CN102250252A - Gastrin-releasing peptide (GRP) guided fusion protein - Google Patents
Gastrin-releasing peptide (GRP) guided fusion protein Download PDFInfo
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- CN102250252A CN102250252A CN2010101733711A CN201010173371A CN102250252A CN 102250252 A CN102250252 A CN 102250252A CN 2010101733711 A CN2010101733711 A CN 2010101733711A CN 201010173371 A CN201010173371 A CN 201010173371A CN 102250252 A CN102250252 A CN 102250252A
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Abstract
The invention relates to a fusion protein which can directionally kill tumor cells. The fusion protein comprises a guiding part composed of gastrin-releasing peptide (GRP) and an effect part composed of cytotoxin, and has a characteristic of directionally reaching and killing tumor cells which overexpress GRP receptors.
Description
One. technical field
The treatment cancer has many means at present, comprising operation, radiation and chemotherapy etc., operation is very effective a kind of means, but be subjected to the restriction of the disease kind and the course of disease, all can't use under a lot of situations, chemicotherapy is the modal means of treatment cancer, but its side effect is very big, effect is not fully up to expectations, substitutes the chemicotherapy method so people attempt to find out the little and biotherapy method efficiently of a kind of side effect.
The present invention relates to a kind of fusion rotein, with the gastrin releasing peptide is the fusion rotein targeting part, can combine with the tumour cell of overexpression gastrin releasing peptide receptor specifically, deletant PE40 with false pseudomonas bacillus exotoxin A, and 5 amino acid mutations of its C-terminal are Lys-Asp-Glu-Leu, be PE40KDEL as fusion rotein effect part, kill tumor cell specifically.
Two. background technology
1. the targeting part of this project fusion rotein: gastrin releasing peptide (GRP):
Gastrin releasing peptide (gastrin releasing peptide, GRP) be the homologue of bombesin-like peptide., be a kind of effective secretogogue or adjusting peptide of gastrointestinal hormone, it is the hormone that exists in the neuroendocrine tissue of the nerve fiber of normal brain, stomach and fetus lung, also secrete GRP in many small cell lung cancer cell strains and the tumor tissues, and be rich in GRP acceptor (GRPR).(preparation of gastrin-releasing peptide precursor fusion rotein and the application in tumor research thereof.China's medicine and clinical, 2007 11 phases, 828-831)
Present studies show that, gastrin releasing peptide receptor produces high expression level in many human tumor cells, in lung cancer, digestive tract cancer and gynecological cancer, gastrin releasing peptide receptor belongs to G albumen lotus root connection acceptor, it is overexpression in tumor tissues, tumor tissues is had important growth effect, and vital role has been brought into play in aspects such as its generation in tumour, development and infiltration.(the progress of gastrin releasing peptide receptor in gynecological tumor.International healthy reproduction/birth control magazine, 2010, V29 (1): 828-831)
2. the toxin moiety of this project fusion rotein: PE40KDEL
False pseudomonas bacillus exotoxin A (PEA) is a kind of polypeptide toxin, and it enters cell by the PEA receptors bind with the target cell surface, and the ADP ribosylation of catalysis elongation factor 2 (EF-2) stops protein synthesis, and causes necrocytosis.The PEA molecule is made up of 3 parts, comprises receptors bind structural domain, transposition (striding film) structural domain and active structure domain.In order to eliminate the non-specific binding of PEA and cell, we remove the receptor domain of PEA molecule, after promptly removing PEA molecule N-terminal 1-252 amino acids, obtained the PE40 molecule, we suddenly change 5 amino acid of C-terminal of PE40 molecule then, sport Lys-Asp-Glu-Leu, obtained active higher toxin moiety, PE40KDEL.
3. being connected of targeting part and toxin moiety:
(His-Met-Ala-Glu-Glu-) two portions are coupled together by peptide bond, order is: targeting part (GRP)-5 peptides-toxin moiety (PE40KDEL), the fusion rotein sequence is seen SEQ ID NO1 by 5 peptides.
