CN1840546A - Recombinant fusion protein with targeted cell for killing tumor - Google Patents
Recombinant fusion protein with targeted cell for killing tumor Download PDFInfo
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- CN1840546A CN1840546A CN 200510059907 CN200510059907A CN1840546A CN 1840546 A CN1840546 A CN 1840546A CN 200510059907 CN200510059907 CN 200510059907 CN 200510059907 A CN200510059907 A CN 200510059907A CN 1840546 A CN1840546 A CN 1840546A
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Abstract
The invention provides a series of fusion proteins comprising human luteinizing hormone releasing factor (GnRH) or its mutant and pseudomonas exotoxin (PE) or its mutant. The medicinal purpose of the GnRH is for the highly effective eradication of tumor cells.
Description
One. technical field
The invention belongs to the guiding fusion rotein field in the biological gene engineering, present cancer remains mortality ratio and occupies second disease, the also not effective especially means of people are treated at present, chemotherapy remains present most common therapeutic method, though certain curative effect is arranged, but its side effect is big, and result of treatment has only the part ideal, the curative effect of this method and efficient that has been easy to generate disadvantages affect such as persister cancer cells during treatment.Targeted therapy is a kind of new treatment means that is expected to replace chemotherapy, the present invention is by selecting for use human interstital-cell-stimulating hormone's releasing hormone and derivative thereof as guiding, the derivative of selecting PE40 (part of removing the I structural domain of false pseudomonas bacillus exotoxin A) for use is as toxin moiety, obtained comparing with the GnRH-PE40 molecule, the active series of genes engineering fusion rotein that improves greatly has the potential using value aspect oncotherapy.
Two. background technology
Targeted therapy is meant that the medicine molecule can guide to arrive cancer cells and play a role, and very low to normal cytotoxicity, so reach the effect of targeted therapy.Immunotoxin or targeted toxin are important developing direction that reaches targeted therapy at present, be to be connected with toxin by specific antibody or cytokine etc., the former is as targeting part, it is part, can guide molecule arrive tumor locus and with this kind receptors bind of the overexpression of tumor cell surface, the latter promptly kills the part of tumour cell as toxin moiety.Existing in the world at present this similar drug listing, i.e. Ontak, indication was intractable cutaneous T cell lymphoma in U.S.'s listing in 1999, its targeting part is an interleukin II, and toxin moiety be diphtheria toxin (
Www.fda.gov).
Selection for targeting part and toxin moiety is an integral part setting up targeted drug, at first, targeting part can be selected hormones, such as human luteinizing hormone's releasing hormone (GnRH), cell growth factor, such as EGF, TGF etc., cytokine, as interleukin II, Interleukin-13, interleukin-6 etc., monoclonal antibodies etc. other.The selection of targeting part is very important, because if targeting part has too much combining with normal cell, toxin moiety will kill normal cell so, human luteinizing hormone's releasing hormone is a targeting part preferably, because it mainly is present in people's the hypophysis, because fusion rotein is the albumen that molecular weight is very big,, just can enter hypophysis and produce toxic side effect so can not pass through hemato encephalic barrier.Toxin moiety uses at present ETA (PE), diphtheria toxin (DT), Ricin etc., ETA (PE) is a class toxin relatively more commonly used, it is made up of 613 amino acid of false pseudomonas bacillus excretory, 3 structural domains are arranged, I plot structure territory is the cell receptor land, and the II district is for striding the film district, and the mediation lps molecule is striden film and entered cell, the III district is the active zone, by the elongation factor EF2 in the ribosylation cell, it is synthetic to stop albumen, cell killing (Allured.V etc.Proc Natl Acad Sci.1986,83,1320-1324).PE40 is the clipped form of PE, has deleted the I district of PE molecule, i.e. 1-252 amino acid of N art end has been eliminated the binding characteristic of the non-special and cell of PE molecule.The someone uses GnRH-PE66 and GnRH-PE40 as targeted toxin, the inoculated tumour of nude mice is subdued, but as a kind of therapeutical agent in the future, these two molecule biologically actives are low, the shortcoming that using dosage is big, each dosage such as every nude mice of GnRH-PE66 is 12.5ug, be equivalent to about 600ug/kg dosage, be clinical trial (the http://www.md.huji.ac.il/depts/humangenetics/lorberbaum/lorberb aum_research.html that can not bear; Med Oncol.1999Apr; 16 (1): 38-45.).The present invention makes its biological activity that significant rising arranged by multipoint mutation and transformation to GnRH-PE40, thereby makes it to become the targeted drug of treatment cancer.
