CN1846785A - Freeze dried composition containing genetic engineering fusion protein - Google Patents
Freeze dried composition containing genetic engineering fusion protein Download PDFInfo
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- CN1846785A CN1846785A CN 200510063136 CN200510063136A CN1846785A CN 1846785 A CN1846785 A CN 1846785A CN 200510063136 CN200510063136 CN 200510063136 CN 200510063136 A CN200510063136 A CN 200510063136A CN 1846785 A CN1846785 A CN 1846785A
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Abstract
The freeze dried composition containing engineered fusion protein has fusion protein of GnRH-PE and its derivatives, and main components including glycine and mannitol. In addition, the composition may also contain surfactant, amino acid and saccharide. The composition is freeze dried for preservation in long term without losing the bioactivity of the GnRH-PE and its derivatives.
Description
One. technical field
The invention belongs to the preservation field of biological product; biological product are the pharmaceutical grade protein of injection mostly; be difficult for stable the preservation; so often adopting freeze dried form preserves; the present invention mainly is a freeze drying protectant of selecting various combination for use, reaches protection GnRH-PE and the bioactive purpose of this class fusion rotein of derivant thereof.
Two. background technology
The freeze drying technology of biological product is exactly that the material that contains a large amount of moisture content is frozen into solid in advance; suitably heating under vacuum condition then makes steam directly distil from solid; and material itself is stayed in the ice shelf when freezing; therefore dried small product size is almost constant; the long preservation that helps albumen or other biological goods; in lyophilizing; selected freeze dried protective agent is crucial; albumin is as a kind of important protective agent; owing to can make sample that good molding is arranged after adding and to the active well protective effect of sample; for a long time often by people selected be conventional freeze drying protectant, as freeze drying protectant as genetic engineering interferon.But because getable human albumin product is blood products in the market; it is subjected to the potential danger of viral pollution bigger; no longer advocated at present and used albumin as the freeze drying protectant of biological product, so utilize novel freeze drying protectant just very important as the albumin succedaneum.
In the present invention, GnRH is meant human interstital-cell-stimulating hormone's releasing factor, what PE40 was meant false pseudomonas bacillus exotoxin A removes the remaining part of domain I, the C art end of GnRH and the N-terminal of PE40, perhaps the N-terminal of the C-terminal of PE40 and GnRH couples together by peptide bond, just formed the GnRH-PE fusion rotein, and various mutant.But this albuminoid is the guiding protein of a class specific killing adenocarcinoma class tumor, now confirm, adenocarcinoma class cell all can overexpression GnRH receptor, after guiding protein enters blood, GnRH by guiding can discern GnRH receptor that such tumor cell shows and can be specifically and receptors bind specifically, and coupled toxin moiety PE and derivant thereof can be killed this cell, thereby this fusion rotein can reach the purpose of kill tumor cell specifically, thereby reaches the purpose of treatment tumor.
GnRH-PE is the non-existent artificial protein of a class occurring in nature, so its stability is not strong.Test shows that GnRH-PE and derivant 4 degree thereof are placed 30 days artifact activity down and just begun to descend, and show that the freeze dried method of needs keeps its biological activity.The present invention has solved the instability problem of GnRH-PE and this albuminoid of derivant thereof by a series of experimental study, and it can be preserved easily, transportation.
Three. summary of the invention:
The present invention is by to GnRH-PE and derivant freeze-dried composition thereof with to the selection of pH; found the freeze-dried composition that is suitable for GnRH-PE and this class fusion rotein of derivant thereof; the albumin that such freeze-dried composition is habitually practised is safe, practical; and it is cheap; biological activity to GnRH-PE and derivant thereof has the better protect effect, for this class fusion rotein of mentioning among the present invention realizes that medicineization provides a good medicament platform.GnRH-PE and derivant thereof are as the possible effective medicine of a class to cancer, and its biological activity has embodied the effect of its specific killing tumor cell, so the present invention is at the positive effect that expection is arranged aspect the tumor cell treatment.