4. the characteristics of fusion rotein of the present invention:
Based on noted earlier, gastrin releasing peptide receptor produces high expression level in many human tumor cells, in lung cancer, digestive tract cancer and gynecological cancer, it is overexpression in tumor tissues, tumor tissues is had important growth effect, and vital role has been brought into play in aspects such as its generation in tumour, development and infiltration.So there are a lot of reports to carry out oncotherapy at present at gastrin-receptor, comprise the analogue and the taxol lotus root connection that use GRP, GRP links to each other with single-chain antibody, (the progress of gastrin releasing peptide receptor in oncotherapy.World Chinese digests magazine, and 2009, V17 (1): 63-67) GRP and signal or metal chelator lotus root connection (improved gastrin releasing peptide compounds.Chinese patent, application number: 200710188657.5).
There is certain limitation in these technology at oncotherapy, such as, chemotherapeutic such as GRP and taxol are the lotus root connection mutually, because the problem of its lotus root connection mode, chemotherapeutic comes off easily, cause drug failure, and chemotherapeutic before arriving tumor locus, whole medicine is encroached on normal cell easily owing to do not have selectivity; Secondly, GRP links to each other with single-chain antibody, because the effect of single-chain antibody does not have the effect of whole antibody strong, so it only plays the effect that suppresses tumour cell, and owing to the rapidity of tumor cell proliferation, can't play the purpose of effective control tumour; GRP and signal or metal chelator lotus root connection mainly is to use as diagnosis at present, as treatment means kill tumor cell efficiently.
Solved the shortcoming of above-mentioned the whole bag of tricks effectively by the present invention, the present invention is to use GRP as guiding, and PE40KDEL is as toxin moiety, efficiently the kill tumor cell.Because the two is to link to each other by 5 peptide peptide bonds, so do not have the problem that medicine comes off; And toxin moiety is only after entering tumour cell, can bring into play the effect of cell killing, and the normal cell surface does not have GRP acceptor or content extremely low, so it is very little for Normocellular toxicity, but specificity kill tumor cell, and because the high-level efficiency of PE40KDEL kill tumor cell can be controlled the development of tumour as treatment means.This patent has been invented the novel targeted drug of a class, is the fusion rotein targeted drug of selecting GRP and cytotoxic little function fragment to form, and it is little that it has a molecular weight, characteristics such as the few and high specificity of consumption.
Three. summary of the invention
Drug targeting in this patent partly comprises GRP, we remove the receptor domain of PEA molecule at the effect part, after promptly removing PEA molecule N-terminal 1-252 amino acids, obtained the PE40 molecule, we suddenly change 5 amino acid of C-terminal of PE40 molecule then, sport Lys-Asp-Glu-Leu, obtained active higher toxin moiety, PE40KDEL.By 5 peptides (His-Met-Ala-Glu-Glu-) two portions are coupled together by peptide bond, the order be:
Targeting part (GRP)-5 peptides-toxin moiety (PE40-KDEL), the fusion rotein aminoacid sequence is seen SEQ ID NO1.
As the fusion rotein among the present invention, be the artificial protein that a class nature does not have, it is characterized by the fusion rotein that GRP and PE40KDEL form, can use a kind of sequence with SEQ ID NO1 in the sequence table, also can use the aminoacid sequence of fusion rotein among the sequence table SEQ ID NO1 is modified, as inserting disappearance, as long as the sequence of suddenling change certain or some amino acid and obtaining is the basic biological activity that keeps original molecule.In other words, also can use adopt its aminoacid sequence substantially with sequence table SEQ ID NO1 in the identical albumen of fusion rotein aminoacid sequence, promptly use aminoacid sequence to have variant or its functional fragment more than or equal to 90% sequence identity.
Another aspect of the present invention relates to a kind of pharmaceutical composition, and it contains fusion rotein of the present invention, and optional pharmaceutically acceptable carrier.
Know that in the present invention term " pharmaceutically acceptable " means the animal that can be used for that pharmacy field generally acknowledges, more particularly can be used for the people's.Term " carrier " refers to thinner, adjuvant (for example (fully or not exclusively) freund's adjuvant), vehicle or be used to holds or the medium of administering therapeutic agent.
