CN101433713A - GnRH-PE mutant fusion protein and uses thereof - Google Patents
GnRH-PE mutant fusion protein and uses thereof Download PDFInfo
- Publication number
- CN101433713A CN101433713A CNA2007101879158A CN200710187915A CN101433713A CN 101433713 A CN101433713 A CN 101433713A CN A2007101879158 A CNA2007101879158 A CN A2007101879158A CN 200710187915 A CN200710187915 A CN 200710187915A CN 101433713 A CN101433713 A CN 101433713A
- Authority
- CN
- China
- Prior art keywords
- gnrh
- mutant
- fusion protein
- sequence
- gly
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the field of medical biology and discloses GnRH-PE mutant fused protein and application thereof. Particularly, the invention relates to novel GnRH-PE mutant fused protein and application thereof of preparing drugs used for treating psoriasis. By utilizing a psoriasis-like animal model of a guinea pig, the inventor finds that the adoption of the GnRH-PE mutant fused protein can effectively treat the psoriasis including psoriasis vulgaris, pustular psoriasis, psoriasis arthropathica and erythrodermic psoriasis.
Description
Invention field
The present invention relates to the GnRH-PE mutant fusion protein, and described mutant fusion protein is used to prepare the purposes for the treatment of psoriatic medicine.
Background technology
Psoriasis is commonly called as psoriasis, is a kind of common chronic dermatosis that is easy to recur with characteristic skin lesion.The silvery white squama in surface, thin film, petechial hemorrhage are three big Clinical symptoms of this disease.The cause of disease that this is sick and pathogeny are still not exclusively clear and definite.Think heredity, dysbolismus in recent years mostly, the key factor that infections, immune dysfunction etc. may be fallen ill, seasonal variations, humidity, psychic trauma or operation etc. also may be brought out this disease.The pathological change of psoriatic tissue mainly shows as the keratinocyte hyper-proliferative, inflammatory cell is assembled and dermal papilla portion vasodilation three big features.Wherein, the keratinocyte hyper-proliferative change that keratin is expressed occurs simultaneously usually with unusual keratinization process.
According to clinical manifestation, psoriasis can be divided into four types usually, i.e. psoriasis vulgaris (psoriasis vulgaris), psoriasis pustulosa (Psoriasis pustulosa), arthropathic psoriasis (psoriasis arthropathica) and erythrodermic psoriasis (erythrodermic psoriasis).
At present psoriasis still there is not specific short.Existing various therapies can only reach short term effect, can not prevent recurrence, mainly comprise:
1) psychotherapy: mainly comprise and freeing one's mind of misgivings, eliminate psychic trauma, avoid various risk factors;
2) external used medicine therapy: external used medicine commonly used has keratoplastics and corticosteroid cream;
3) constitutional treatment: do not consider to use constitutional treatment for the external used medicine curative effect is good enough, comprise and adopt immunosuppressant, retinoic acid, antibiotic (penicillin and novel penicillin), corticosteroid etc.; With
4) physiotherapy: comprise balneation, phototherapy and photochemotherapy.
Utilize Cavia porcellus psoriasiform animal model, the GnRH-PE mutant fusion protein is adopted in the beat all discovery of the inventor, can effectively treat psoriasis, comprises psoriasis vulgaris, psoriasis pustulosa, arthropathic psoriasis and erythrodermic psoriasis.
Summary of the invention
The present invention relates to the GnRH-PE mutant fusion protein, and described mutant fusion protein is used to prepare the purposes for the treatment of psoriatic medicine.
The GnRH-PE fusion rotein is made up of human luteinizing hormone's releasing factor (GnRH) and Pseudomonas exotoxin A (PE), is the biological target tropism's medicine that becomes known for treating tumor.Wherein GnRH is the little peptide of being made up of 10 aminoacid, its sequence is expressed as with trigram: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly, wherein the first amino acids residue is a pyroglutamic acid, and aminoacid sequence is shown in SEQ ID NO:1 in the sequence table.Pseudomonas exotoxin A (PE) is known to be that (GENEBANK, gi:151215), its concrete aminoacid sequence is shown in SEQ ID NO:2 in the sequence table for a protein molecule of being made up of 613 aminoacid.
Concrete, in described fusion rotein, GnRH is as the targeting part of target medicine, and PE is as the toxin moiety of target medicine.
Kill and wound canceration T cell activity in order to improve described fusion rotein, based on described GnRH-PE fusion rotein, respectively at the PE exploitation of the GnRH of above-mentioned targeting part and toxin moiety and prepared the mutant (referring to Chinese invention patent " the genetic engineering recombiant protein of the special kill tumor cell of a kind of energy ", patent No. ZL03137587.1) of a series of fusion rotein.
The composition of GnRH-PE mutant fusion protein and kind
In the present invention, described GnRH-PE mutant fusion protein is made up of GnRH mutant and PE mutant, and wherein said mutant fusion protein is selected from:
1) the N-end of the C-end of GnRH mutant and the PE mutant mutant fusion protein LLP that links to each other and form with peptide bond by joint sequence, i.e. GnRH mutant-joint sequence-PE mutant.Choose wantonly, in described mutant fusion protein LLP, the C-end of described PE mutant can also contain 2-5 and be selected from-XYEL or-repeated fragment of REDLK, that is:
The n of GnRH mutant-joint sequence-PE mutant-(XYEL), or
The n of GnRH mutant-joint sequence-PE mutant-(REDLK), wherein n is the integer of 2-5;
2) the N-end of the C-end of PE mutant and the GnRH mutant mutant fusion protein PLL that links to each other and form with peptide bond by joint sequence, i.e. PE mutant-joint sequence-GnRH mutant.Choose wantonly, in described mutant fusion protein PLL, the C-end of described GnRH mutant can also contain 2-5 and be selected from-XYEL or-repeated fragment of REDLK, that is:
The n of PE mutant-joint sequence-GnRH mutant-(XYEL) or
The n of PE mutant-joint sequence-GnRH mutant-(REDLK), wherein n is the integer of 2-5;
3) have 1) or 2) described in variant or its functional fragment of one or several amino acid whose insertion in the mutant fusion protein aminoacid sequence, disappearance and/or replacement, prerequisite is to keep 1 basically) or 2) described in the biologic activity of mutant fusion protein; And
4) with 1) or 2) described in the mutant fusion protein aminoacid sequence have variant or its functional fragment more than or equal to 90% sequence homogeneity, prerequisite is to keep 1 basically) or 2) described in the biologic activity of mutant fusion protein.
