CN1377887A

CN1377887A - Polypeptide for inhibiting growth and migration of blood vessel endothelial cell and endothelial stem cell and its preparing method and use

Info

Publication number: CN1377887A
Application number: CN01108179A
Authority: CN
Inventors: 刘建宁; 陈于红
Original assignee: Nanjing Landing Science And Technology Co Ltd; Molecular Medicine Institute Of Nanjing University
Current assignee: Nanjing Landing Science And Technology Co Ltd; Molecular Medicine Institute Of Nanjing University
Priority date: 2001-04-04
Filing date: 2001-04-04
Publication date: 2002-11-06
Anticipated expiration: 2021-04-04
Also published as: CN1220699C

Abstract

The present invention relates to the technological field of gene engineering to produce polypeptide medicine and chemosynthesizing polypeptide medicine. Through chemosynthesis of HSIFTPETNPRAGLEK polypetide, chemosynthesis of the gene of the polypeptide, high-efficiency expression in colibacillus and separation to purify, polypeptide medicine for inhibiting growth and migrating of blood vessel endothelial cell and endothelial stem cell is prepared. The polypeptide medicine is used to treat diseases relating to the growth of tumor cell and other endothelial cells.

Description

Suppress polypeptide of growth of vascular endothelial cell and endothelial stem cell and migration and its production and use

The present invention relates to a kind of polypeptide that suppresses vascular endothelial cell and endothelial stem cell growth and migration and its production and use.

1972, Judah Folkman has proposed the hypothesis of " tumor growth depends on angiogenic action ", along with some stimulate vasculogenesis and the discovery of checking molecule, closely-related somatomedin takes place (as VEGF with tumour more particularly, FGF, HGF, TGF β etc.) to the influence of blood vessel generation and the discussion of mechanism thereof, for above-mentioned hypothesis provides strong evidence.If can suppress the vasculogenesis around the tumour, just can prevent the tumour diffusion effectively, and force tumour constantly to reduce because of generating new cell.

1994, people such as O`Reily extract a kind of vasculogenesis and check material in serum of suffering from Lewies Lung Carcinomas mouse and urine, called after angiostatin, its molecular weight are 38KD, with preceding four the kringle structural domain homology degree of Profibrinolysin up to 98%.Experiment shows that but this albumen specificity suppresses the merisis of endotheliocyte in external and the body, and to epithelial cell and not effect of epidermic cell.By external injection angiostatin, can suppress the in-house vasculogenesis of primary tumor and metastatic tumor, thereby suppress growth of tumor.People such as Yihai Cao find that Profibrinolysin (kringle1-3) structural domain has the effect that the inhibition endotheliocyte stronger than kringle1-4 structural domain (angiostatin) generates, the concentration (ED50) that they reach maximum restraining effect one half is respectively 70nM, 135nM.1997, they reported Profibrinolysin kringle5 again and have had the function that capillary endothelial cells that stronger inhibition bFGF excites generates, and its ED50 is 50nM.

Subsequently, people have successively reported the different kringle of Profibrinolysin and have had the endothelial cell growth restraining effect more or less, and its active order is kringle1-5＞kringle5＞kringle1-3＞kringle1-4＞kringle3＞kringle2.

Angiostatin, kringle5 etc. are in the U. S. application patent, and wherein angiostatin has been used to clinical experiment, but result and unsatisfactory.Because it is just effective when it must use high dosage.Its curative effect and endostatin have synergetic property when share.Therefore, for oncotherapy, suddenly wait to develop the new formulation that has stronger angiogenesis suppression action than angiostatin and kringle5.

The objective of the invention is to experimental result, propose a kind of polypeptide that suppresses vascular endothelial cell and endothelial stem cell growth and migration and its production and use according to our existing plasminogen Kringle5 enzymatic fragment.

Technology contents of the present invention comprises:

1) utilize the preparation of chemical synthesis and reverse hplc purification process to have 16 peptides that can suppress vascular endothelial cell and endotheliocyte stem cell growth and migration, its sequence is HSIFTPETNPRAGLEK.

2) prepare the gene of above-mentioned peptide section with chemical synthesis, be cloned into coli expression carrier, carry out amalgamation and expression, purified and enzyme is cut the back and is obtained target polypeptides.