These pharmaceutical carriers can be sterile liquids, such as water and oil, comprise being derived from oil, animal, plant or synthetic oil, such as peanut oil, soybean oil, mineral oil, sesame oil, like that.When intravenously was used medicinal compositions, water was preferred carrier.Salt brine solution and aqueous dextrose and glycerine solution also can be used as liquid carrier, especially for Injectable solution.Suitable pharmaceutical excipient comprises starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talcum, sodium-chlor, milk powder, glycerine, propylene, ethylene glycol, water, ethanol, like that.If desired, composition can also comprise the wetting or emulsifying agent such as the hyaluronate sodium of lesser amt, or the pH buffer reagent.These compositions can be taked solution, suspension, milk sap, tablet, pill, capsule powder, slowly-releasing prescription, suchlike form.
In one embodiment of the invention, described pharmaceutical composition is the freeze dried injection form, wherein contains the fusion rotein of 0.01%-0.2%, and 5% N.F,USP MANNITOL, and pharmaceutically acceptable carrier.
Those of ordinary skills know, and described fusion rotein can be by the method for dna recombinant expression or the method preparation of chemosynthesis.In the present invention, described fusion rotein is to obtain by the recombinant expressed method of genetically engineered.
The structure of example one recombinant fusion protein grain and the acquisition of engineering bacteria
With the SEQ ID NO1 aminoacid sequence that designs is template, according to central dogma, selects the codon of intestinal bacteria preference, obtains the pairing nucleotide sequence of fusion rotein, re-uses full gene synthesis technology and obtains the above-mentioned purpose gene fragment.The recombinant technology of knowing by those of ordinary skills obtains recombination carrier pUC-GRP2, and the gene fragment of inserting carrier is carried out sequencing.After verifying the exactness of its gene order, amplification is preserved, and cuts down target gene fragment from this carrier, inserts in the expression vector recombinant plasmid called after pGRP2.Use CaCl2 method transformed into escherichia coli BL21DE3 then, be coated on and contain antibiotic screening plate culture medium surface, selecting positive bacteria drops on when being cultured to OD600 for 0.6-0.8 in the liquid nutrient medium and to add IPTG 1.0mM and induce 3-4 hour centrifugal collection thalline, a tangible protein expression band occurs during the 12%SDS-PAGE electrophoresis behind the broken bacterium of 8M urea, expression amount is 40%.Monoclonal antibody immunity trace calibrating through natural type PE, show positive reaction, again recombinant expression vector is imported in the expressive host bacterium, through culture expression, hydrophobic chromatography and ion exchange chromatography obtain target protein GRP-PE40KDEL, survey the activity that the method for living is measured the killing tumor cell of GRP-PE40KDEL, the engineering bacteria called after pGRP2/BL21DE3 that efficiently expresses fusion rotein that is obtained with the MTT cell.
The expression of example two target proteins
1) shaking bottle expresses: will be as the genetic engineering bacterium pGRP2/BL21DE3 of above-mentioned preparation, in the LB substratum, shake a bottle incubated overnight (37 ℃ 200rpm), were seeded in the LB substratum in 1: 30 by volume again, cultivate after 3 hours for 37 ℃, add 0.1mMIPTG and induced 4 hours.Collect thalline through the SDS-PAGE electrophoretic analysis, find fusion rotein (43KD) based on solubility expression, expression amount accounts for 40% of bacterial protein.
2) large scale fermentation is expressed:
A. substratum is formed:
A) seed liquor substratum (LB):
Tryptones: 10 grams per liters, yeast powder: 5 grams per liters, sodium-chlor: 10 grams per liters.
B) go up a jar substratum (15 liters):
Sodium phosphate dibasic: 250 grams, potassium primary phosphate: 110 grams, sodium-chlor: 13. grams, ammonium chloride: 30 grams, Tryptones: 120 grams.
Above composition is sterilized in jar together; After sterilizing separately, following composition adds fermentor tank.
Sal epsom: 30 grams, glucose: 450 grams, feed supplement (500 grams per liter): glucose.
Fermentation and abduction delivering:
I) seed culture:
Draw from the plate identified and to get bacterial classification, be inoculated among 50 milliliters of (250 milliliters triangular flasks) LB, these 50 milliliters of seed liquor are transferred among 700 milliliters of LB after 36 ℃, 200 rev/mins, 8 hours, 36 ℃, 200 rev/mins, overnight incubation.
