CN108314727B - Membrane type metalloprotease inhibiting protein and its use - Google Patents

Abstract

The invention belongs to the technical field of biological medicine, and claims a membrane type metalloprotease inhibiting protein T1^PrαTACEThe invention also discloses a membrane type metalloprotease inhibiting protein T1^PrαTACECan be used for preparing medicaments which take MT1-MMP or TACE endoprotease as targets.

Description

Membrane type metalloprotease inhibiting protein and its use

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to a membrane type metalloprotease inhibitory protein with a function of inhibiting the proliferation of human renal carcinoma cells.

Background

Membrane Type Matrix metalloproteinases (Membrane Type 1-Matrix MetalloProteinase, MT1-MMP) and the TNF- α Converting Enzyme TACE (TNF- α Converting Enzyme, TACE) are the two of the most widely studied endoproteases at present. The two proteases are positioned on the surface of a cell membrane, and can regulate the concentration of extracellular proteins and other bioactive substances under the condition of zinc ion combination, so that the growth, proliferation, invasion and metastasis of cancer cells are promoted. For normal cells, the enzymatic activities of MT-MMP and TACE are tightly regulated and inhibited by cellular endogenous metalloprotease inhibiting proteins (TIMPs). Four types of TIMP are currently known, including TIMP-1, TIMP-2, TIMP-3 and TIMP-4. These four types of typing correspond to the regulation and inhibition of a diverse range of metalloproteinases [ literature: 1 ].

TIMP-1 was first isolated and purified from human lymphoblasts in 1980. Originally named erythrocyte-enhancing factor (EPA). Subsequent studies have shown that TIMP-1 is a glycosylated secreted protein with a relative molecular mass of about 21kDa and has no inhibitory effect on MT1-MMP and TACE. Meanwhile, the research shows that TIMP-1 can enhance the activity of cancer cells as a cell growth factor.

Disadvantages of the existing protein (i.e., original TIMP-1): (1) original TIMP-1 is a soluble secreted protein. There has been no evidence that TIMP-1 can interact with MT1-MMP and TACE, whether intracellular or extracellular. (2) TIMP-1 had no inhibitory effect on MT1-MMP and TACE. Relevant Experimental determination, TIMP-1 is directed against K of MT1-MMP or TACE_i ^appAre all greater than 150 nM. (3) TIMP-1 can enhance cell activity, greatly limiting its value in cancer research.

Disclosure of Invention

The invention aims to solve the problem of providing a modified membrane type metalloprotease inhibiting protein T1 according to the metalloprotease inhibiting protein TIMP in the prior art^PrαTACE。

In a first aspect, the invention provides a modified membrane-type metalloprotease inhibiting protein T1^PrαTACEThe amino acid sequence is shown in Seq ID No. 1.

In a second aspect, the present invention provides the above-mentioned membrane-type metalloprotease inhibiting protein T1^PrαTACEThe application of the compound in preparing a medicine for treating tumors with MT1-MMP or TACE endoprotease as potential targets.

In a third aspect, the invention provides a medicament, a pack thereofMembrane-containing metalloprotease inhibiting protein T1^PrαTACEAnd a pharmaceutically acceptable excipient.

In a preferred technical scheme of the invention, the excipient is a carrier, a solvent, an emulsifier, a dispersant, a wetting agent, an adhesive, a stabilizer, a colorant and a perfume.

In the preferable technical scheme of the invention, the medicine is injection, tablets, capsules, granules, drops, granules or ointment.

In the technical scheme, the medicinal composition for treating the target spots by using the MT1-MMP or TACE endoprotease can pass through protein T1 with the purity of more than 98 percent (mass percentage)^PrαTACEAdding general medicinal auxiliary components, and making into tablet, capsule, granule, drop, lyophilized extract, granule, ointment or injection.

Pharmaceutical formulations and administration

According to conventional methods known in the art for preparing various dosage forms, T1^PrαTACEThe proteins may be formulated as solutions, emulsions, suspensions, dispersions, or inclusion complexes such as cyclodextrins in suitable pharmaceutical solvents or carriers, or as pills, tablets, lozenges, suppositories, sachets, dragees, granules, powders, reconstituted powders, or capsules along with solid carriers. The pharmaceutical compositions of the embodiments may be administered by a suitable route of delivery, for example, oral, parenteral, rectal, nasal, topical, or ocular routes, or by inhalation. Preferably, the composition is formulated for intravenous or oral administration.