In the present invention, described joint sequence is one section sequence of being made up of 0-20 amino acid residue.Concrete, between described GnRH mutant and PE mutant, can not use joint sequence, also can select as required to use by 3 amino acid residues, 5 amino acid residues, 10 amino acid residues, 15 amino acid residues, even 20 joint sequences that amino acid residue is formed.Preferably, described joint sequence can be the sequence of (representing with trigram) as follows:
-His-Met-Ala-Glu-Glu-(SEQ?ID?NO：3)；
-Gly-Ser-Gly-Gly-Gly-(SEQ?ID?NO：4)；
-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-(SEQ?IDNO：5)。
In the context of the invention, among described fragment-XYEL, X represents lysine (Lys) or arginine (Arg), and Y represents aspartic acid (Asp), glutamine (Gln) or glutamic acid (Glu), and E represents glutamic acid (Glu), and L represents leucine (Leu); The aminoacid sequence fragment of described fragment-REDLK for adopting single-letter to represent, wherein, R represents arginine (Arg), and E represents glutamic acid (Glu), and D represents aspartic acid (Asp), and L represents leucine (Leu), and K represents lysine (Lys);
In the present invention, described GnRH mutant is meant that its amino terminal first amino acids replaces to glutamic acid (Glu), and optional, the 6th mutant that sports alanine (Ala) or tryptophan (Trp) or leucine (Leu) by glycine (Gly) of its amino terminal.
In the present invention, described GnRH-PE mutant fusion protein can further be modified under the prerequisite that keeps or keep basically its biologic activity, be included in and insert one or more other residues between two residues of parent's protein amino acid sequence, and deletion from aminoacid sequence, replace one or more residues.
Concrete, in the present invention, human luteinizing hormone's releasing factor (GnRH) amino terminal first amino acids by described its aminoacid sequence of GnRH mutant that pyroglutamic acid replaces to glutamic acid is: Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly (SEQ IDNO:6); After the 6th of its amino terminal substituted by alanine (Ala) or tryptophan (Trp) or leucine (Leu) respectively by glycine (Gly), its aminoacid sequence was respectively:
Glu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-Gly(SEQ?ID?NO：7)
Glu-His-Trp-Ser-Tyr-Trp-Leu-Arg-Pro-Gly(SEQ?ID?NO：8)
Glu-His-Trp-Ser-Tyr-Leu-Leu-Arg-Pro-Gly(SEQ?ID?NO：9)
In the present invention, described PE mutant comprises:
1) Pseudomonas exotoxin A (PE) is removed the deletion mutant PE40 that forms after the N-terminal 1-252 amino acids, referring to SEQ ID NO:10 in the sequence table; Choose wantonly, 5 aminoacid-Arg-Glu-Asp-Leu-Lys of the C-end of described PE40 (promptly-RDELK) can be replaced by-XYEL;
2) with the 253-364 position of Pseudomonas exotoxin A (PE), the two sections aminoacid sequences in 381-613 position link to each other by former order and obtain albumen PE38, referring to SEQ ID NO:11 in the sequence table; Choose wantonly, 5 aminoacid-Arg-Glu-Asp-Leu-Lys of the C-end of described PE38 (promptly-RDELK) can be replaced by-XYEL;
3) with the 280-364 position of Pseudomonas exotoxin A (PE), the two sections aminoacid sequences in 381-613 position link to each other by former order and obtain albumen PE35, referring to SEQ ID NO:12 in the sequence table; Choose wantonly, 5 aminoacid-Arg-Glu-Asp-Leu-Lys of the C-end of described PE35 (promptly-RDELK) replace with-XYEL;
4) the fragment albumen PE37 of the 280-613 amino acids sequence of Pseudomonas exotoxin A (PE) is referring to SEQ ID NO:13 in the sequence table; Choose wantonly, 5 aminoacid-Arg-Glu-Asp-Leu-Lys of the C-end of described PE37 (promptly-RDELK) can be replaced by-XYEL;
5) with the 253-358 position of Pseudomonas exotoxin A (PE), the two sections aminoacid sequences in 366-613 position link to each other by former order and obtain albumen PE39, referring to SEQ IDNO:14 in the sequence table; Choose wantonly, 5 aminoacid-Arg-Glu-Asp-Leu-Lys of the C-end of described PE39 (promptly-RDELK) can be replaced by-XYEL.
Concrete, " variant " is meant the polypeptide that has following feature at least described in the present invention: the peptide sequence of (1) and GnRH-PE mutant fusion protein has about 80% or above amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity.
" percentage (%) amino acid sequence identity " when being defined in two sequences of parallelism, the percent of the amino acid residue identical with the amino acid residue of the aminoacid sequence of described GnRH-PE mutant fusion protein in the candidate sequence.
In order to measure % aminoacid homogeneity, the sequence parallelism is also introduced the room in order to obtain maximum % sequence homogeneity where necessary; Conservative substitution is not considered to the part of sequence homogeneity.The aminoacid sequence parallelism method of measuring percentage homogeneity is well known to a person skilled in the art.The computer software that common use can openly obtain as BLAST, BLAST2, ALIGN or Megalign (DNASTAR) software, comes the parallelism peptide sequence.Those skilled in the art can be identified for the suitable parameter of parallelism, are included in and obtain the required any algorithm of maximum parallelism on the sequence total length that is compared.
When the parallelism aminoacid sequence, the % amino acid sequence identity of specific amino acids sequence A and specific amino acids sequence B (perhaps can be expressed as the specific amino acids sequence A that has or comprise certain % amino acid sequence identity with the specific amino acids sequence B) can followingly calculate:
% amino acid sequence identity=X/Y100
The X number that to be sequence parallelism program by A and B or algorithm parallelism marked for the amino acid residue of identical match wherein, Y is the amino acid residue sum among the B.
If aminoacid sequence A is uneven in length in the length of aminoacid sequence B, then the % amino acid sequence identity of A and B will be not equal to the % amino acid sequence identity of B and A.
