaken for 1min, and then the mixture is drained; repeating the steps for 8 times until piperidine is completely removed;

(5) the amino acid to be incorporated (i.e. Fmoc protected valine) and coupling reagent were weighed on an electronic balance: dissolving 4 times of amino acid, 3.9 times of HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate) and 4 times of HOBT (1-hydroxybenzotriazole) in 4ml of DMF, mixing until completely dissolved, adding into a solid phase reactor, fully mixing with resin, and oscillating for five minutes by a shaking table;

(6) adding DIEA (N, N-diisopropylethylamine) with the molar weight 8 times that of the resin, fully mixing, placing in a shaking table, and reacting for 2 hours in a timing manner;

(7) sequentially accessing Fomc-protected proline, leucine, arginine, serine, leucine, alanine, arginine, phenylalanine, leucine, alanine, glycine, isoleucine and phenylalanine (purchased from Gill Biochemical Shanghai Co., Ltd.), and repeating the operation steps 2-6 when one amino acid is connected;

(8) the Kaiser test, ninhydrin with ammonia or primary amine produces a purple-red complex, the Kaiser reagent comprising: 6% ninhydrin ethanol solution; 80% phenol ethanol solution; 2% 0.001M KCN pyridine solution, taking a small amount of resin in (6) after the reaction is completed and resin in (2), adding 2-3 drops of each of three components in Kaiser reagent, heating at 100 ℃ for 1-2min, if the solution is blue or red brown, indicating that free amino groups exist, otherwise, indicating that the connection is complete;

(9) after the peptide chain is jointed, the resin is washed and deprotected twice by piperidine;

(10) washing the resin with DMF 10 times (4 ml each time); the resin was washed with DCM 10 times, 4ml each time;

(11) drying the sample in vacuum;

(12) after the sample is dried, transferring the resin into a chicken heart bottle, installing a magnetic stirrer, fixing the chicken heart bottle, slowly adding a mixed cutting reagent (trifluoroacetic acid, ultrapure water, benzylthioether, phenol, ethylene dithiol, 82.5:5:5:5:2.5), adding magnetons, fully stirring, and reacting at room temperature for 12 hours;

(13) after the reaction is finished, transferring the reactant into a solid phase reactor, reacting the resin which is not transferred in the solid phase reactor, standing for 10min in this way, flushing a heart-shaped flask with TFA (trifluoroacetic acid), pouring all the resin and the solution into the solid phase reactor, filtering the mixture under nitrogen flow, placing the filtrate in a round-bottom flask, and drying under nitrogen flow;

(14) blowing the sample in the round-bottom flask to be viscous, removing nitrogen, pouring about 20ml of glacial ethyl ether into the round-bottom flask to precipitate the polypeptide, fully scattering insoluble substances, then balancing, placing in a refrigerated centrifuge, centrifuging at 4 ℃ at 8000rpm/min for 15min, discarding supernatant, dissolving in 20ml of glacial ethyl ether again, scattering, and centrifuging; repeating the operation for 3 times, and vacuum drying the precipitate to obtain a crude polypeptide product;

(15) analyzing the purity of the crude polypeptide by using analytical HPLC, and purifying by using preparative HPLC;

(16) the purified target polypeptide is identified by ESI high resolution mass spectrometry, the mass spectrum is shown in figure 1, the molecular weight of the synthesized LSAP-1 polypeptide is 1729.70, and the amino acid sequence is shown in SEQ ID NO. 1.

Example 2: preparation of LSAP-2 Polypeptides

The synthesis of the LSAP-2 polypeptide adopts a solid-phase synthesis method, and the steps for synthesizing 0.25 millimole polypeptide are as follows:

(1) activating resin: weighing Wang resin (purchased from Gill Biochemical Shanghai, Inc.) connected with Fmoc-protected lysine, pouring into a clean and anhydrous solid phase reactor, adding 5ml DCM (dichloromethane) for dissolving and activating, and standing overnight;

(5) the amino acid to be incorporated (i.e. Fmoc-protected aspartic acid) and the coupling reagent were weighed on an electronic balance: dissolving 4 times of amino acid, 3.9 times of HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate) and 4 times of HOBT (1-hydroxybenzotriazole) in 4ml of DMF, mixing until completely dissolved, adding into a solid phase reactor, fully mixing with resin, and oscillating for five minutes by a shaking table;

(7) subsequently, sequentially accessing Fomc-protected glycine, arginine, glycine, isoleucine, leucine, glycine, valine, proline, leucine, arginine, serine, leucine, alanine, arginine, phenylalanine, leucine, alanine, glycine, isoleucine and phenylalanine (purchased from Gill Biochemical Shanghai Co., Ltd.), and repeating the operation steps 2-6 when each amino acid is connected;

(11) drying the sample in vacuum;

(16) the purified target polypeptide is identified by ESI high resolution mass spectrometry, the mass spectrum is shown in figure 2, the molecular weight of the synthesized LSAP-2 polypeptide is 2469.45, and the amino acid sequence is shown in SEQ ID NO. 2.