For oral administration, the drug may be provided in solid form, such as tablets or capsules, or formulated as a solution, emulsion or suspension. Oral tablets may include the active ingredient mixed with pharmaceutically acceptable compatible excipients such as diluents, disintegrants, binding agents, lubricants, sweeteners, flavoring agents, coloring agents and preservatives. Suitable inert fillers include sodium and calcium carbonate, sodium and calcium phosphate, lactose, starch, sugar, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, and the like. Exemplary liquid oral excipients include ethanol, glycerol, water, and the like. Exemplary disintegrants include starch, polyvinylpyrrolidone (PVP), sodium starch glycolate, microcrystalline cellulose and alginic acid. Binders may include starch and gelatin. The lubricant, if present, may be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract, or may be enteric coated. Oral formulations may exist in the following forms: in the form of discrete units, such as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient; in the form of a powder or granules; in the form of a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or in the form of an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be formulated as a bolus, electuary or paste.

Oral capsules include hard and soft gelatin capsules. To prepare hard gelatin capsules, the active ingredient may be mixed with a solid, semi-solid or liquid diluent. The preparation of soft gelatin capsules can be achieved by mixing the active ingredients with: water, an oil such as peanut oil or olive oil, liquid paraffin, a mixture of mono-and diglycerides of short chain fatty acids, polyethylene glycol 400, or propylene glycol.

Tablets may be made by compression or molding, and optionally one or more accessory ingredients may be added to the tablets. Tableting may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, and optionally incorporating therein a binder (e.g. povidone, gelatin, hydroxypropylmethylcellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate, crospovidone, croscarmellose sodium), surfactant or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so that the active ingredient therein is slowly or controllably released, for example by using hydroxypropylmethyl cellulose in varying proportions to achieve the desired release profile.

The oral liquid may have the following form: suspensions, solutions, emulsions, or syrups, or can be lyophilized or presented as a dry product that can be reconstituted with water or other suitable carrier before being administered. Such liquid compositions may optionally comprise the following: pharmaceutically acceptable excipients such as suspending agents (e.g., sorbitol, methyl cellulose, sodium alginate, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, etc.); non-aqueous carriers such as oils (e.g., almond oil or fractionated coconut oil), propylene glycol, ethanol or water; preservatives (e.g., methyl or propyl p-hydroxybenzoic acid or sorbic acid); wetting agents such as lecithin; and-if desired-a flavouring or colouring agent.

For parenteral administration, including intravenous, intramuscular, intraperitoneal, intranasal or subcutaneous routes, the compositions may be prepared in the form of: a sterile aqueous solution or suspension buffered to an appropriate pH and isotonicity, or a parenterally acceptable oil. Suitable aqueous carriers include ringer's solution and isotonic sodium chloride. Such forms may be presented as follows: unit dose forms, such as ampoules or disposable injection devices; multi-dose forms, such as vials from which appropriate doses may be withdrawn; or in solid form or preconcentrate useful for preparing injectable formulations. Formulations suitable for parenteral administration, including intravenous administration, include the following forms: aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type previously described.

Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or appropriate amount of an active ingredient.

Dosage and route of administration

T1 for administration to an individual (e.g., human) as described herein^PrαTACEThe dosage may vary with the particular composition, method of administration, and the particular stage of the condition. In an amount sufficient to produce the desired reaction. In some embodiments, the amount of the composition is a therapeutically effective amount. In some embodiments, the amount of the composition is a prophylactically effective amount. In some embodiments, T1^PrαTACEThe total amount in the composition is below a level that induces a toxicological effect (i.e., the level is above a clinically acceptable toxicity level), or at a level where potential side effects can be controlled or tolerated when the composition is administered to an individual.

In some embodiments, protein T1^PrαTACEThe amount in the medicament is in any one of the following ranges: from about 0.5mg to about 5mg, from about 5mg to about 10mg, from about 10mg to about 15mg, from about 15mg to about 20mg, from about 20mg to about 25mg, from about 20mg to about 50mg, from about 25mg to about 50mg, from about 50mg to about 75mg, from about 50mg to about 100mg, from about 75mg to about 100mg, from about 100mg to about 125 mg.