In the preferred embodiment of the invention, described GnRH-PE mutant fusion protein is selected from GnRH-PE40, referring to SEQ ID NO:15 in the sequence table; GnRH-PE40KDEL is referring to SEQ ID NO:16 in the sequence table;
6Ala-GnRH-PE40KDEL is referring to SEQ ID NO:17 in the sequence table; GnRH-PE39KDEL is referring to SEQ ID NO:18 in the sequence table; And GnRH-PE38, referring to SEQ ID NO:19 in the sequence table.
The construction strategy of described GnRH-PE mutant fusion protein
Mutant fusion protein of the present invention can easily produce with gene mutation well known in the art and recombination method.Concrete, described mutant fusion protein can prepare with (fixed point) mutation, alanine scanning and the PCR mutation of method well known in the art such as oligonucleotide-mediation.Can select mutation or other known technology to be carried out direct mutagenesis, box mutation, restriction by the DNA that cloned, with the DNA sequence of the variant that produces the GnRH-PE mutant fusion protein.
The nucleotide sequence of coding GnRH mutant can read the frame endomixis with the nucleotide sequence of coding PE or its mutant.The gene of described encoding mutant body fusion rotein also can be synthetic by routine techniques, comprises automatic dna synthesizer.Also can adopt the pcr amplification that uses anchor primer, this anchor primer produces complementary jag between two successive genetic fragments, can anneal then, and amplification again produces chimeric gene sequence.By will producing described mutant fusion protein to being used for the known in the art multiple expression vector that target protein is expressed as gene sub-clone in reading frame of the encoding mutant body fusion rotein of above-mentioned acquisition.
Concrete, for obtaining GnRH-PE mutant fusion protein of the present invention, take following tactful human luteinizing hormone's releasing factor (GnRH) and false pseudomonas bacillus exotoxin A (PE) to transform respectively.
(1), to the transformation of the GnRH part of fusion rotein, comprising:
1. change the primary pyroglutamic acid of the N-terminal of natural GnRH into glutamic acid;
2. optional, N-terminal the 6th amino acids of the GnRH mutant that will obtain according to step 1 replaces with respectively and is Ala or Trp or Leu.
3. optional, the GnRH mutant C-terminal that obtains in step 1 or 2 is connected-(XYEL) n or-(REDLK) n, wherein n is the integer of 2-5.
(2), the transformation of the false pseudomonas bacillus exotoxin A of fusion rotein part:
1.PE40 and the structure of mutant
Pseudomonas exotoxin A (PE) is removed N-terminal 1-252 amino acids to be obtained deletion mutant and is called PE40.As required, can be further 5 aminoacid-Arg-Glu-Asp-Leu-Lys of the C-end of described PE40 (promptly-RDELK) be replaced with-XYEL.
2.PE38 and the structure of mutant
With the 253-364 position of Pseudomonas exotoxin A (PE), the two sections aminoacid sequences in 381-613 position link to each other by former order, obtain albumen PE38.As required, 5 aminoacid-Arg-Glu-Asp-Leu-Lys of the C-end of described PE38 (promptly-RDELK) can be replaced with-XYEL.
3.PE35 and the structure of mutant
With the 280-364 position of Pseudomonas exotoxin A (PE), the two sections aminoacid sequences in 381-613 position link to each other by former order and obtain albumen PE35.As required, can be further 5 aminoacid-Arg-Glu-Asp-Leu-Lys of the C-end of described PE35 (promptly-RDELK) be replaced with-XYEL.
4.PE37 and the structure of mutant
Pseudomonas exotoxin A (PE) is removed the deletion mutant that N-terminal 1-279 amino acids obtains, and promptly the 280-613 amino acids sequence fragment of Pseudomonas exotoxin A (PE) is called albumen PE37.As required, 5 aminoacid-Arg-Glu-Asp-Leu-Lys of the C-end of described PE37 (promptly-RDELK) can be replaced with-XYEL.
5.PE39 and the structure of mutant
With the 253-358 position of Pseudomonas exotoxin A (PE), the two sections aminoacid sequences in 366-613 position link to each other by former order and obtain albumen PE39.As required, 5 aminoacid-Arg-Glu-Asp-Leu-Lys of the C-end of described PE39 (promptly-RDELK) can be replaced by-XYEL.
The present invention relates to described GnRH-PE mutant fusion protein and is used for the treatment of psoriatic purposes on the other hand.
The present invention relates to described GnRH-PE mutant fusion protein and is used to prepare the purposes for the treatment of psoriatic medicine on the other hand.
In the present invention, described " treatment " be included in make after the administration described psoriatic remission, effectively, the situation of produce effects and reverse.Concrete, according to general Baker method (people such as BakerBS, Br J Dermatol, 1992,126:105 110) formulation histological score standard, that is: typical psoriatic lesion scoring is between 4.0～10.0, the judgement of carrying out according to following standard: greater than 7.0 patient, scoring is judged to be the psoriasis remission less than 5.0 after all medications for scoring before the medication, scoring is less than 3.0 after all medications, it is effective to be judged to be the psoriasis symptom, and scoring is judged to be psoriasis symptom produce effects less than 2.0 after all medications, scoring is judged to be the psoriasis symptom and reverses less than 1.0 after all medications.
Accordingly, treatment effective dose of the present invention is meant the amount that has required curative effect when according to the character of experimenter's disease or disease and the order of severity particular subject being used, for example will alleviate, effectively, produce effects and the described psoriatic amount of reverse
Among the present invention, judge that by adopting following two kinds of animal models described GnRH-PE mutant fusion protein treats psoriatic effect.
Model one:
Adopt conventional animal model---the Cavia porcellus psoriasiform animal model that is used to estimate the curing psoriasis effect in the prior art, estimate GnRH-PE mutant fusion protein of the present invention and treat psoriatic effect (referring to " keratin K14 in the Cavia porcellus psoriasiform animal model; 16; 17 changes of expression level ", Fan Pingshen, Liao Wenjun, Liu Yufeng, the 22nd the 24th phase of volume of The Fourth Military Medical University's journal (newly) calendar year 2001 December).