Ii) ferment and the abduction delivering process:
During cultured seed liquid (OD600=5-8) added jar, mix up each parameter, 36 ℃, 150 change, dissolved oxygen 100%, begin fermentation; Beginning adds 2 hours with 2.5 fast streams after 5 hours, adds 800 milliliters of feed supplements approximately, adds 0.5 gram IPTG and induces (five hours).
Example three target protein purifying:
(1) hydrophobic chromatography
Adopt hydrophobic chromatoghaphy medium (Octyl Sepharose 4Fast Flow), balance liquid A adopts 25mM Tris-HCl, pH8,1M ammonium sulfate is after the balance, split sample on the centrifugal supernatant of bacterium (mending ammonium sulfate) to 1M, behind 5 column volumes of A balance, linear gradient elution, 0-20 column volume, 1-0.1M ammonium sulfate is collected the fusion rotein peak.
(2) anion-exchange chromatography
Adopt Source 30Q anion-exchange chromatography medium, balance liquid A is 25mM Tris-HCl, pH8, balance, the samples with water dilution that previous step obtains is gone up sample, balance, linear gradient elution for 4 times, 0-20 column volume, 0-0.5M NaCl collects purpose fusion rotein peak.
(3) anion-exchange chromatography
Adopt Q Sepharose High Performance chromatography media, balance liquid is 20mM PB, pH7.4, the samples with water dilution that previous step obtains is gone up sample for 3 times, adopts 20mM PB, pH7.4 after the balance, 0-1M NaCl, the gradient elution of 0-10 column volume is collected the target protein elution peak.
Example four determinations of activity (mtt assay)
Number with viable cell in the cell proliferation of MTT colorimetric method for determining, determine that with this fusion rotein kills and wounds the vigor of various tumour cells, selected tumour cell comprises SPC (human lung adenocarcinoma cell), MGC (gastric carcinoma cells), PC-3 (Human Prostate Cancer Cells) and A549 (human lung carcinoma cell).Get 3 of 96 porocyte culture plates, every hole inoculating cell number is 1 * 104,20 holes of every kind of cell repeated inoculation of every culture plate, 37 ℃, cultivate under the 5%CO2 condition, the fusion protein sample that adds proportional diluted then respectively, continue to cultivate 24,48 and 72h after, every hole adds 5g/L MTT 20 μ l and cultivates 4h again, go supernatant liquor to add methyl-sulphoxide 200 μ l then, culture plate is placed on the 10min that vibrates on the micro oscillator, after treating that MTT is dissolved fully by cell reduction formation Jia Keli, use microplate reader immediately, wavelength 570nm measures its absorbancy, calculates the target protein activity by the criterion calculation formula then.Above-mentioned experiment repeats 3 times, calculates its average IC50 value.The result: see Table 1,
Fusion rotein cytoactive in table 1. example
| Cell IC50 | SPC (ug/mL) | A549 (ug/mL) | MGC (ug/mL) | PC-3 (ug/mL) |
| Fusion rotein | 0.52 | 0.08 | ＞100 | 9.75 |
From table, can see that this fusion rotein is for the kill capability difference of different sorts tumour cell, and is the strongest to the A549 effect, and the MGC cell is not had effect.
GRP receptor determination on example five cytolemma (immunohistochemical methods method)
1. experiment material:
Tumor cell culture: SPC, A549, MGC, PC-3.
The one anti-monoclonal antibody that adopts mouse-anti GRP acceptor, working concentration 1-2g/ml.
The two anti-sheep anti-mouse iggs that adopt.Working dilution 1: 200-1: 1000.
0.01M PBS and the 0.01M PBS that contains 0.3%Triton X-100.
2. experimental design: see Table 2.：
The experimental design of table 2.GRP receptor determination
| Grouping | SPC | A549 | MGC | PC-3 |
| Negative control group | Not adding one resists | Not adding one resists | Not adding one resists | Not adding one resists |
| Experimental group | Adding one resists | Adding one resists | Adding one resists | Adding one resists |
3. experimental procedure:
1) with cell with 4% Paraformaldehyde 96 fix 4 ℃ 1 hour.