3) prepare the gene of above-mentioned peptide section with chemical synthesis.Its coding strand and complementary strand 3 ' end increases ATG and CAT respectively, obtains the tandem gene of this peptide section after connecting, and is cloned into pET31b, is the expressive host bacterium with BLR (DE3) LysS, and expression product obtains 16 peptide monomers through the cracking of bromination nitrile.

4) utilize cell and mouse transplanting tumor animal model mensuration biological activitys such as Bovine capillary endothelial cells (BCE), Calf pulmonaryarterial endothelial cells (CPAE), Human Umbilical Vein EndothelialCells (HUVEC), Endothelial progenitor cells cells such as (EPC).

The invention solves the big shortcomings of consumption such as Angiostatin, be used for the treatment of the relevant disease of endothelial cell growth clinically, comprise solid tumor, obesity, diabetes, congee shape arteriosclerosis etc. and associating or assisting therapy in chemotherapy, radiotherapy, effect is very good.

Embodiments of the invention:

1, suppress the synthetic of growth of endotheliocyte and endothelial stem cell and transfer polypeptide:

Chemical synthesis prepares HSIFTPETNPRAGLEK peptide section, and synthetic product is through C ₄The reverse chromatography purification of post HPLC, its HPLC purity are greater than 95%, and its molecular weight of mass spectroscopy is 1800 dalton.

2, the structure that suppresses growth of endotheliocyte and endothelial stem cell and transfer polypeptide tandem gene:

Two complementary strands of chemosynthesis 16 peptide genes, its sequence are respectively 5 ' CAC TCCATC TTC ACT CCg gAA ACT AAC CCg CgT gCT ggT CTg gAA AAAATg, 3 ' 5 ' TTT TTC CAg ACC AgC ACg Cgg gTT AgT TTC Cgg AgT gAA gAT ggA gTgCAT 3 '

Obtain encoding after above-mentioned two chain warp phosphorylations, the annealing gene of 16 peptides adds T ₄Dna ligase connects product obtains different sizes respectively through 1.5% agarose gel electrophoresis 16 peptide tandem genes.

16 peptide tandem genes are connected with the pET31b (+) that cuts through the AlwNI enzyme, connect product and change NovaBlue over to, through NdeI and XhoI enzyme cut screen positive colony.

3, suppress the expression and the inclusion body preparation of growth of endotheliocyte and endothelial stem cell and transfer polypeptide tandem gene:

Change the above-mentioned recombinant plasmid that contains tandem repeat locus over to expressive host bacterium BLR (DE3) lysS, the picking mono-clonal is in LB (containing 100 μ g/ml Amp), and 37 ℃ of shake overnight incubation are got the bacterium that spends the night and inserted in the fresh LB substratum with 2% ratio, 250rpm, 37 ℃ are cultured to OD ₆₀₀0.3～0.5, add IPTG to final concentration 0.5mM, continue to cultivate OD 3-6 hour ₆₀₀Reach about 2.0.Centrifugal, collection thalline add 1 * binding buffer liquid (5mM imidazoles, 40mMTrisHCl pH7.9,500mM NaCl) in 10ml/g thalline ratio, and carrying out ultrasonic bacteria breaking is to the no longer sticking silk fabric of bacterium liquid, 12000g, 4 ℃ of centrifugal 10min, collecting precipitation.

4, suppress the purifying and the CNBr cracking of growth of endotheliocyte and endothelial stem cell and transfer tandem polypeptide.

With the binding buffer liquid that contains the 6M Guanidinium hydrochloride inclusion body that suspends again, 12000rpm4 ℃ of centrifugal 10min removes insolubles, get Ni-NTA post on the supernatant, use binding buffer liquid (containing the 6M Guanidinium hydrochloride) successively, lavation buffer solution (20mM Tris-HCl, pH7.9,0.5MNaCl, 16mMimidazole, the 6M Guanidinium hydrochloride) each 8 bed volume washing.Use elution buffer (20mMTris-HCl, pH7.9,0.5MnaCl, 300mM imidazoles, 6M Guanidinium hydrochloride) wash-out at last, collect elution peak.To the water dialysed overnight, 2000g4 ℃ of centrifugal 10min, collecting precipitation.