Model two:
Flakey skin (fsn/fsn) sudden change mouse model.Described model is U.S. Jackson laboratory newfound a kind of spontaneous autosomal recessive gene mutant mice strain in 1985, and this mice shows as hypochrosis when just being born, and squamous canescence speckle appears in whole body or part soon.And find that pathological characters such as hyperkeratosis, acanthosis, subcorneal pustule and corium telangiectasis 2 weeks after birth occur, 3～4 all skin lesion merge diffusion, it takes place relevant in the infiltration of corium with amount lymphocyte and a spot of neutrophilic granulocyte greatly, macrophage, show the histopathology feature very similar [China J Lepr Skin Dis.Jan2006 to psoriasis, Vol.22, No.1; Pathogenic function of IL-1b in psoriasiformskin lesions of flaky skin (fsn/fsn) mice; Clin Exp Immunol 2001; 123:505-510].
The preparation of embodiment 1 GnRH-PE mutant code gene
1, the acquisition of GnRH-PE40 encoding gene
Aminoacid sequence according to known GnRH mutant and PE40 mutant, select for use the full gene of escherichia coli preference codon to synthesize the GnRH-PE40 DNA sequence, obtain the GnRH-PE40 nucleotide sequence, for making things convenient for the gene recombinaton operation, simultaneously add restriction enzyme site NdeI (CATATG), EcoRI (GAATTC), in 3 ' the terminal modified termination codon that goes up at N-terminal and C-terminal corresponding to described polypeptide.
According to this area common method, referring to as " molecular cloning (second edition): lab guide " (1992, cold spring harbor laboratory), will be as the GnRH-PE40 DNA sequence and screening plasmid pBSK (the Stratagene company of above-mentioned preparation, article No. 212205) handles with restricted enzyme NdeI, EcoRI, behind each sample concentration, 1:1 adds the T4 linked system in molar ratio as calculated.Be pSK-GnRH-PE40 through transforming, filter out the recombiant plasmid that obtains comprising above-mentioned GnRH-PE40 nucleotide sequence, ordering, the order-checking back is standby.
2, the acquisition of other mutant code genes of GnRH-PE
According to this area common method, referring to as " molecular cloning (second edition): lab guide " (1992, cold spring harbor laboratory), with the GnRH-PE40 gene is template, adopt PCR method,, obtain the encoding gene of other GnRH-PE mutant by the disappearance and the mutation method of conventional DNA sequence.Is that the site is inserted among the sequencing vector pBSK (Stratagene company, article No. 212205) in proper order with described encoding gene fragment with NdeI, EcoRI, and the order-checking back is standby.
The preparation of embodiment 2 GnRH-PE mutant fusion proteins
Among the present invention, the preparation of GnRH-PE mutant fusion protein can be finished by the following method: the described sequencing vector that obtains from embodiment 1 is taken off target gene fragment with NdeI, EcoRI double digestion, insert in the expression vector, again recombinant expression carrier is imported in the expressive host bacterium, through culture expression, hydrophobic chromatography, chelating chromatography, ion-exchange chromatography obtain destination protein.Below be that example describes with GnRH-PE mutant fusion protein GnRH-PE40.Other fusion rotein all can make according to similar approach.
1, the preparation of recombiant plasmid PET20-GnRH-PE40
Respectively with the carrier cutting-out described antigen-4 fusion protein gene of corresponding restricted enzyme from embodiment 1 preparation, again with this antigen-4 fusion protein gene fragment with through the big fragment of recovery of Nde I and EcoR I double digestion plasmid PET20 (NOVAGEN), 18 ℃ in T4 ligase system two sections target gene fragment connect.The gained recombinant screen is differentiated the recombiant plasmid that is correctly connected, called after PET20-GnRH-PE40 through enzyme action and sequencing.
2. contain the preparation of the genetic engineering bacterium of recombiant plasmid PET20-GnRH-PE40
Use CaCl then
2Method transformed into escherichia coli RBL21 (NOVAGEN company) is coated on the LB flat board that contains 100 μ g/ml ampicillin, selects positive bacterium colony, differentiates the transformant that contains above-mentioned recombiant plasmid PET20-GnRH-PE40.
Cultivate described transformant when OD600 is 0.6-0.8 in the LA fluid medium, add IPTG 0.1mM and induce 3-4 hour centrifugal collection thalline, occur the protein band about a 43KD behind the broken bacterium of 8M carbamide during 15% SDS-PAGE electrophoresis, expression is 35%.Monoclonal antibody immunity trace calibrating through PE shows positive reaction.Acquisition efficiently expresses the proteic genetic engineering bacterium of GnRH-PE40, called after PET20-GnRH-PE40/RBL21.
3.GnRH-PE40 expression and purification
1) shaking bottle expresses: will be as the genetic engineering bacterium PET20-GnRH-PE40/RBL21 that contains the GnRH-PE40 protein gene of above-mentioned preparation, in the LB culture medium that contains 100 μ g/ml ampicillin, shake (37 ℃ of bottle incubated overnight, 200rpm), 1:30 is seeded in the LB culture medium that contains 100 μ g/ml ampicillin 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 to contain GnRH-PE40 (43KD) based on solubility expression, expression accounts for 35% of bacterial protein.
2) large scale fermentation is expressed:
A. culture medium is formed:
A) seed liquor culture medium (LA):
Soy peptone: 10 grams per liters, yeast powder: 5 grams per liters, sodium chloride: 10 grams per liters, kanamycin: 50 mcg/ml.
B) culture medium (15 liters):
Sodium hydrogen phosphate: 315 grams, potassium dihydrogen phosphate: 100 grams, sodium chloride: 10.05 grams, ammonium chloride: 50 grams, soy peptone: 90 grams.
Above composition is sterilized in jar together; After sterilizing separately, following composition adds fermentation tank.
Magnesium sulfate: 15 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 strain, be inoculated among 50 milliliters of (250 milliliters triangular flasks) LK, these 50 milliliters of seed liquor are transferred among 700 milliliters of LK 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=3-4) 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 1 gram IPTG and induces (four hours).
3) step of purification:
I) broken bacterium: take out 200 frozen gram thalline of pie, use the broken bacterium buffer (20mM Tris-HCl, pH8,1mM EDTA) of 1600mL to dissolve fully, ice bath stirred 15 minutes.Centrifugal 10000rpm, 20min collects supernatant.
Ii) mend salt:
The volume of measuring samples, adding and making it to reach final concentration into ammonium sulfate is 0.3M, adding sodium chloride, to make it to reach final concentration be 1M.