In some embodiments, T1^PrαTACEThe amount of protein administered in the medicament is at least about 0.1mg/kg, 0.5mg/kg, 1mg/kg, 2.5mg/kg, 5mg/kg, 7.5mg/kg, 10mg/kg, 15mg/kg or 20mg/kg per day.

T1^PrαTACEThe effective dose of the protein can vary according to the administration mode, the age and weight of the patient, the severity of the disease condition and other relevant factors, and the recommended dose is 10-2000 mg/time and 1-2 times per day when the protein is orally administered; the recommended dose for parenteral administration is 5-500 mg/time, 1 time per day.

Although protein T1^PrαTACEThe original basic protein structure of TIMP-1 is retained, but the "MMP-binding ridge" (the actual binding site to the target MMP) and the structure of the carboxyl terminal are greatly edited and improved. Thereby leading the improved T1^{Pr αTACE}It also has inhibitory effect on MT1-MMP and TACE. In addition, the experimental data show that T1^PrαTACECan effectively inhibit human kidney cancer cells (Caki-1) in miceTumor formation effect in vivo.

Protein T1 of the present invention^PrαTACEHas the advantages that:

(1) original TIMP-1 had no significant inhibitory effect on MT1-MMP and TACE. And T1^PrαTACEIt has very high inhibiting effect. Determined by experiments, K_i ^appCan be as low as 7.70nM (inhibiting MT1-MMP) and 0.14nM (inhibiting TACE). Unlike the original water-soluble TIMP-1, newly developed T1^PrαTACESuccessfully expressed in cells and embedded into the surface of cell membranes. Subsequent immunofluorescence experiments further showed that T1 was located on the membrane surface^PrαTACEIt can be combined with MT1-MMP or TACE more easily.

(2) The experiment of a mouse xenogeneic cell tumor model shows that the model is different from original TIMP-1, T1^PrαTACEDoes not promote cell growth (i.e., loses the function of enhancing cell activity).

(3) Model experiments of mouse transplantation of human renal carcinoma cells (Caki-1) further showed that T1^PrαTACEHas the functions of effectively inhibiting the growth and the tumor formation of human kidney cancer cells in a mouse body.

Drawings

FIG. 1 shows T1^PrαTACEAnd amino acid sequence schematic of original TIMP-1.

FIG. 2 shows T1^PrαTACESchematic of the mechanism of inhibition of MT1-MMP and TACE is shown in T1^PrαTACEThe mechanism of action of (c). T1^PrαTACEProteins can be broadly divided into two relatively independent and functionally distinct domains.

(1) The first is an inhibitory domain, which can interact with MT1-MMP (K)_i ^app7.70nM, Table 1) and TACE (K)_i ^app0.14nM, Table 1) tightly bound to the active site of the enzyme, and efficiently inhibited the activity of metalloproteases by preventing the substrate from being enzymatically cleaved.

(2) The second is a transmembrane domain, comprising a GPI anchor, which allows free TIMP to intercalate into the cell membrane surface, closer to (also located on the membrane surface) MT1-MMP and TACE.

FIG. 3 is a diagram of a (cell membrane) non-penetrating immunofluorescence assay, in which FIG. 3A shows T1^PrαTACEAnd MT1-MMP in the cell membraneSurface-bound non-penetrating immunofluorescence maps; FIG. 3B is T1^PrαTACEAnd TACE bind to each other on the cell membrane surface in a non-penetrating immunofluorescence pattern.

FIG. 4 shows T1^PrαTACEAnd the effect of TIMP-1 on the inhibition of MT1-MMP enzymolysis gelatin.

FIGS. 5A,5B are changes in the concentrations of extracellular active TNF-. alpha.and HB-EGF.

FIG. 6 shows TIMP-1 and T1^PrαTACEEffect of expression of (a) on the tumorigenic volume of cancer cells in NOD/SCID mice (xenogenous cell tumorigenic model).

FIG. 7 shows T1 of the present invention^PrαTACESchematic amino acid sequence.

Detailed Description

The following describes embodiments of the present invention with reference to the drawings.