Three. summary of the invention:
The present invention is by the transformation to GnRH-PE40, and making it biological activity has had significant raising.At first, aspect the transformation of PE molecule, by removing the 1-252 of former PE molecule, two sections aminoacid sequences of 365-380, two sections aminoacid sequences of remaining 253-364 and 381-613 link to each other by former order, become PE38; Remove the 1-279 amino acids of PE molecule, be left the 280-613 part, and 287 Cys are sported Ser or Ala or Gly, be PE37; Remove two sections aminoacid sequences of 1-279 and 365-380, remaining 280-364, two sections aminoacid sequences of 381-613 link to each other by former order, and 287 Cys is sported Ser or Ala or Gly, become PE35; PE38, PE37, the activity of the molecule of PE35 is all having raising than PE40 in varying degrees.And by with PE40, PE38, PE37,5 amino acid Arg-Glu-Asp-Leu-Lys of PE35 C-terminal replace with Lys-Asp-Glu-Leu, are the delay sequence of endoplasmic reticulum, can strengthen combining of molecule and endoplasmic reticulum, make active raising.And aspect targeting part, what we used is that first amino acids is the GnRH of Glu, and when the 6th of GnRH sported Ala, Trp perhaps during Leu, has strengthened itself and the combining of the acceptor of GnRH.
As GnRH-PE among the present invention and derivative thereof, be the artificial protein that a class nature does not have, it is characterized by the fusion rotein that human interstital-cell-stimulating hormone's releasing hormone GnRH or its mutant and PE40 or its mutant are formed, can use a kind of fusion rotein 1 in the sequence table that has, 2,3,4,5 sequence, also can use fusion rotein in the sequence table 1,2,3,4,5 aminoacid sequence 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 in fusion rotein 1,2,3,4, the albumen that 5 aminoacid sequences are identical.
The method those of skill in the art that obtain fusion rotein among the present invention can realize by the method for conventional molecular cloning, gene order synthetic by the full gene of multi-disc section and can synthesize target protein in conjunction with the method for PCR, cut means such as connection then by enzyme gene is connected into expression vector, be transformed into corresponding host then, express fermentation.Ferment the back by centrifugal collection thalline, method smudge cellses such as mechanical process or osmotic pressure, by a series of chromatography methods commonly used, as ion-exchange, gel-filtration and hydrophobic chromatography etc., purifying finally obtains target protein.The bioactive mensuration mtt assay of target protein, at first pair cell digests and collects, and cell is diluted to 6-8 * 10 with the RPMI RPMI-1640
4The cell suspension of individual/milliliter is inoculated in 96 orifice plates, the 100ul/ hole, and 37 degree were cultivated 4 hours.Contrast adds the 100ul nutrient solution.Sample is diluted to 100ul/mL with the RPMI RPMI-1640 earlier, as starter hole, presses the dilute sample that concerns in 2.5 times in every hole and last hole then, each adds the every hole 100ul of sample of dilution, and 37 degree were cultivated 24 hours, take out,, measure and calculate IC with microplate reader 570nm with mtt assay dyeing
50Value.
Four. embodiment:
Below the example purpose be further to set forth the present invention, and can not constitute the await the reply restriction of claim of patent of the present invention.
One. embodiment
Example one.
Protein sequence according to fusion rotein 1, principle according to the host's of different expression systems preference codon, design the prokaryotic expression gene order and the eukaryotic expression gene order of fusion rotein 1, synthesize gene order by full gene synthetic method in conjunction with the method for PCR, through after the sequence verification, goal gene is connected into expression vector, pET plasmid after the main application enhancements of protokaryon, eukaryotic system mainly adopts pIC9K plasmid series, the commentaries on classics of recombinant plasmid after the checking is imported in the host bacterium, through expressing, fermentation obtains the purpose thalline, then by behind mechanical means or the broken bacterium of osmometry, the centrifuging and taking supernatant, the method purifying by hydrophobic chromatography and ion exchange chromatography obtains fusion rotein 1.By electrophoresis, HPLC, mass spectrum, what methods such as N-terminal determined amino acid sequence were determined to obtain is target protein.Carry out the cytoactive test at last.
Example two.