Iii) Phenyl Sepharose HP chromatography:
The chromatographic column diameter is 50mm, high 20cm, the about 200mL of medium, 280nm monitors, and uses 50mM Tris-HCl, 0.3M NH3 (SO4) 2,1M NaCl, pH8 carries out balance to chromatographic column, and the sample of previous step is gone up sample, and then with above-mentioned solution equilibria to baseline, use 50mMTris-HCl, 0.5M NaCl, pH8 carries out eluting, collects the albumen eluting peak.Above flow velocity is 20mL/min.
Iv) Chelating Sepharose Fast Flow chromatography
Buffer A: 20mM PB, pH7.4,0.5M NaCl
Buffer B: 20mM PB, pH7.4,0.5M NaCl, 50mM imidazoles.
The chromatographic column diameter is 35mm, high 20cm, the about 100mL of medium, 280nm monitoring.
At first washing, pass through chromatographic column with 1% copper-bath then, treat that medium is in conjunction with after the complete copper ion, washing, 5 column volumes of buffer A balance, the sample of previous step is directly gone up sample then, reequilibrate is to baseline afterwards, carry out the linear gradient eluting then, 100%A is to 100%B, 20 column volumes.Collect the destination protein eluting peak.Above flow velocity is 15mL/min.
V) Q Sepharose HP chromatography
Buffer A: 20mM PB, pH7.4.
Buffer B: 20mM PB, pH7.4,0.5M NaCl.
The chromatographic column diameter is 26mm, high 20cm, about 60 milliliters of medium.
The buffer A balance, last sample, and then balance carry out the linear gradient eluting afterwards, and 100%A is to 100%B, 10 column volumes.Collect the destination protein eluting peak.Above flow velocity is 15mL/min.
By above step, the 200g thalline can obtain the pure product of about 200mg destination protein.
4). quality testing:
Detect by SDS-PAGE electrophoresis and reversed-phase HPLC, the destination protein purity that obtains is greater than 95%.
The preparation of embodiment 3 other mutant fusion proteins of GnRH-PE
According to the foregoing description 2 preparation methoies, make up and obtained to efficiently express the proteic genetic engineering bacterium of GnRH-PE40KDEL, 6Ala-GnRH-PE40KDEL, GnRH-PE39KDEL and GnRH-PE38 respectively, called after PET20-
6Ala-GnRH-PE40KDEL/RBL21, PET20-GnRH-PE39KDEL/RBL21, PET20-GnRH-PE38/RBL21, PET20-GnRH-PE40KDEL/RBL21.
Utilize the said gene engineering bacteria, obtain purity greater than 95% destination protein: GnRH-PE40KDEL,
6Ala-GnRH-PE40KDEL, GnRH-PE39KDEL and GnRH-PE38.
Embodiment 4 GnRH-PE mutant fusion protein GnRH-PE40 are to psoriatic therapeutic effect
1, material
70 healthy adult Cavia porcelluss (body constitution amount 300～400g, male and female are not limit), 5% propranolol Emulsion is prepared by the Beijing Northland Biotechnology Limited Liability Company, and test specimen GnRH-PE40 is according to embodiment 1 described method preparation.
2, method
2.1, Cavia porcellus psoriasiform model preparation
Laboratory animal is divided into totally two groups of model group and model control group at random, 60 laboratory animals of model group wherein, use the evenly outer ears butt skin that is coated with of 5% propranolol Emulsion, every day 4 times, be coated with for 4 weeks continuously (referring to " the propranolol coating causes the psoriasiform pathological change of Cavia porcellus ear ", China's skin magazine, 1991,24 (2): 9697; AK auto Ab is to the influence of Cavia porcellus psoriasiform model, and March the 16th in 2002 rolled up for the 2nd phase by Chinese skin cypridology magazine: 73-5).10 laboratory animals of model control group are smeared ears butt skin with simple Emulsion, every day 4 times, are coated with for 4 weeks continuously.Get model group and 6 Cavia porcellus auricles of model control group specimen weekly respectively at random, paraffin embedding, HE dyeing, tissues observed changes.
HE dyeing: will be with the fixed specimen dehydration of 10% formalin, paraffin section is made in embedding behind the transparent waxdip, and conventional H E dyes. tissues observed finding one by one under light microscopic.Method with reference to Baker is formulated the histological score standard, that is: typical psoriatic lesion scoring sees Table 1 between 4.0～10.0.
Table 1 histological score method
2.2, GnRH-PE mutant fusion protein GnRH-PE40 is to the treatment of Cavia porcellus psoriasiform animal pattern
48 Cavia porcelluss in the model group are divided into 4 groups at random, 12 every group.Use the fusion rotein of following 4 kinds of variable concentrations respectively: GnRH-PE40 30ug/kg, GnRH-PE40 60ug/kg, GnRH-PE40 120ug/kg, solvent (phosphate buffer of 5% mannitol, 20mM, pH value 7-8) 1ml/ are only.Adopt the administration of intravenous injection mode, 1 time/day, continuous 15 days.Preceding 0 day of administration, administration the 15th are got the skin of pinna specimen respectively and are stored to be checked.
3 results
3.1 collect specimen HE coloration result in the Cavia porcellus psoriasiform Preparation of model process
The horny layer that normal model matched group guinea pig skin is as seen poor, granular layer is about 1～3 layer, about 3～5 layers of prickle cell layer, basal layer is the monolayer cylindrical cell. true epidermis has a common boundary more flat, and intradermal is dispersed in the minority monocyte infiltration.
Model group through propranolol coating 3 after week most of specimen all see in horny layer hyperkeratosis, epidermal area acanthosis, the skin corium mastoid process cell infiltration arranged, the part specimen is seen extensively or kitchen range shape parakeratosis, granular layer attenuation or disappearance, the trochanterellus extension is bar-shaped, stretch on the mastoid process and be the change of pestle shape, telangiectasis.
We pass through with reference to the Baker methods of marking, every part of specimen is carried out the histological score analysis one by one. application organizes point system (typical psoriatic lesion scoring is between 4.0～10.0) scoring obtains, group specimen histological score is 0.47 ± 0.18 before the coating, it is 7.23 ± 0.71 that 4 weeks of coating are organized the specimen histological score, histological score was before and after the simple Emulsion of normal model matched group was smeared: 0.47 ± 0.18,0.38 ± 0.24.Learn result by statistics, group and the normal model matched group significant difference (P＜0.01) of comparing before group and the coating behind the coating, and matched group is coated with and organizes zero difference after group and matched group are coated with simple Emulsion before the simple Emulsion, sees Table 2.