Example 3: preparation of LSAP-3 Polypeptides

The synthesis of the LSAP-3 polypeptide adopts a solid-phase synthesis method, and in order to synthesize 0.25 millimole of polypeptide, the steps are as follows:

(7) subsequently, sequentially accessing Fomc-protected glycine, arginine, aspartic acid, leucine, glycine, isoleucine, leucine, glycine, valine, proline, leucine, arginine, serine, leucine, alanine, arginine, phenylalanine, leucine, alanine, glycine, isoleucine and phenylalanine (purchased from Gill Biochemical Shanghai Co., Ltd.), and repeating the operation steps 2-6 when each amino acid is connected;

(11) drying the sample in vacuum;

(16) the purified target polypeptide is identified by ESI high resolution mass spectrometry, the mass spectrum is shown in figure 3, the molecular weight of the synthesized LSAP-3 polypeptide is 2811.30, and the amino acid sequence is shown in SEQ ID NO. 3.

Example 4: self-assembled forms of polypeptide LSAP-3

The self-assembled form of the polypeptide LSAP-3 is observed by a transmission electron microscope, and the operation steps are as follows:

(1) preparing LSAP-3 solution with concentration of 40 μ M, standing overnight at room temperature for self-assembly, and preparing 20mg/mL phosphotungstic acid (PTA) with pH adjusted to 6.0-7.0 by 0.1M sodium hydroxide;

(2) taking out a copper mesh special for TEM, dropping a polypeptide sample on the copper mesh to form a water drop, standing for 1min, sucking a small amount of liquid on the edge of the copper mesh by using filter paper, enabling the copper mesh to be in a semi-wet state, then dropping a drop of phosphotungstic acid dye solution, dyeing for about 1.5min, sucking dry by using the filter paper, and then placing the copper mesh in a culture dish for drying;

(3) the self-assembled state of the LSAP-3 polypeptide is observed by a transmission electron microscope, and as shown in FIG. 4, the solution containing the LSAP-3 polypeptide can be placed overnight at room temperature to form the nano-micelle by self-assembly.

Referring to the amino acid sequence of the LSAP-3 polypeptide, the N end is hydrophobic, the C end is hydrophilic, the polypeptide has amphipathy, and the LSAP-3 polypeptide forms self-assembly polypeptide micelles, so that the LSAP-3 polypeptide with self-assembly capability has higher stability in human serum.

Example 5: activity assays for LSAP-1 polypeptides, LSAP-2 polypeptides, and LSAP-3 polypeptides

The cell activity test of the polypeptide adopts an MTT method for detection, and the test steps are as follows:

(1) preparing a solvent: preparation of MTT solution: 5 mg/mL of PBS (phosphate buffer, from Gibco Co.) was used^-1MTT (thiazole blue, purchased from Sigma) was wrapped with tinfoil paper, stirred at room temperature for 30 minutes to aid dissolution, filtered with a 0.22 μ M filter membrane after complete dissolution, and finally stored at 4 ℃ in the dark; preparing a triple dissolving solution: 10g of SDS (sodium dodecyl sulfate), 5mL of isobutanol and 0.12mL of 10M hydrochloric acid, and dissolving the mixture by using double distilled water to prepare 100mL of solution; preparation of a culture medium: 90% DMEM medium (available from Gibco) and 10% FBS;

DMEM medium was used to culture Hela cells (human cervical cancer cell line, purchased from Shanghai cell Bank of Chinese academy of sciences)

(2) Adding 1mL of pancreatin (purchased from Sigma company) to digest when the Hela cells in the culture bottle grow to more than 80%, placing at 37 ℃, culturing in a 5% carbon dioxide incubator, observing the completion of cell digestion under a mirror, adding 4mL of culture medium, stopping, and centrifuging at the rotating speed of 900rpm/min for 4 min; discarding the supernatant, adding a culture medium, and gently blowing to disperse the cells in the culture medium uniformly;