2) use PBS rinsing cell three times
3) use PBS rinsing cell then with handling cell 30min. under the 0.3%Triton X-100PBS room temperature.
4) cell and is resisted (1: 200, PBS) hatch 4 ℃ of 40h.
5) use PBS rinsing cell three times.
6) with fluorescein-labeled sheep anti-mouse antibody IgG (1: 200, PBS) incubated at room 2 hours.
7) use PBS rinsing cell three times.
8) use the confocal laser microscopic.
4. experimental result:
Negative control group and MGC experimental group: do not observe laser labelling.
A549 group: very strong laser labelling.
PC-3 group: more weak laser labelling.
Experimental result shows: this project fusion rotein is by GRP and cell surface GRP receptors bind kill tumor cell, tumor cell surface GRP acceptor is many more, the ability of fusion rotein killer cell is strong more, and tumor cell surface GRP acceptor is few more, and the ability of fusion rotein killer cell is weak more.
Sequence table
＜110〉the military veterinary institute of Military Medical Science Institute
＜120〉gastrin releasing peptide guiding fusion rotein
<160>1
<210>1
<211>392
<212>PRT
<213>SEQ?ID?NO：1
<400>1
Val?Pro?Leu?Pro?Ala?Gly?Gly?Gly?Thr?Val?Leu?Thr?Lys?Met?Tyr
1 5 10 15
Pro?Arg?Gly?Asn?His?Trp?Ala?Val?Gly?His?Leu?Met?His?Met?Ala
20 25 30
Glu?Glu?Gly?Gly?Ser?Leu?Ala?Ala?Leu?Thr?Ala?His?Gln?Ala?Cys
35 40 45
His?Leu?Pro?Leu?Glu?Thr?Phe?Thr?Arg?His?Arg?Gln?Pro?Arg?Gly
50 55 60
Trp?Glu?Gln?Leu?Glu?Gln?Cys?Gly?Tyr?Pro?Val?Gln?Arg?Leu?Val
65 70 75
Ala?Leu?Tyr?Leu?Ala?Ala?Arg?Leu?Ser?Trp?Asn?Gln?Val?Asp?Gln
80 85 90
Val?Ile?Arg?Asn?Ala?Leu?Ala?Ser?Pro?Gly?Ser?Gly?Gly?Asp?Leu
95 100 105
Gly?Glu?Ala?Ile?Arg?Glu?Gln?Pro?Glu?Gln?Ala?Arg?Leu?Ala?Leu
110 115 120
Thr?Leu?Ala?Ala?Ala?Glu?Ser?Glu?Arg?Phe?Val?Arg?Gln?Gly?Thr
125 130 135
Gly?Asn?Asp?Glu?Ala?Gly?Ala?Ala?Asn?Ala?Asp?Val?Val?Ser?Leu
140 145 150
Thr?Cys?Pro?Val?Ala?Ala?Gly?Glu?Cys?Ala?Gly?Pro?Ala?Asp?Ser
155 160 165
Gly?Asp?Ala?Leu?Leu?Glu?Arg?Asn?Tyr?Pro?Thr?Gly?Ala?Glu?Phe
170 175 180
Leu?Gly?Asp?Gly?Gly?Asp?Val?Ser?Phe?Ser?Thr?Arg?Gly?Thr?Gln
185 190 195
Asn?Trp?Thr?Val?Glu?Arg?Leu?Leu?Gln?Ala?His?Arg?Gln?Leu?Glu
200 205 210
Glu?Arg?Gly?Tyr?Val?Phe?Val?Gly?Tyr?His?Gly?Thr?Phe?Leu?Glu
215 220 225
Ala?Ala?Gln?Ser?Ile?Val?Phe?Gly?Gly?Val?Arg?Ala?Arg?Ser?Gln
230 235 240
Asp?Leu?Asp?Ala?Ile?Trp?Arg?Gly?Phe?Tyr?Ile?Ala?Gly?Asp?Pro
245 250 255
Ala?Leu?Ala?Tyr?Gly?Tyr?Ala?Gln?Asp?Gln?Glu?Pro?Asp?Ala?Arg
260 265 270
Gly?Arg?Ile?Arg?Asn?Gly?Ala?Leu?Leu?Arg?Val?Tyr?Val?Pro?Arg
275 280 285
Ser?Ser?Leu?Pro?Gly?Phe?Tyr?Arg?Thr?Ser?Leu?Thr?Leu?Ala?Ala
290 295 300
Pro?Glu?Ala?Ala?Gly?Glu?Val?Glu?Arg?Leu?Ile?Gly?His?Pro?Leu
305 310 315
Pro?Leu?Arg?Leu?Asp?Ala?Ile?Thr?Gly?Pro?Glu?Glu?Glu?Gly?Gly
320 325 330
Arg?Leu?Glu?Thr?Ile?Leu?Gly?Trp?Pro?Leu?Ala?Glu?Arg?Thr?Val
335 340 345
Val?Ile?Pro?Ser?Ala?Ile?Pro?Thr?Asp?Pro?Arg?Asn?Val?Gly?Gly