Materials (I) and (II)

1. Laboratory animal

NOD-SCID mouse (Beijing Witonglihua laboratory animal technology Co., Ltd.)

2. Drugs and reagents:

2.1 drug puromycin

Reagents TIMP-1 antibody (R & D), MT1-MMP antibody (Abcam), TACE antibody (Abcam), Alexa Fluor488 anti-mouse secondary antibody (Invitrogen), Alexa Fluor555 anti-rabbit secondary antibody (Invitrogen), DAPI blocking tablet (Invitrogen), 38% paraformaldehyde (Sigma), Green fluorescent gelatin (ThermoScientific: Oregon green 488), lipofectamine transfection kit (Invitrogen), propylene glycol methyl ether acetate (PMA), phosphate substrate (Sigma), TNF-alpha Elisa kit (Sino Bio), EcoR I endonuclease (ThermoScientific), Xho I endonuclease (ThermoCiientific), Apa I endonuclease (ThermoScientic), Nde I endonuclease (ThermoScientic), related gene sequences and primers were synthesized from biology.

2.3 instruments LS-55 fluorescence spectrometer (Perkinelmer Life Sciences), C1-Si laser confocal microscope (Nikon), upright fluorescence microscope (Nikon), inverted fluorescence microscope (Nikon), multifunctional microplate reader (Thermoscientific).

Second, example

(I) T1^PrαTACEInhibitory effects on MT1-MMP and TACE:

according to the literature methods:

1.Lee MH,Rapti M,Knauper V,Murphy G.Threonine 98,the pivotal residue of tissue inhibitor of metalloproteinases(TIMP)-1in metalloproteinase recognition.J.Biol.Chem.2004；279(17):17562-69.

lee MH, Rapti M, Murphy G.undercut the surface epitopes inhibitor of methacrylic-1 inactive agar membrane type 1-matrix methacrylic, J.biol.chem.2003; 278(41):40224-30. the concrete operation is as follows:

1. MT1-MMP (R & D, 918-MP-010) and TACE (R & D, 930-ADB-010) proteins were purchased from R & D.

2. The synthetic TIMP-1 gene sequence (GenBank: S68252.1) was prepared and PCR amplified using the following primers (synthetic) forward: 5' -GAACCATATGTGCACCTGTGTACCACCCCACCCA-3' (Nde I cleavage site is shown in Seq ID No. 3), reverse primer: 5' -TCAACTGCTCGAGTTAATGATGATGATGATGATGATGATGGGCTATCTGGGACCGCAGGGACTG-3' (Xho I cleavage site is shown in Seq ID No. 4) to obtain a PCR product.

3. Preparation of T1 for the same purpose^PrαTACESequence, forward primer: 5' -GAACCATATGTGCACCTGTTCCCCACCCCACCCA-3', as shown in Seq ID No.5, the reverse primer was identical to the TIMP-1 sequence to obtain a PCR product.

4. The two PCR products were digested with Nde I and Xho I, and inserted into pRSET-c expression vector (Invitrogen) digested with the same enzymes, respectively, to obtain recombinant plasmids carrying the target genes. The recombinant plasmid was sequenced (Shanghai Biotech Co., Ltd.) and the inserted target gene was not mutated.

5. Recombinant plasmids after sequencing TIMP-1 and T1^PrαTACEThe two target proteins are obtained by transforming into BL21(DE3) pLys competent cells respectively and using an E.coli protein expression system.

6. MT1-MMP and TACE are used as target proteins respectively, and the two purified proteins are subjected to kinetic analysis by an LS-55 fluorescence spectrometer.

As a result: as shown in table 1 below:

TABLE 1

T1^PrαTACEThe inhibitory effect on MT1-MMP and TACE is far stronger than that of the original TIMP-1

Table 1 shows T1^PrαTACEAnd the inhibitory effect of primary TIMP-1 on metalloprotease MT1-MMP and TACE, respectively. The same assay was used for all experiments, i.e., measuring fluorescent polypeptide substrate (Mca-K-P-L-G-L-Dpa-A-R-NH2, R) using LS-55 fluorescence spectroscopy&D Systems) to calculate the corresponding suppression effect. T1 whether directed to MT1-MMP or TACE^PrαTACE(K_i ^app7.70nM and 0.14nM, respectively) are much stronger than the original TIMP-1 (K)_i ^appAre all made of>150nM)。