Protein sequence according to fusion rotein 3, principle according to the host's of different expression systems preference codon, design the prokaryotic expression gene order and the eukaryotic expression gene order of fusion rotein 1, synthesize gene order in conjunction with the method for PCR by full gene synthetic method.Through after the sequence verification, goal gene is connected into expression vector, pET plasmid after the main application enhancements of protokaryon, eukaryotic system mainly adopts pIC9K plasmid series, recombinant plasmid after the checking changeed importing in the host bacterium, through expression, fermentation obtains the purpose thalline, then by behind mechanical means or the broken bacterium of osmometry, the centrifuging and taking supernatant, the method purifying by hydrophobic chromatography and ion exchange chromatography obtains fusion rotein 3.By electrophoresis, HPLC, mass spectrum, what methods such as N-terminal determined amino acid sequence were determined to obtain is target protein.Carry out the cytoactive test at last.
Example three.
On the basis of the gene order of fusion rotein 1, the method of application PCR sudden change is removed 4 amino acid Lys-Asp-Glu-Leu of its C-terminal, change Arg-Glu-Asp-Leu-Lys, by having obtained fusion rotein GnRH-PE40 with example 1 similar method, this is a reference substance.By electrophoresis, HPLC, mass spectrum, what methods such as N-terminal determined amino acid sequence were determined to obtain is target protein.Carry out the cytoactive test at last.
Example four.
On the basis of the gene order of fusion rotein 1, the method for using the PCR sudden change transform it gene order of fusion rotein 2 as, by having obtained fusion rotein 2 with example 1 similar method.By electrophoresis, HPLC, mass spectrum, what methods such as N-terminal determined amino acid sequence were determined to obtain is target protein.Carry out the cytoactive test at last.
Example five.
On the basis of the gene order of fusion rotein 3, the method for using the PCR sudden change transform it gene order of fusion rotein 4 as, by having obtained fusion rotein 4 with example 1 similar method.By electrophoresis, HPLC, mass spectrum, what methods such as N-terminal determined amino acid sequence were determined to obtain is target protein.Carry out the cytoactive test at last.
Example six.
On the basis of the gene order of fusion rotein 3, the method for using the PCR sudden change transform it gene order of fusion rotein 5 as, by having obtained fusion rotein 5 with example 1 similar method.Carry out the cytoactive test.
Example six.
On the basis of the gene order of fusion rotein 2, the method for using the PCR sudden change makes its 7th Gly into Ala, by having obtained Ala with example 1 similar method
7-fusion rotein 2.By electrophoresis, HPLC, mass spectrum, what methods such as N-terminal determined amino acid sequence were determined to obtain is target protein.Carry out the cytoactive test at last.
Example seven.
On the basis of the gene order of fusion rotein 4, the method for using the PCR sudden change is changed its 327 Gly and is Ala, by having obtained Ala with example 1 similar method
327-fusion rotein 4.By electrophoresis, HPLC, mass spectrum, what methods such as N-terminal determined amino acid sequence were determined to obtain is target protein.Carry out the cytoactive test at last.
The result of table 1. determination of activity:
| Cell IC 50Fusion rotein | SPC (ug/mL) | HL60 (ug/mL) | MGC (ug/mL) | MCF-7 (ug/mL) |
| The GnRH-PE40 contrast | 26.5 | 19.8 | 56.9 | 5.2 |
| Fusion rotein 1 | 4.8 | 15.4 | 18.7 | 1.5 |
| Fusion rotein 2 | 4.6 | 19.1 | 10.3 | 0.48 |
| Fusion rotein 3 | 16.8 | 22.4 | 15.6 | 5.1 |
| Fusion rotein 4 | 5.8 | 11.2 | 25.4 | 0.22 |
| Fusion rotein 5 | 2.5 | 8.7 | 23.8 | 0.14 |
| Ala 7-fusion rotein 2 | 2.2 | 5.2 | 6.8 | 0.11 |
| Ala 327-fusion rotein 4 | 1.4 | 2.1 | 20.1 | 0.08 |
As can be seen from the above table, the various derivatives of GnRH-PE40 are compared with GnRH-PE40, and the biological activity of killing and wounding of different tumour cells has been had raising in various degree.