350 355 360
Asp?Leu?Asp?Pro?Ser?Ser?Ile?Pro?Asp?Lys?Glu?Gln?Ala?Ile?Ser
365 370 375
Ala?Leu?Pro?Asp?Tyr?Ala?Ser?Gln?Pro?Gly?Lys?Pro?Pro?Lys?Asp
380 385 390
Glu?Leu
392
Claims (5)
1. a genetic engineering fusion protein has the aminoacid sequence shown in the SEQ ID NO1.
2. polynucleotide sequence, its fusion rotein as claimed in claim 1 of encoding.
3. the fusion rotein in the claim 1 is the pharmaceutical composition of main component, mainly attacks all kinds of tumour cells of gastrin releasing peptide receptor unconventionality expression.
4. the host cell of the recombinant vectors in the claim 3 comprises bacterium, yeast and mammalian cell.
5. the fusion rotein in the claim 1 is to use the genetically engineered recombinant expression vector to obtain through conversion, fermentation and purifying.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109422810A (en) * | 2017-08-24 | 2019-03-05 | 孙立春 | The exploitation and application of full source of people or humanization bombesin receptor GRPR monoclonal antibody drug or diagnostic reagent |
| CN111500612A (en) * | 2020-03-30 | 2020-08-07 | 扬州大学 | Soluble porcine gastrin releasing peptide fusion protein expression vector and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1738650A (en) * | 2003-01-13 | 2006-02-22 | 伯拉考成像股份公司 | Improved gastrin releasing peptide compounds |
| CN1830489A (en) * | 2006-04-11 | 2006-09-13 | 中国药科大学 | Tumour polypeptide vaccine based on gastrin release peptide |
| CN1861194A (en) * | 2006-04-11 | 2006-11-15 | 中国药科大学 | Anti gastrin-releasing peptide nucleic acid vaccine and its prepn. method |
| CN101433713A (en) * | 2007-11-15 | 2009-05-20 | 北京诺思兰德生物技术有限责任公司 | GnRH-PE mutant fusion protein and uses thereof |
-
2010
- 2010-05-17 CN CN2010101733711A patent/CN102250252A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1738650A (en) * | 2003-01-13 | 2006-02-22 | 伯拉考成像股份公司 | Improved gastrin releasing peptide compounds |
| CN1830489A (en) * | 2006-04-11 | 2006-09-13 | 中国药科大学 | Tumour polypeptide vaccine based on gastrin release peptide |
| CN1861194A (en) * | 2006-04-11 | 2006-11-15 | 中国药科大学 | Anti gastrin-releasing peptide nucleic acid vaccine and its prepn. method |
| CN101433713A (en) * | 2007-11-15 | 2009-05-20 | 北京诺思兰德生物技术有限责任公司 | GnRH-PE mutant fusion protein and uses thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109422810A (en) * | 2017-08-24 | 2019-03-05 | 孙立春 | The exploitation and application of full source of people or humanization bombesin receptor GRPR monoclonal antibody drug or diagnostic reagent |
| CN111500612A (en) * | 2020-03-30 | 2020-08-07 | 扬州大学 | Soluble porcine gastrin releasing peptide fusion protein expression vector and application thereof |
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Application publication date: 20111123 |