(II) T1^PrαTACEAnd MT1-MMP (or TACE) are combined and aggregated on the surface of a cell membrane:

1. preparing a packaging lentivirus carrying a target protein coding gene:

a. the TIMP-1 gene sequence (GenBank: S68252.1) was prepared for transfecting cells and PCR amplified with the following primers, forward: 5' -GCAGCAGAATTCACCATGGCCCCCTTTGAGCCCCTGGCT-3' (EcoR I cleavage sites have been marked, as shown in Seq ID No. 6), reverse primer: 5' -TCAACTGGGGCCCTTAGGCTATCTGGGACCGCAGGGACTG-3' (Apa I cleavage site is indicated, as shown in Seq ID No, 7), to obtain a PCR product.

b. Preparation of T1 for the same purpose^PrαTACESequence, forward primer identical to TIMP-1, reverse primer: sequentially, 5' -GGCCTGAGATTCCCTCTCGTACTGGGCTATCTGGGACCGCAGGGACTG-3 as shown in Seq ID No. 8; 5'-CACAGGTGGGGAGGAGAAGAGGACCATGCTCGATCCTCTCTGGTAATAGGCCTGAGATTCCCTCTCGTACTG-3'; as shown in Seq ID No. 9; (iii) 5' -TAAACGGGCCCTCATCCCACTATTAGGAAGATGAGGAAAGAGATCAGGAGGATCACAGGTGGGGAGGAGAAGAGGAC-3', as shown in Seq ID No. 10; the PCR product is obtained by the sequential action of 1 forward primer and 3 reverse primersA compound (I) is provided.

c. Carrying out enzyme digestion on the obtained two PCR products by EcoR I and Apa I, and respectively connecting the two PCR products with a pLVX-puro (Takara) expression vector subjected to the same enzyme digestion to obtain a recombinant plasmid carrying a target gene. The recombinant plasmid was sequenced and the inserted target gene was not mutated.

d. After sequencing, a large amount of the target DNA was extracted with Maxiprep plasmid extraction kit (AXYGEN) and purified for use.

e.0.5ml of sterile ultrapure water was dissolved in Lenti-X virus packaging reagent (Takara 631276) and 10. mu.g of TIMP-1 and T1 were taken^PrαTACEThe DNA was mixed with the respective Lenti-X virus packaging reagents.

f. The above mixed solutions were added to Lenti-X293T cells seeded on 10cm cell culture dishes, respectively, and cultured at 37 ℃ under 5% carbon dioxide for 3 days, followed by collection of the supernatant culture solution.

g. Infecting CaKi-1 cells with a lentivirus culture containing a target gene, and screening the cells with puromycin antibiotic to obtain stable expression TIMP-1 and T1^PrαTACECaKi-1 cells.

2.T1^PrαTACEThe interaction and aggregation with MT1-MMP (or TACE) on the surface of cell membrane

According to the literature methods:

jiang B, Zhang Y, Liu J, et al, engineering, molecular type 1-matrix metalloproteinase (MT1-MMP) with a Tissue Inhibitor of Metalloproteinase (TIMP) -2using the haemotaxin domain of the protease as a carrier: a target associated with a protease inhibitor inhibition. on target,2017,8(14): 22685. The specific operation is as follows:

(1) inoculation with stably expressed TIMP-1 and T1^PrαTACEThe CaKi-1 cells are put on a cavity type glass slide, cultured for 2 days at the temperature of 37 ℃ under the condition of 5 percent of carbon dioxide,

(2) cells were fixed in 4% paraformaldehyde for 10 min, then washed 3 times with PBS,

(3) the slides were blocked with PBS containing 5% BSA, left at room temperature for 2 hours,

(4) two antibodies TIMP-1+ MT1-MMP/TIMP-1+ TACE from different sources are added on the glass slide, and incubated overnight at 4 ℃,

(5) the slides were rinsed 3 times with PBS for 5 minutes each,

(6) simultaneously adding two secondary antibodies with different fluorescence, incubating for 2 hours at room temperature,

(7) after 3 times of PBS immersion cleaning, the sealing piece is ready for use,

(8) the prepared slide was observed with a confocal laser microscope.