Dissolve above-mentioned precipitation with 6ml 80%formic acid.Change the 50ml round-bottomed flask over to, add 0.2gCNBr, nitrogen is saturated, and 18-22hr is stirred in sealing, with 28 ℃ of evaporates to dryness of rotary evaporation liquid, uses 40% CH of small volume as far as possible ₃CN/60% H ₂O/0.1%TFA stirs 1 hour with dissolution precipitation, and 4 ℃ of centrifugal 10min of 12000g get supernatant, with 0.22 μ M membrane filtration, with RP-HPLC signing polypeptide purity.

5, the purifying that suppresses clone, expression and the expression product of growth of endotheliocyte and endothelial stem cell and transfer polypeptide:

The gene of chemical synthesis coding 16 peptides, its 5 ' and 3 ' end increases NcoI and EcoRI site respectively, being cloned among the pET30a, is the host bacterium with BL21 (DE3), behind 0.5mM IPTG abduction delivering, collect thalline, add 1 * binding buffer liquid (5mM imidazoles, 40mMTrisHCl pH7.9,500mM NaCl) in 10ml/g thalline ratio, carrying out ultrasonic bacteria breaking is to the no longer sticking silk fabric of bacterium liquid, 12000g, 4 ℃ of centrifugal 10min get Ni-NTA post on the supernatant, use binding buffer liquid successively, lavation buffer solution (20mMTris-HCl, pH7.9,0.5MNaCl, 16mM imidazole) each 8 bed volume washing.Use at last elution buffer (20mM Tris-HCl, pH7.9,0.5MnaCl, the 300mM imidazoles) wash-out, collect elution peak.To the water dialysed overnight, the enteropeptidase enzyme is cut, oppositely the chromatography purification target polypeptides.

6, suppress growth of endotheliocyte and endothelial stem cell and the bioactive mensuration of transfer polypeptide:

1. Bovine capillary endothelial cells (BCE) assay method: get well-grown BCE cell, through PBS washing, trysinization, with containing the suspension that the DMEM+1%GPS nutrient solution is made into 25,000/ml, add 24 orifice plates, every hole 0.5mL, 37 ℃, 10%CO ₂Cultivated under the condition 24 hours, and abandoned supernatant liquor behind the cell attachment, add 0.5mL DMEM+5% calf serum+10%GPS and testing sample, cultivate after 20 minutes for 37 ℃, add bFGF to final concentration 1ng/ml or VEGF to final concentration 25ng/ml.After 72 hours, the XTT method is surveyed viable count.

2. Calf pulmonary arterial endothelial cells (CPAE) assay method: method is the same.

3. Human Umbilical Vein Endothelial Cells (HUVEC) assay method: the cell that will grow in EBM+2% calf serum substratum adds to 96 orifice plates, and every hole 0.1mL (50,000Cells), 37 ℃, 10%CO ₂Cultivated 24 hours under the condition, abandon supernatant liquor behind the cell attachment, add 0.1mL EBM+2% calf serum+4ng/mL bFGF or 100ng/mL VEGF, continue to cultivate 48 hours, abandon supernatant, add 0.1mL EBM+2% calf serum and testing sample, cultivate after 20 minutes for 37 ℃, add 0.1ml substratum (EBM+2% calf serum+2ng/mL bFGF or 50ng/mL VEGF), after 24 hours, the XTT method is surveyed viable count.

More than the measurement result of three kinds of methods show that 16 peptides all are better than Angiostatin to three kinds of cell inhibiting effects.

4. Endothelial progenitor cells (EPC) external test method: get Balb ' c mouse, subcutaneous injection B16 cell 1 * 10 ⁶, can be observed the swollen lump of about 1cm about 10 days.Get its medullary cell, cultivate with the RPMI-1640 that contains VEGF, IGF, BFGF, after 12 days, harvested cell, PBS washing, counting.The antibody (1 μ g/mL) that adds anti-mouse FLK-1 was placed 1 hour for 4 ℃, and is centrifugal, and free antibodies is removed in the PBS washing.Kind property Beads 1 μ L with protein G separates FLK-1+ cell and counting.The result shows control group and adds in the medullary cell of 1.2 μ M Angiostatin, 1.2 μ M, 16 peptides that the ratio of FLK-1+ is respectively 9.6%, 2.7%, and 1.3%.