The histological score of table 2 Cavia porcellus psoriasis model and matched group sample
Grouped organizing compares P before and after learning the scoring coating
The model group coating is preceding 0.47 ± 0.18-
That matched group is coated with simple Emulsion is preceding 0.42 ± 0.05-
Behind the model group coating 7.23 ± 0.71＜0.01
Matched group is coated with behind the simple Emulsion 0.38 ± 0.24 0.47
Above-mentioned experimental result shows, the psoriasis model of utilizing Cavia porcellus successfully to set up can be used for treating the recruitment evaluation of psoriasis medicine.
3.2 the GnRH-PE40 that adopts variable concentrations is to respectively organizing specimen HE coloration result before and after the treatment of Cavia porcellus psoriasis model
The GnRH-PE40 that adopts variable concentrations is to the model group Cavia porcellus intravenous drug in the Cavia porcellus psoriasis model after 15 days, in GnRH-PE40 60ug/kg group and the 120ug/kg processed group specimen, horny layer parakeratosis phenomenon disappears substantially, and only an other places has parakeratosis to change.The attenuation of epidermal area prickle cell layer, 1～2 layer of granular layer cell of most appearance, stretch on trochanterellus extension and the mastoid process, telangiectasis etc. obviously alleviates, and the minority cell infiltration is only arranged in the skin corium mastoid process.The GnRH-PE4030ug/kg processed group is similar to aforementioned change, but change is lighter, and blank group histology does not have obvious change.Histological score saw Table 3 before and after each organized administration.
Histological score before and after the GnRH-PE40 administration of table 3 variable concentrations
Group is handled the news commentary divisional processing P that marks after 15 days
GnRH-PE40 30ug/kg group 7.33 ± 0.24 4.38 ± 0.63 0.000781
GnRH-PE40 60ug/kg organizes 7.38 ± 0.73 2.15 ± 0.75 8.58E-06
GnRH-PE40 120ug/kg organizes 7.46 ± 0.46 1.42 ± 0.53 6.38E-10
Blank group 7.42 ± 0.39 6.88 ± 0.37 0.267819
By The above results as can be known, 30ug/kg group, 60ug/kg group and 120ug/kg group GnRH-PE40 all have the effect of improving the psoriasis symptom, and along with administration concentration increases, the effect of improving the psoriasis symptom also obviously strengthens.
Embodiment 5, GnRH-PE mutant fusion protein
6Ala-GnRH-PE40KDEL, GnRH-PE40KDEL, GnRH-PE39KDEL, GnRH-PE38 are to psoriatic therapeutic effect
1, material
80 healthy adult Cavia porcelluss (body constitution amount 300～400g, male and female are not limit), 5% propranolol Emulsion is prepared by the Beijing Northland Biotechnology Limited Liability Company, test specimen GnRH-PE mutant fusion protein
6Ala-GnRH-PE40KDEL, GnRH-PE40KDEL, GnRH-PE39KDEL, GnRH-PE38 prepare according to embodiment 1 described method respectively.
2, method
2.1 Cavia porcellus psoriasiform model preparation
Described with reference to embodiment 3, laboratory animal is divided into totally two groups of model group and model control group at random.Set up Cavia porcellus psoriasiform model according to the mode that embodiment 3 describes, confirm that by histological score described animal model is suitable for the assessment of curing psoriasis effect.
2.2, the application program of GnRH-PE mutant fusion protein
60 Cavia porcelluss in the model group are divided into 5 groups at random, 12 every group, use following 5 kinds of GnRH-PE mutant fusion proteins respectively:
6Ala-GnRH-PE40KDEL60ug/kg, GnRH-PE40KDEL 60ug/kg, GnRH-PE39KDEL 60ug/kg, GnRH-PE38 60ug/kg, solvent 1ml/ are only.Adopt the administration of intravenous injection mode, 1 time/day, continuous 15 days.Before the administration, administration gets the skin of pinna specimen and stores to be checked in the time of the 15th day.
3 results
3.1 the HE coloration result of Cavia porcellus psoriasis model collect specimen
The horny layer that normal model matched group guinea pig skin is as seen poor, granular layer is about 1～3 layer, about 3～5 layers of prickle cell layer, basal layer is the monolayer cylindrical cell. true epidermis has a common boundary more flat, and intradermal is dispersed in the minority monocyte infiltration.
Model group through propranolol coating 3 after week most of specimen all see in horny layer hyperkeratosis, epidermal area acanthosis, the skin corium mastoid process cell infiltration arranged, the part specimen is seen extensively or kitchen range shape parakeratosis, granular layer attenuation or disappearance, the trochanterellus extension is bar-shaped, stretch on the mastoid process and be the change of pestle shape, telangiectasis.
We carry out the histological score analysis by with reference to the Baker methods of marking one by one to every part of specimen.Application organizes point system (typical psoriatic lesion scoring is between 4.0～10.0) scoring obtains, the specimen histological score is 0.35 ± 0.28 before the model group coating, it is 7.81 ± 0.62 that 4 weeks of coating are organized the specimen histological score, histological score was respectively before and after the normal model matched group was coated with simple Emulsion: 0.41 ± 0.32,0.47 ± 0.11, learn result by statistics, organize before group and the coating behind the coating and the normal model matched group significant difference (P＜0.01) of comparing, and matched group is coated with before the simple Emulsion group and organizes zero difference after being coated with simple Emulsion with matched group, sees Table 4.
The histological score of table 4 Cavia porcellus psoriasis model and matched group sample
Grouped organizing compares P before and after learning the scoring coating
The model group coating is preceding 0.35 ± 0.28-
That matched group is coated with simple Emulsion is preceding 0.41 ± 0.32-
Behind the model group coating 7.81 ± 0.62＜0.01
Matched group is coated with behind the simple Emulsion 0.47 ± 0.11 0.46
Above-mentioned experimental result shows, the psoriasis model of utilizing Cavia porcellus successfully to set up can be used for treating the recruitment evaluation of psoriasis medicine.