Sequence table
＜110〉Beijing Nuo Silande Bioisystech Co., Ltd
＜120〉has the recombination fusion protein of targeted cell for killing tumor
<140>
<141>
<160>5
<210>1
<211>376
<212>PRT
＜213〉warm albumen 1
<400>1
Met Glu His Trp Ser Tyr Gly Leu Arg Pro Gly His Met Ala Glu
1 5 10 15
Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His
20 25 30
Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp
35 40 45
Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala
50 55 60
Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val
65 70 75
Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly
80 85 90
Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr
95 100 105
Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly
110 115 120
Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr
125 130 135
Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly
140 145 150
Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu
155 160 165
Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn
170 175 180
Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu
185 190 195
Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala
200 205 210
Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp
215 220 225
Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala
230 235 240
Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly
245 250 255
Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser
260 265 270
Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro
275 280 285
Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro
290 295 300
Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg
305 310 315
Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val
320 325 330
Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp
335 340 345
Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala
350 355 360
Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Lys Asp Glu
365 370 375
Leu
376
<2l0>2
<211>361
<212>PRT
＜213〉fusion rotein 2
<400>2
Met Glu His Trp Ser Tyr Gly Leu Arg Pro Gly His Met Ala Glu
1 5 10 15
Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His
20 25 30
Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp
35 40 45
Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala
50 55 60
Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val
65 70 75
Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly
80 85 90
Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr
95 100 105
Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly
110 115 120
Asn Asp Glu Ala Gly Ala Ala Asn Gly Pro Ala Asp Ser Gly Asp
125 130 135
Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly
140 145 150
Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp
155 160 165
Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg
170 175 180
Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala
185 190 195
Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu
200 205 210
Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu
215 220 225
Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg
230 235 240
Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser
245 250 255
Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu
260 265 270
Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu
275 280 285
Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu
290 295 300
Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile
305 310 315
Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu
320 325 330
Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu
335 340 345
Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu
350 355 360
Lys
361
<210>3
<211>376
<212>PRT
＜213〉fusion rotein 3
<400>3
Met Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His
1 5 10 15
Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp
20 25 30
Glu Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala
35 40 45
Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val
50 55 60
Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly
65 70 75
Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr
80 85 90
Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly
95 100 105
Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val Ser Leu Thr
110 115 120
Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly
125 130 135
Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu
140 145 150
Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn
155 160 165
Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu
170 175 180
Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala
185 190 195
Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp
200 205 210
Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala
215 220 225
Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly
230 235 240
Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser
245 250 255
Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro
260 265 270
Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pr0
275 280 285
Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg
290 295 300
Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val
305 310 315
Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp
320 325 330
Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala
335 340 345
Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Glu His Trp Ser
350 355 360
Tyr Gly Leu Arg Pro Gly Pro Gly Lys Pro Pro Arg Glu Asp Leu
365 370 375
Lys
376
<210>4
<211>349
<212>PRT
＜213〉fusion rotein 4
<400>4
Met Gly Trp Glu Gln Leu Glu Gln Ser Gly Tyr Pro Val Gln Arg
1 5 10 15
Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val
20 25 30
Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly
35 40 45
Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu
50 55 60
Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln
65 70 75
Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val
80 85 90
Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala Gly Pro Ala
95 100 105
Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala
110 115 120
Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly
125 130 135
Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln
140 145 150
Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe
155 160 165
Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg
170 175 180
Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly
185 190 195
Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp
200 205 210
Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val
215 220 225
Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu
230 235 240
Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His
245 250 255
Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu
260 265 270
Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg
275 280 285
Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val
290 295 300
Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala
305 310 315
Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Glu
320 325 330
His Trp Ser Tyr Gly Leu Arg Pro Gly Pro Gly Lys Pro Pro Arg
335 340 345
Glu Asp Leu Lys
349
<210>5
<211>333
<212>PRT
＜213〉fusion rotein 5
<400>5
Met Gly Trp Glu Gln Leu Glu Gln Ser Gly Tyr Pro Val Gln Arg
1 5 10 15
Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val
20 25 30
Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly
35 40 45
Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu
50 55 60
Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln
65 70 75
Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Gly Pro Ala Asp
80 85 90
Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu
95 100 105
Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr
110 115 120
Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu
125 130 135
Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu
140 145 150
Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser
155 160 165
Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp
170 175 180
Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala
185 190 195
Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro
200 205 210
Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala
215 220 225
Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro
230 235 240
Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly
245 250 255
Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr
260 265 270
Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly
275 280 285
Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile
290 295 300
Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Glu His
305 310 315
Trp Ser Tyr Gly Leu Arg Pro Gly Pro Gly Lys Pro Pro Arg Glu
320 325 330
Asp Leu Lys
333
Claims (11)
1. one group of serial fusion rotein of forming by human interstital-cell-stimulating hormone's releasing hormone (GnRH) or its mutant and false pseudomonas bacillus exotoxin A (PE) or its mutant.