As a result: (cell membrane) non-penetrating immunofluorescence assay employing stable expression of TIMP-1 and T1^PrαTACEThe CaKi-1 cell line of (1). The original TIMP-1 was soluble in water and thus not present on the cell membrane surface. As shown in fig. 3A and 3B, T1^PrαTACEIt is only present on the surface of the cell membrane and binds and aggregates with MT1-MMP or TACE on the membrane surface.

(III) T1^PrαTACEInhibiting MT1-MMP from disintegrating animal gelatin matrix:

according to the literature methods:

jiang B, Zhang Y, Liu J, et al, engineering, molecular type 1-matrix metalloproteinase (MT1-MMP) with a Tissue Inhibitor of Metalloproteinase (TIMP) -2using the haemotaxin domain of the protease as a carrier: a target associated with a protease inhibitor inhibition. on target,2017,8(14): 22685. The specific operation is as follows:

1. the successful lentivirus packaging was used to infect HT1080 cells, and stable expression of TIMP-1 and T1 was obtained in the same manner^PrαTACEThe presence of HT1080 cells in a human,

2. coating the cavity type glass slide with 0.5mg/ml fluorescent gelatin, then placing for 2 hours at room temperature,

gently rubbing and washing the slide with PBS for 2 times, then fixing gelatin with 4% paraformaldehyde for 10 minutes,

4. the washing step was repeated 5 times to ensure sufficient removal of paraformaldehyde,

5. inoculation with Stable expression of TIMP-1 and T1, respectively^PrαTACEThe HT1080 cells of (1) were cultured for 1 day,

6. the cells were fixed in the same manner, and then washed 3 times with PBS,

7. cold methanol penetrated the cell membrane for 20 minutes (at low temperature), PBS washed 3 times,

8. after blocking the slides with blocking solution containing 5% BSA + 0.3% Triton X-100, MT1-MMP antibody was incubated overnight at 4 ℃,

9. incubating the fluorescent secondary antibody for 2 hours at room temperature, sealing for later use,

10. observed and photographed with an upright fluorescence microscope.

As a result: this experiment was performed to enable stable expression of TIMP-1 and T1^PrαTACEThe human fibrosarcoma HT1080 cells were used as samples. It was seeded on a fluorescent gelatin-based slide glass and incubated overnight. The experimental result shows that T1 is stably expressed as shown in FIG. 4^PrαTACEThe cell has significantly inhibited the ability of gelatin to enzymolyze, much lower than that of TIMP-1 expressing cells.

(IV) comparison with original TIMP-1, T1^PrαTACECan inhibit TACE more efficiently, thereby effectively blocking activation and release of TNF-alpha and HB-EGF by (TACE):

according to the literature methods:

duan J X, Rapti M, Tsigkou A, Lee MH. expanding the activity of tissue inhibitors of metallic enzymes (TIMP) -1against surface-treated metallic enzymes by the replacement of its C-terminal domain: indications for anti-cancer effects [ J ]. PloS one,2015,10(8): e 0136384. The specific operation is as follows:

1. three groups of HT1080 cells were seeded into 24-well plates, control, TIMP-1 (Stable expression) and T1^{Pr αTACE}Stably expressed) groups, each group of cells was inoculated into 3 wells, and the number of cells per well was 1 × 10⁵And cultured overnight.

2. 0.1. mu.g of HB-EGF/TNF-. alpha.plasmid was transfected and cultured for 2 days.

3. Old culture medium was aspirated, and then fresh culture medium was added to one well of each group of cells, culture medium containing PMA (200ng/ml) was added to the other well, and the cells were cultured for 3 hours, and the same treatment was performed on the three groups of cells.

4. The culture medium (containing HB-EGF/TNF-. alpha.) from each well was collected and stored for further use.

5. A culture solution containing HB-EGF (-PMA/+ PMA 200ng/ml) was added to diethanolamine buffer with an alkaline phosphate substrate, which was then transferred to a 96-well plate and left at 37 ℃ until color change.

6. And reading the absorbance of the HB-EGF by a microplate reader.

The concentration of TNF- α (-PMA/+ PMA 200ng/ml) was measured by TNF- α ELISA kit (Sino Bio).