As GnRH-PE that uses among the present invention and derivant 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 false pseudomonas bacillus exotoxin A (PE) or its mutant are formed, so can use a kind of albumen with aminoacid sequence shown in sequence 1 or the sequence 5, also can use and a kind of sequence 1 or sequence 5 aminoacid sequences be modified, as inserting, disappearance, the sequence of suddenling change certain or some aminoacid and obtaining is as long as keep its biological activity.In other words, also can adopt shown with sequence 1 and the sequence 5 substantially identical albumen of aminoacid of its aminoacid sequence, promptly except aminoacid sequence shown in sequence 1 and the sequence 5, comprise that also aminoacid sequence shown in sequence 1 and the sequence 5 is carried out part to be inserted, disappearance is suddenlyd change certain or some aminoacid and the sequence that obtains.Such as sequence 3 is to add a Met by the N-terminal in sequence 1, the 6th Gly is sported Ala, after the 10th amino acids of sequence 1, inserted the catenation sequence of His-Met-Ala-Glu-Glu, C-terminal in sequence 1 removes Arg-Glu-Asp-Leu-Lys, replaces with Lys-Asp-Glu-Leu and the sequence that obtains.It is worthy of note that the sequence that the present invention lists only is to further specify of the present invention, and do not constitute the await the reply restriction of claim the present invention.
The method that obtains GnRH-PE and derivant thereof can realize by the method for molecular cloning, the gene order of can be synthetic and can synthesizing destination protein in conjunction with the method for PCR by the full gene of multi-disc section, by means such as enzyme action connections gene is connected into the purpose carrier then, as pET etc., be transformed into corresponding host bacterium then, express.Express the back by the collection thalline, the broken host bacterium of methods such as Mechanical Method or osmotic pressure, by the chromatography method of routine, as ion-exchange chromatography, gel permeation chromatography and hydrophobic chromatography etc. are purified to destination protein.Bioactive mensuration to destination protein adopts mtt assay, at first the Hela cell is digested 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 culture fluid.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.Lyophilizing procedure Selection-45 ℃ freezing 4 hours, evacuation then, programming rate be controlled at 2 degree/hour, after sample temperature surpasses 0 degree, heat up be controlled at 8 degree/hour, be up to 20 degree and kept 6 hours.Lyophilizing finishes.
Aspect the main protective agent of GnRH-PE and derivant thereof, tried out mannitol, glycine, the mixture of mannitol and glycine is using the concentration between the 3%-20% to find that it all has reasonable effect aspect lyophilizing skeleton forming.Lyophilizing has the phenomenon of subsiding when concentration is less than 3%, and considers the waste when protective agent concentration is high, and preferred protective agent concentration has been fixed on 5%.Aspect about pH, the pH of its freeze-dried composition is less than in 7.0 before lyophilizing, find after the sample lyophilizing and be not clear after the dissolving, but micro white, and decline has also appearred in the activity of sample, and the pH that considers finished product should not be greater than pH7.5, and we are chosen as 7.0-7.5 with the pH scope, preferred 7.25.
In addition, the protective agent in the freeze-dried composition can also contain at least a following material: monosaccharide or derivatives thereof, aminoacid, disaccharides, surfactant.Wherein the monosaccharide or derivatives thereof comprises glucose, fructose, sorbitol, dextran; Aminoacid comprises alanine, arginine, lysine; Disaccharides is a sucrose, lactose, trehalose, maltose; Surfactant comprises Tween-80 and Tween-20.
Three. the specific 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.
Example one.
The prescription of freeze-dried composition 1 is, the 2mL dosage form, and the albumen 1mg of sequence 3, the main protective agent of lyophilizing is a mannitol, 100mg, pH are 7.25.Carry out lyophilizing.
Example two.
The prescription of freeze-dried composition 2 is, the 2mL dosage form, and the albumen 1mg of sequence 3, the main protective agent of lyophilizing is a mannitol, and 120mg also contains 0.004%Tween-80, and pH is 7.30.Carry out lyophilizing.