5. assay method in Endothelial progenitor cells (EPC) body: get Balb ' c mouse, subcutaneous injection B16 cell 1 * 10 ⁶, tail vein injection testing sample, continuous 5 days.Anesthetized mice is got peripheral blood from eye socket, handles 15 minutes with erythrocyte cracked liquid, and is centrifugal, PBS washed twice, counting.The antibody that adds fluorescently-labeled anti-mouse FLK-1, CD45 was placed 1 hour for 4 ℃, and is centrifugal, and free antibodies is removed in the PBS washing.Add stop buffer, Facs measures the cell count of FLK-1+/CD45+.The result shows: 0.4% is the FLK-1+/CD45+ cell in the normal mice peripheral blood, and behind tumor mouse and intravenous injection Angiostatin, 16 peptides, the ratio of FLK-1+/CD45+ is respectively 1.0%, 0.3% and 0.35% in the peripheral blood.

6. mouse entity knurl assay method: get Balb ' c mouse, subcutaneous implantation B16 cell 1 * 10 ⁶, the tail vein injection testing sample in continuous three weeks, is observed the tumor growth situation.

Claims

1, suppress the polypeptide of growth of vascular endothelial cell and endothelial stem cell and migration, it is characterized in that: its aminoacid sequence is HSIFTPETNPRAGLEK.

2, the polypeptide of growth of inhibition vascular endothelial cell according to claim 1 and endothelial stem cell and migration is characterized in that having 60% above homology and similar bioactive other similar synthetic polypeptide.

3, a kind of polypeptide preparation method who suppresses vascular endothelial cell and endothelial stem cell growth and migration is characterized in that preparing this polypeptide by chemical synthesis process.

4, the polypeptide preparation method of growth of inhibition vascular endothelial cell according to claim 3 and endothelial stem cell and migration is characterized in that chemical synthesis process obtains the gene of this peptide section, and wherein 3 of coding strand ' end increases ATG, and 3 of complementary strand ' end increases CAT; Use T ₄DNA ligase connect to obtain the tandem gene of this peptide section, and is cloned into PET31b, changes the plysS with BLR (DE3) over to, the picking mono-clonal to LB (containing 100ug/ml Amp), 37 ℃ of overnight incubation, after transferring by 2% ratio, 37 ℃ of shakes are cultured to OD ₆₀₀About 0.5, add IPTG to final concentration 0.5mM, continue to cultivate 4 hours.

5, the polypeptide preparation method of growth of inhibition vascular endothelial cell according to claim 4 and endothelial stem cell and migration, it is characterized in that described nutrient solution, carrying out ultrasonic bacteria breaking, extract inclusion body, under the sex change condition, carry out the Ni-NTA affinity chromatography, elutriant is after dialysis, and the cracking of bromination nitrile is with the access method extraction target polypeptides of quenching.

6, the polypeptide of growth of inhibition vascular endothelial cell according to claim 1 and endothelial stem cell and migration, the gene that it is characterized in that this peptide of chemosynthesis, be cloned into prokaryotic expression carrier, carry out N (C) and hold the amalgamation and expression that contains oligo-histidine or GST etc., expression product obtains target polypeptides after enzyme is cut.

7, the polypeptide of growth of inhibition vascular endothelial cell according to claim 1 and endothelial stem cell and migration is characterized in that utilizing Bovine capillary endothelial cells (BCE), Calf pulmonary arterial endothelial cells (CPAE), Human UmbilicalVein Endothelial Cells (HUVEC), Endothelial progenitor cells cells such as (EPC) and mouse to carry out its biological activity determination.

8, a kind of polypeptide purposes that suppresses vascular endothelial cell and endothelial stem cell growth and migration, it is characterized in that treating the relevant disease of endothelial cell growth as active substance, comprise solid tumor, obesity, diabetes, congee shape arteriosclerosis etc. and associating or assisting therapy in chemotherapy, radiotherapy with the polypeptide of claim 1.