As a result: changes in the concentrations of extracellular active TNF-. alpha.and HB-EGF indicate that T1^PrαTACEThe inhibition effect on TACE is obviously better than that of the original TIMP-1.

The in vitro experiment uses the polypeptide capable of stably expressing T1^PrαTACEAnd TIMP-1 HT1080 cells as samples, and the concentration changes of extracellular TNF-alpha and HB-EGF were detected, thereby reflecting the intensity of TACE enzyme activity in each cell line. The two groups of experimental results show that T1 is combined^PrαTACEThe inhibition effect on TACE is stronger than that of the original TIMP-1.

(V) T1^PrαTACEThere was no effect (in the vector experiments) to promote cell growth:

materials: t175 flask (Corning), cell counting slide (Invitrogen), 1ml syringe

Reagent: 0.4% Trypan blue solution (Sigma), DMEM

The instrument comprises the following steps: automatic cell counter (Invitrogen), vernier caliper, balance

The method comprises the following steps:

1. culture of blank CaKi-1 cells stably expressing Primary TIMP-1 and T1^PrαTACECaKi-1 cells until the number of cells required for the experiment is reached.

2. The cells were collected separately and then stained and counted with a 0.4% trypan blue solution.

DMEM resuspending cells to a concentration of 2X10⁶/ml。

Randomly dividing NOD/SCID mice into 3 groups, control group, TIMP-1 group, and T1^PrαTACEGroups of 8 mice each,

5.0.1 ml/position was injected subcutaneously into the abdomen of mice, one in each of the left and right abdomen.

6. Mice were cultured continuously for 35 days, and the tumor size was measured twice a week (caliper measurement).

Mice were sacrificed after 7.35 days and the tumors weighed.

As a result: as shown in FIG. 6, 6 of 8 mice implanted with TIMP-1 cells formed different volumes (20 mm in volume)³To 248mm³In between). But in the control group and implanted with T1^PrαTACENo cell neoplasia (. about.p.) was found in mice with cells<0.05)。T1^PrαTACEHas no effect of promoting cell growth, and is different from original TIMP-1.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited by the foregoing examples, which are provided to illustrate the principles of the invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention, which is also intended to be covered by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