Example three.
The prescription of freeze-dried composition 3 is, the 2mL dosage form, and the albumen 1mg of sequence 4, the main protective agent of lyophilizing is a glycine, and 100mg also contains 0.004%Tween-80, and pH is 7.20.Carry out lyophilizing.
Example four.
The prescription of freeze-dried composition 4 is, the 1mL dosage form, and the albumen 1mg of sequence 5, the main protective agent of lyophilizing is mannitol 50mg, also contains 2% dextran, pH is 7.25.Carry out lyophilizing.
Example five.
The prescription of freeze-dried composition 5 is, the 2mL dosage form, and the albumen 1mg of sequence 6, the main protective agent of lyophilizing is a glycine, and 90mg also contains 3%Arg, and pH is 7.15.Carry out lyophilizing.
Example six.
The prescription of freeze-dried composition 6 is, the 2mL dosage form, and the albumen 1mg of sequence 2, the main protective agent of lyophilizing is glycine 50mg, mannitol 50mg, pH are 7.20.Carry out lyophilizing.
The result:
| The freeze-dried composition sequence number | IC before the lyophilizing 50 (ug/mL) | IC after the lyophilizing 50(ug/mL) | 37 degree are placed 7 days IC after the lyophilizing 50(ug/mL) |
| 1 | 5.02 | 5.52 | 5.81 |
| 2 | 4.86 | 5.48 | 6.03 |
| 3 | 6.58 | 6.21 | 5.87 |
| 4 | 4.39 | 4.46 | 4.95 |
| 5 | 9.87 | 8.36 | 10.09 |
| 6 | 8.85 | 9.18 | 9.32 |
As can be seen from the above table, lyophilizing prescription of the present invention has been protected the biological activity of destination protein effectively.
Sequence table
＜110〉Beijing Nuo Silande Bioisystech Co., Ltd
＜120〉contain the freeze-dried composition of genetic engineering fusion protein
<140>
<141>
<160>6
<210>1
<211>371
<212>PRT
＜213〉sequence 1
<400>1
Glu His Trp Ser Tyr Gly Leu Arg Pro Gly Gly Gly Ser Leu Ala
1 5 10 15
Ala Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe
20 25 30
Thr Arg His Arg Gln Pro Arg Gly Tro Glu Gln Leu Glu Gln Cys
35 40 45
Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg
50 55 60
Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala
65 70 75
Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln
80 85 90
Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser
95 100 105
Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala
110 115 120
Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly
125 130 135
Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg
140 145 150
Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val
155 160 165
Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu
170 175 180
Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val
185 190 195
Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe
200 205 210
Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg
215 220 225
Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala
230 235 240
Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala
245 250 255
Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr
260 265 270
Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val
275 280 285
Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile
290 295 300
Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly
305 310 315
Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro
320 325 330
Thr Asp Pro Asn Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile
335 340 345
Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser
350 355 360
Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys
365 370 371
<210>2
<211>361
<212>PRT
＜213〉sequence 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 Ala Ala Arg Ser Gln Asp Leu
200 205 210
Asp Ala Ile Trg 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〉sequence 3
<400>3
Met Glu His Trp Ser Tyr Ala 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
<210>4
<211>360
<212>PRT
＜213〉sequence 4
<400>4
Met Glu His Trp Ser Tyr Ala Leu Arp 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 Tvr 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 Ala 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 Lys Asp Glu Leu
350 355 360
<210>5
<211>348
<212>PRT
＜213〉sequence 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 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 Ala Leu Arg Pro Gly Pro Gly Lys Pro Pro Lys
335 340 345
Asp Glu Leu
348
<210>6
<211>332
<212>PRT
＜213〉sequence 6
<400>6
Met Gly Ttp 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 Ala Leu Arg Pro Gly Pro Gly Lys Pro Pro Lys Asp
320 325 330
Glu Leu
332
Claims (11)
1. freeze-dried composition that contains genetic engineering fusion protein.Fusion rotein wherein is meant that first amino acids changes the human interstital-cell-stimulating hormone's releasing hormone GnRH of Glu or the fusion rotein of its mutant and false pseudomonas bacillus exotoxin A (PE) or its mutant composition into.