(3) counting the number of cells by a cell counter, and adjusting the cell concentration to 5X 10 by adding a culture medium⁴mL^-1；

(4) Plating cell suspension, adding cell in a volume of 100 μ L/well and a cell number of 5000/well, and standing with 5% CO₂Incubating in an incubator at 37 ℃ for 16-18 hours;

(5) after the incubation is finished, polypeptide drugs with different concentrations are prepared by serum-free culture medium, added into a 96-well plate, and incubated with cells for 24 hours. Only adding culture medium (without cells) into a blank group, only adding culture medium into cells of a control group, and adding polypeptide medicines with different concentrations into an experimental group;

(6) after the reaction was completed, the medium was aspirated. Previously prepared MTT was diluted ten-fold with phenol red free whole medium and then 100. mu.L was added per well, wherein the blank was not added with MTT. Putting the 96-well plate into a cell culture box;

(7) after 4 hours of reaction, 100. mu.L of the triple reagent (10% SDS + 5% isobutanol +0.012mol/L HCl) was added to each well, mixed on a shaker at room temperature for 12-24 hours, OD at 570nm was measured with a microplate reader, and IC of each cell was calculated using SPSS software₅₀Values, as shown in table 1.

TABLE 1 toxicity of LSAP-1, LSAP-2, LSAP-3 polypeptides on Hela cells

Example 6: specificity of LSAP-3 polypeptides for MMP2/9

The specificity of the polypeptide LSAP-3 is detected by adopting an MTT method, and the detection steps are as follows:

(1) preparing a solvent: the preparation of MTT solution, triple lysis solution and culture medium is described in example 5.

The MMP2/9 inhibitor SB-3CT (available from Sigma-Aldrich) was used to inhibit MMP2/9 enzyme activity secreted by Hela cells.

(5) after the incubation is finished, polypeptide drugs with different concentrations are prepared by serum-free culture medium, added into a 96-well plate, and incubated with cells for 24 hours. The blank group is only added with culture medium (without cells), the polypeptide group is only added with culture medium to the control cells, the polypeptide group is added with polypeptide drugs with different concentrations, the inhibition group is added with 10 mu M SB-3CT to the control cells, and the inhibition group is added with polypeptide with different concentrations and 10 mu M SB-3 CT;

(7) after 4 hours of reaction, 100. mu.L of the triple reagent (10% SDS + 5% isobutanol +0.012mol/L HCl) was added to each well, mixed on a shaker at room temperature for 12-24 hours, and then measured for OD at 570nm with a microplate reader to calculate the cell viability.

Cell survival (%) ═ (OD)_{Polypeptide group}-OD_{Blank group})/(OD_{Control group}-OD_{Blank group})

As shown in fig. 5, the activity of polypeptide LSAP-3 was also inhibited in the case of MMP2/9 inhibition at lower concentrations (<8 μ M), thus demonstrating that the activity of polypeptide LSAP-3 can be further activated by MMP2/9, increasing its activity on tumor cells.

SEQUENCE LISTING

<110> Beijing university of chemical industry

<120> polypeptide with anti-tumor activity and application thereof

<130>

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 16

<212> PRT

<213> Artificial sequence

<400> 1

Phe Leu Gly Ala Leu Phe Arg Ala Leu Ser Arg Leu Leu Pro Val Gly

1 5 10 15

<210> 2

<211> 23

<212> PRT

<213> Artificial sequence

<400> 2

Phe Leu Gly Ala Leu Phe Arg Ala Leu Ser Arg Leu Leu Pro Val Gly

1 5 10 15

Leu Ile Gly Arg Gly Asp Lys

20

<210> 3

<211> 26

<212> PRT

<213> Artificial sequence

<400> 3

Phe Leu Gly Ala Leu Phe Arg Ala Leu Ser Arg Leu Leu Pro Val Gly

1 5 10 15

Leu Ile Gly Leu Leu Asp Arg Gly Asp Lys

20 25

Claims

1. A polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3.

2. A polynucleotide encoding the polypeptide of claim 1.

3. A vector comprising the polynucleotide of claim 2.

4. A host cell comprising the polynucleotide of claim 2 or the vector of claim 3.

5. The method for producing a polypeptide according to claim 1, which comprises using a solid phase synthetic resin as a starting material and carrying out Fmoc chemical synthesis.

6. A pharmaceutical composition comprising a therapeutically effective amount of the polypeptide of claim 1 as an active ingredient and a pharmaceutically acceptable carrier.

7. The use of the polypeptide of claim 1 in the preparation of a medicament for the treatment of cervical cancer.