2. according to claim 1, the aminoacid sequence of said fusion rotein 1 is:
Met-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Lys-Asp-Glu-Leu。
3. according to claim 1, the aminoacid sequence of said fusion rotein 2 is:
Met-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys。
4 according to claim 1, and said fusion rotein 3 sequences are:
Met-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys。
5 according to claim 1, and said fusion rotein 4 sequences are:
Met-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Ser-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Ala-Asp-Val-Val-Ser-Leu-Thr-Cys-Pro-Val-Ala-Ala-Gly-Glu-Cys-Ala-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-ValArg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys。
6 according to claim 1, and said fusion rotein 5 sequences are:
Met-Gly-Trp-Glu-Gln-Leu-Glu-Gln-Ser-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asn-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Glu-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Glu-Ala-Gly-Ala-Ala-Asn-Gly-Pro-Ala-Asp-Ser-Gly-Asp-Ala-Leu-Leu-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Leu-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Glu-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Glu-Glu-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Leu-Glu-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Val-Arg-Ala-Arg-Ser-Gln-Asp-Leu-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Leu-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Glu-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asn-Gly-Ala-Leu-Leu-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Leu-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Leu-Thr-Leu-Ala-Ala-Pro-Glu-Ala-Ala-Gly-Glu-Val-Glu-Arg-Leu-Ile-Gly-His-Pro-Leu-Pro-Leu-Arg-Leu-Asp-Ala-Ile-Thr-Gly-Pro-Glu-Glu-Glu-Gly-Gly-Arg-Leu-Glu-Thr-Ile-Leu-Gly-Trp-Pro-Leu-Ala-Glu-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Leu-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Glu-Gln-Ala-Ile-Ser-Ala-Leu-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Pro-Gly-Lys-Pro-Pro-Arg-Glu-Asp-Leu-Lys。
7 according to claim 2, and the 7th Gly of fusion rotein 1 can replace with Ala, Trp or Leu.
8 according to claim 3, and the 7th Gly of fusion rotein 2 can replace with Ala, Trp or Leu; Can remove simultaneously the Arg-Glu-Asp-Leu-Lys of C-terminal, replace with Lys-Asp-Glu-Leu.
9. according to claim 4, the 367th Gly of fusion rotein 3 can replace with Ala, Trp or Leu; Can remove simultaneously the Arg-Glu-Asp-Leu-Lys of C-terminal, replace with Lys-Asp-Glu-Leu.
10. according to claim 5, the 341st Gly of fusion rotein 4 can replace with Ala, Trp or Leu; Can remove simultaneously the Arg-Glu-Asp-Leu-Lys of C-terminal, replace with Lys-Asp-Glu-Leu.
11. according to claim 5, the 311st Gly of fusion rotein 5 can replace with Ala, Trp or Leu; Can remove simultaneously the Arg-Glu-Asp-Leu-Lys of C-terminal, replace with Lys-Asp-Glu-Leu.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101469031A (en) * | 2007-12-27 | 2009-07-01 | 合肥丽湖医药科技开发有限公司 | High cell toxicity targeting fusion protein |
| WO2010022639A1 (en) * | 2008-08-25 | 2010-03-04 | 北京博翱泰生物技术有限公司 | Target-specific double-mutant fusion protein |
| CN101433713B (en) * | 2007-11-15 | 2011-10-12 | 北京诺思兰德生物技术股份有限公司 | GnRH-PE mutant fusion protein and uses thereof |
| CN103864938A (en) * | 2014-03-24 | 2014-06-18 | 北京博翱泰生物技术有限公司 | Target-specificity double-mutant fused protein and preparation process thereof |
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2005
- 2005-04-01 CN CN 200510059907 patent/CN1840546A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101433713B (en) * | 2007-11-15 | 2011-10-12 | 北京诺思兰德生物技术股份有限公司 | GnRH-PE mutant fusion protein and uses thereof |
| CN101469031A (en) * | 2007-12-27 | 2009-07-01 | 合肥丽湖医药科技开发有限公司 | High cell toxicity targeting fusion protein |
| WO2010022639A1 (en) * | 2008-08-25 | 2010-03-04 | 北京博翱泰生物技术有限公司 | Target-specific double-mutant fusion protein |
| CN103864938A (en) * | 2014-03-24 | 2014-06-18 | 北京博翱泰生物技术有限公司 | Target-specificity double-mutant fused protein and preparation process thereof |
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