2. in the claim 1, the mutant of GnRH can comprise that the 6th amino acids sports Ala, the molecule of Trp or Leu.
3. in the claim 1, the mutant of PE can comprise and remove the 1-252 amino acids, and a section of remaining 253-613 amino acids is PE40; Can comprise and remove 1-252, 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, are PE38; Can comprise the 1-279 amino acids of removing the PE molecule, be left the 280-613 part, and 287 Cys are sported Ser or Ala or Gly, be PE37; Can comprise and remove 1-279 and two sections aminoacid sequences of 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, are PE35; Can comprise PE, PE40, PE38, PE37, PE35 molecule remove 5 aminoacid Arg-Glu-Asp-Leu-Lys of C-terminal, and replace to the molecule of Lys-Asp-Glu-Leu.
4. in the claim 1, except fusion rotein, its freeze drying protectant main component is a mannitol in the freeze-dried composition.
5. in the claim 1, except fusion rotein, its freeze drying protectant main component is a glycine in the freeze-dried composition.
6. in the claim 1, except fusion rotein, the main component of its freeze drying protectant is the mixture of mannitol and glycine in the freeze-dried composition.
7. in the claim 4, the concentration of used mannitol is 3%-20%, preferred 5%.
8. in the claim 5, the concentration of used glycine is 3%-20%, preferred 5%.
9. in the claim 6, the total concentration of the used mannitol and the mixture of glycine is 3%-20%, preferred 5%.
10. in the claim 2,3,4,5,6, the pH of freeze-dried composition is 7.0-7.5, preferred 7.25.
11. in the claim 2,3,4,5,6, the protective agent in the freeze-dried composition can also contain at least a following material: the monosaccharide or derivatives thereof, amino acids, disaccharides, surfactant-based.Wherein the monosaccharide or derivatives thereof comprises glucose, fructose, sorbitol, dextran; Amino acids comprises alanine, arginine, lysine; Disaccharides comprises sucrose, lactose, trehalose, maltose; Surfactant-based Tween-80 and the Tween-20 of comprising.
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| CN101195822B (en) * | 2006-12-08 | 2012-08-08 | 湖南康都制药有限公司 | High-endophilicity targeting amalgamation protein |
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| 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 |
| CN110563835A (en) * | 2018-06-05 | 2019-12-13 | 内蒙古伊利实业集团股份有限公司 | Lactoferrin freeze-drying protective agent and application thereof |
| CN111195234A (en) * | 2018-11-16 | 2020-05-26 | 鲁南制药集团股份有限公司 | Recombinant FGF21-Fc fusion protein freeze-dried powder preparation |
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2005
- 2005-04-05 CN CN 200510063136 patent/CN1846785A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101195822B (en) * | 2006-12-08 | 2012-08-08 | 湖南康都制药有限公司 | High-endophilicity targeting amalgamation protein |
| CN102813933A (en) * | 2012-08-30 | 2012-12-12 | 青岛康地恩药业股份有限公司 | Chicken infectious bursal disease egg yolk antibody cryoprotectant |
| 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 |
| CN110563835A (en) * | 2018-06-05 | 2019-12-13 | 内蒙古伊利实业集团股份有限公司 | Lactoferrin freeze-drying protective agent and application thereof |
| CN111195234A (en) * | 2018-11-16 | 2020-05-26 | 鲁南制药集团股份有限公司 | Recombinant FGF21-Fc fusion protein freeze-dried powder preparation |
| CN111195234B (en) * | 2018-11-16 | 2022-08-26 | 鲁南制药集团股份有限公司 | Recombinant FGF21-Fc fusion protein freeze-dried powder preparation |
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