<110> university of west Crossware Ph

<120> membrane-type metalloprotease inhibitory protein and use thereof

<160> 10

<210> 1

<211> 245

<212> PRT

<213> Artificial Synthesis of T1Pr α TACE

<400> 1

Met Ala Pro Phe Glu Pro Leu Ala Ser Gly Ile Leu Leu Leu Leu Trp

1 5 10 15

Leu Ile Ala Pro Ser Arg Ala Cys Thr Cys Ser Pro Pro His Pro Gln

20 25 30

Thr Ala Phe Cys Asn Ser Asp Leu Val Ile Arg Ala Lys Phe Val Gly

35 40 45

Thr Pro Glu Val Asn Gln Gly Pro Phe Gly Thr Gln Arg Tyr Glu Ile

50 55 60

Lys Met Thr Lys Met Tyr Lys Gly Phe Gln Ala Leu Gly Asp Ala Ala

65 70 75 80

Asp Ile Arg Phe Val Tyr Thr Pro Ala Met Glu Ser Leu Cys Gly Tyr

85 90 95

Phe His Arg Ser His Asn Arg Ser Glu Glu Phe Leu Ile Ala Gly Lys

100 105 110

Leu Gln Asp Gly Leu Leu His Ile Thr Leu Cys Ser Phe Val Ala Pro

115 120 125

Trp Asn Ser Leu Ser Leu Ala Gln Arg Arg Gly Phe Thr Lys Thr Tyr

130 135 140

Thr Val Gly Cys Glu Glu Cys Thr Val Phe Pro Cys Leu Ser Ile Pro

145 150 155 160

Cys Lys Leu Gln Ser Gly Thr His Cys Leu Trp Thr Asp Gln Leu Leu

165 170 175

Gln Gly Ser Glu Lys Gly Phe Gln Ser Arg His Leu Ala Cys Leu Pro

180 185 190

Arg Glu Pro Gly Leu Cys Thr Trp Gln Ser Leu Arg Ser Gln Ile Ala

195 200 205

Gln Tyr Glu Arg Glu Ser Gln Ala Tyr Tyr Gln Arg Gly Ser Ser Met

210 215 220

Val Leu Phe Ser Ser Pro Pro Val Ile Leu Leu Ile Ser Phe Leu Ile

225 230 235 240

Phe Leu Ile Val Gly

245

<210> 2

<211> 738

<212> DNA

<213> Artificial Synthesis

<400> 2

atggccccct ttgagcccct ggcttctggc atcctgttgt tgctgtggct gatagccccc 60

agcagggcct gcacctgttc cccaccccac ccacagacgg ccttctgcaa ttccgacctc 120

gtcatcaggg ccaagttcgt ggggacacca gaagtcaatc agggtccgtt cggcacccag 180

cgttatgaga tcaagatgac caagatgtat aaagggttcc aagccttagg ggatgccgct 240

gacatccggt tcgtctatac ccccgccatg gagagtctct gcggatactt ccacaggtcc 300

cacaaccgca gcgaggagtt tctcattgct ggaaaactgc aggatggact cttgcacatc 360

actctctgca gtttcgtggc tccctggaac agcctgagct tagctcagcg ccggggcttc 420

accaagacct acactgttgg ctgtgaggaa tgcacagtgt ttccctgttt atccatcccc 480

tgcaaactgc agagtggcac tcattgcttg tggacggacc agctcctcca aggctctgaa 540

aagggcttcc agtcccgtca ccttgcctgc ctgcctcggg agccagggct gtgcacctgg 600

cagtccctgc ggtcccagat agcccagtac gagagggaat ctcaggccta ttaccagaga 660

ggatcgagca tggtcctctt ctcctcccca cctgtgatcc tcctgatctc tttcctcatc 720

ttcctaatag tgggatga 738

<210> 3

<211> 34

<212> DNA

<213> Artificial Synthesis

<400> 3

gaaccatatg tgcacctgtg taccacccca ccca 34

<210> 4

<211> 64

<212> DNA

<213> Artificial Synthesis

<400> 4

tcaactgctc gagttaatga tgatgatgat gatgatgatg ggctatctgg gaccgcaggg 60

actg 64

<210> 5

<211> 34

<212> DNA

<213> Artificial Synthesis

<400> 5

gaaccatatg tgcacctgtt ccccacccca ccca 34

<210> 6

<211> 39

<212> DNA

<213> Artificial Synthesis

<400> 6

gcagcagaat tcaccatggc cccctttgag cccctggct 39

<210> 7

<211> 40

<212> DNA

<213> Artificial Synthesis

<400> 7

tcaactgggg cccttaggct atctgggacc gcagggactg 40

<210> 8

<211> 48

<212> DNA

<213> Artificial Synthesis

<400> 8

ggcctgagat tccctctcgt actgggctat ctgggaccgc agggactg 48

<210> 9

<211> 72

<212> DNA

<213> Artificial Synthesis

<400> 9

cacaggtggg gaggagaaga ggaccatgct cgatcctctc tggtaatagg cctgagattc 60

cctctcgtac tg 72

<210> 10

<211> 77

<212> DNA

<213> Artificial Synthesis

<400> 10

taaacgggcc ctcatcccac tattaggaag atgaggaaag agatcaggag gatcacaggt 60

ggggaggaga agaggac 77

Claims (5)

1. Membrane type metalloprotease inhibiting protein T1^PrαTACEThe amino acid sequence is shown in Seq ID No. 1.

2. The membrane-type metalloprotease inhibiting protein T1 according to claim 1^PrαTACEThe application of (1) is characterized by being used for preparing a medicine for treating tumors with MT1-MMP or TACE endoprotease as a potential target.

3. A medicament comprising the membrane-type metalloprotease inhibiting protein T1 according to claim 1^PrαTACEAnd a pharmaceutically acceptable excipient.

4. The pharmaceutical of claim 3, wherein the excipient is a carrier, solvent, emulsifier, dispersant, wetting agent, binder, stabilizer, colorant, or fragrance.

5. The medicament of claim 3, wherein the medicament is an injection, a tablet, a capsule, a granule, a drop, a granule or an ointment.

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