3.2 the GnRH-PE mutant fusion protein is respectively organized specimen HE coloration result before and after being applied to the Cavia porcellus psoriasis model
According to before this 2,2 the joint described in application program, the GnRH-PE mutant fusion protein was applied to the model group Cavia porcellus after 15 days,
6In the specimen of Ala-GnRH-PE40KDEL 60ug/kg group, GnRH-PE40KDEL 60ug/kg group, GnRH-PE 39KDEL 60ug/kg group and GnRH-PE38 60ug/kg group, horny layer parakeratosis phenomenon disappears substantially, and only an other places has parakeratosis to change.The attenuation of epidermal area prickle cell layer, 1～2 layer of granular layer cell of most appearance, stretch on trochanterellus extension and the mastoid process, telangiectasis etc. obviously alleviates, and the minority cell infiltration is only arranged in the skin corium mastoid process.Blank group histology does not have obvious change.Histological score saw Table 5 before and after each organized administration.
Table 5 is respectively organized administration front and back histological score
Group is handled the news commentary divisional processing P that marks after 15 days
6Ala-GnRH-PE4OKDEL organizes 7.57 ± 0.58 1.43 ± 0.37 1.49297E-10
GnRH-PE40KDEL organizes 7.37 ± 0.58 1.62 ± 0.35 1.24225E-09
GnRH-PE39KDEL organizes 8.08 ± 0.75 1.51 ± 0.55 4.14948E-09
GnRH-PE38 organizes 7.62 ± 0.49 2.32 ± 0.81 1.67254E-06
Blank group 7.70 ± 0.65 6.49 ± 0.88 0.163981
By The above results as can be known, GnRH-PE mutant fusion protein
6All the have clear improvement effect of psoriasis symptom of Ala-GnRH-PE40KDEL, GnRH-PE40KDEL, GnRH-PE39KDEL, GnRH-PE38.
Embodiment 5, GnRH-PE mutant fusion protein GnRH-PE40,6Ala-GnRH-PE40KDEL, GnRH-PE40KDEL, GnRH-PE39KDEL, GnRH-PE38 are to the therapeutic effect of flakey skin (fsn/fsn) sudden change mouse model
1, material
36 healthy adult flakey skins (fsn/fsn) sudden change Mus (male and female are not limit), test specimen GnRH-PE40,
6Production provides according to embodiment by the Beijing Northland Biotechnology Limited Liability Company for Ala-GnRH-PE40KDEL, GnRH-PE40KDEL, GnRH-PE39KDEL, GnRH-PE38.
2, sample is to the treatment of model
36 Cavia porcelluss in the model group are divided into 6 groups at random, 6 every group.Correspond respectively to following 6 kinds of processing schemes: GnRH-PE40 60ug/kg,
6Ala-GnRH-PE40KDEL, GnRH-PE40KDEL 60ug/kg, GnRH-PE39KDEL 60ug/kg, GnRH-PE38 60ug/kg, solvent 1ml/ are only.Adopt the administration of intravenous injection mode, 1 time/day, continuous 15 days.Preceding 0 day of administration, administration were got the skin of pinna specimen on the 15th day respectively and are stored to be checked.Paraffin embedding, HE dyeing, tissues observed changes.
HE dyeing: will be with the fixed specimen dehydration of 10% formalin, paraffin section is made in embedding behind the transparent waxdip, and conventional H E dyes. tissues observed finding one by one under light microscopic.Method with reference to Baker is formulated the histological score standard.
Respectively organize specimen HE coloration result before and after the treatment of 3 models
Model group Cavia porcellus intravenous drug is after 15 days, GnRH-PE40 60ug/kg group,
6Following phenomenon nearly all occurs in Ala-GnRH-PE40KDEL60ug/kg group, GnRH-PE40KDEL60ug/kg group, GnRH-PE39KDEL 60ug/kg group and the GnRH-PE38 60ug/kg group specimen: the parakeratosis phenomenon disappears substantially, and only an other places has parakeratosis to change.The prickle cell layer attenuation, 1～2 layer of granular layer cell of most appearance, stretch on trochanterellus extension and the mastoid process, telangiectasis etc. obviously alleviates, and the minority cell infiltration is only arranged in the mastoid process.Blank group histology does not have obvious improvement, in horny layer hyperkeratosis, epidermal area acanthosis, the skin corium mastoid process a large amount of cell infiltration is arranged, granular layer attenuation or disappearance, and the trochanterellus extension is bar-shaped, stretches on the mastoid process to be the change of pestle shape, telangiectasis.Histological score saw Table 6 before and after each organized administration.
Table 6 is respectively organized administration front and back histological score
| Group | Mark during processing | Handle scoring after 15 days | P |
| GnRH-PE40 | 9.13±0.81 | 1.55±0.39 | 2.26519E-12 |
| 6The Ala-GnRH-PE40KDEL group | 8.66±0.48 | 1.47±0.51 | 2.48357E-13 |
| The GnRH-PE40KDEL group | 8.99±0.38 | 1.82±0.39 | 1.11022E-16 |
| The GnRH-PE39KDEL group | 8.47±0.73 | 1.55±0.35 | 1.38062E-11 |
| The GnRH-PE38 group | 8.62±0.79 | 2.12±0.46 | 3.05394E-09 |
| The blank group | 8.70±0.65 | 9.49±0.88 | 0.261516492 |
Sequence table
＜110〉Beijing Northland Biotechnology Limited Liability Company
＜120〉GnRH-PE mutant fusion protein and uses thereof
<130>IDC070102
<160>19
<170>PatentIn?version?3.3
<210>1
<211>10
<212>PRT
＜213〉people
<400>1
<210>2
<211>613
<212>PRT
＜213〉people
<400>2
<210>3
<211>5
<212>PRT
＜213〉artificial sequence
<400>3
<210>4
<211>5
<212>PRT
＜213〉artificial sequence
<400>4
<210>5
<211>11
<212>PRT
＜213〉artificial sequence
<400>5
<210>6
<211>10
<212>PRT
＜213〉artificial sequence
<400>6
<210>7
<211>10
<212>PRT
＜213〉artificial sequence
<400>7
<210>8
<211>10
<212>PRT
＜213〉artificial sequence
<400>8
<210>9
<211>10
<212>PRT
＜213〉artificial sequence
<400>9
<210>10
<211>361
<212>PRT
＜213〉artificial sequence
<400>10
<210>11
<211>345
<212>PRT
＜213〉artificial sequence
<400>11
<210>12
<211>318
<212>PRT
＜213〉artificial sequence
<400>12
<210>13
<211>334
<212>PRT
＜213〉artificial sequence
<400>13
<210>14
<211>354
<212>PRT
＜213〉artificial sequence
<400>14
<210>15
<211>376
<212>PRT
＜213〉artificial sequence
<400>15
<210>16
<211>375
<212>PRT
＜213〉artificial sequence
<400>16
<210>17
<211>375
<212>PRT
＜213〉artificial sequence
<400>17
<210>18
<211>368
<212>PRT
＜213〉artificial sequence
<400>18
<210>19
<211>360
<212>PRT
＜213〉artificial sequence
<400>19
Claims (9)
1, the GnRH-PE mutant fusion protein is used to prepare the purposes for the treatment of psoriatic medicine, and wherein said GnRH-PE mutant fusion protein is selected from:
1) the N-end of the C-end of GnRH mutant and the PE mutant fusion rotein LLP that links to each other and form with peptide bond by joint sequence, i.e. GnRH mutant-joint sequence-PE mutant; With
2) the N-end of the C-end of PE mutant and the GnRH mutant mutant fusion protein PLL that links to each other and form with peptide bond by joint sequence, i.e. PE mutant-joint sequence-GnRH mutant;
3) have 1) or 2) described in the variant of one or several amino acid whose insertion in the mutant fusion protein aminoacid sequence, disappearance and/or replacement, prerequisite is that described variant keeps 1 basically) or 2) described in the biologic activity of mutant fusion protein; And
4) with 1) or 2) described in the mutant fusion protein aminoacid sequence have variant more than or equal to 90% sequence homogeneity, prerequisite is that described variant keeps 1 basically) or 2) described in the biologic activity of mutant fusion protein;
Wherein
Described GnRH mutant is meant that its amino terminal first amino acids replaces to human luteinizing hormone's releasing factor mutant of glutamic acid (Glu);
Described joint sequence is made up of 0-20 aminoacid, as needs, can not use described joint sequence, or select to use by 3 amino acid residues, 5 amino acid residues, 10 amino acid residues, 15 amino acid residues, even 20 joint sequences that amino acid residue is formed;
Described PE mutant is selected from:
A) Pseudomonas exotoxin A (PE) is removed the deletion mutant PE40 that forms after the N-terminal 1-252 amino acids;
B) with the 253-364 position of Pseudomonas exotoxin A (PE), the two sections aminoacid sequences in 381-613 position link to each other by former order and obtain albumen PE38;
C) with the 280-364 position of Pseudomonas exotoxin A (PE), the two sections aminoacid sequences in 381-613 position link to each other by former order and obtain albumen PE35;
D) with the fragment albumen PE37 of the 280-613 amino acids sequence of Pseudomonas exotoxin A (PE); With
E) with the 253-358 position of Pseudomonas exotoxin A (PE), the two sections aminoacid sequences in 366-613 position link to each other by former order and obtain albumen PE39.
2, the described purposes of claim 1, wherein said joint sequence is selected from:
-His-Met-Ala-Glu-Glu-(SEQ?ID?NO：3)；
-Gly-Ser-Gly-Gly-Gly-(SEQ ID NO:4); With
-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-(SEQ?IDNO：5)。
3, the described purposes of claim 1, among the wherein said fusion rotein LLP, the C-end of described PE mutant can also contain 2-5 and be selected from-XYEL or-repeated fragment of REDLK, that is:
The n of GnRH mutant-joint sequence-PE mutant-(XYEL), or
The n of GnRH mutant-joint sequence-PE mutant-(REDLK),
Wherein n is the integer of 2-5;
Among described fragment-XYEL, X represents lysine (Lys) or arginine (Arg), and Y represents aspartic acid (Asp), glutamine (Gln) or glutamic acid (Glu), and E represents glutamic acid (Glu), and L represents leucine (Leu); The aminoacid sequence fragment of described fragment-REDLK for adopting single-letter to represent, wherein, R represents arginine (Arg), and E represents glutamic acid (Glu), and D represents aspartic acid (Asp), and L represents leucine (Leu), and K represents lysine (Lys).
4, the described purposes of claim 1, among the wherein said fusion rotein PLL, the C-end of described GnRH mutant can also contain 2-5 and be selected from-XYEL or-repeated fragment of REDLK, promptly
The n of PE mutant-joint sequence-GnRH mutant-(XYEL) or
The n of PE mutant-joint sequence-GnRH mutant-(REDLK),
Wherein n is the integer of 2-5;
Described fragment-XYEL and-definition such as claim 2 of REDLK described in.
5, the described purposes of claim 1, wherein said GnRH mutant further the 6th glycine of its amino terminal (Gly) optional sport alanine (Ala), tryptophan (Trp) or leucine (Leu).
6, the described purposes of claim 1, wherein said PE mutant further sport-XYEL at 5 aminoacid-Arg-Glu-Asp-Leu-Lys of its C-end.
7, the described purposes of claim 1, wherein said GnRH-PE mutant fusion protein be selected from GnRH-PE40,
6Ala-GnRH-PE40KDEL, GnRH-PE40KDEL, GnRH-PE39KDEL and GnRH-PE38.
8, the described purposes of claim 1, wherein said psoriasis is selected from psoriasis vulgaris, psoriasis pustulosa, arthropathic psoriasis and erythrodermic psoriasis.
9, GnRH-PE mutant fusion protein, it is selected from:
1)GnRH-PE39KDEL(SEQ?ID?NO：18)；
2) have 1) described in the variant of one or several amino acid whose insertion in the mutant fusion protein aminoacid sequence, disappearance and/or replacement, prerequisite is that described variant keeps 1 basically) described in the biologic activity of mutant fusion protein; And
3) with 1) described in the mutant fusion protein aminoacid sequence have variant more than or equal to 90% sequence homogeneity, prerequisite is that described variant keeps 1 basically) described in the biologic activity of mutant fusion protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007101879158A CN101433713B (en) | 2007-11-15 | 2007-11-15 | GnRH-PE mutant fusion protein and uses thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007101879158A CN101433713B (en) | 2007-11-15 | 2007-11-15 | GnRH-PE mutant fusion protein and uses thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101433713A true CN101433713A (en) | 2009-05-20 |
| CN101433713B CN101433713B (en) | 2011-10-12 |
Family
ID=40708454
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007101879158A Active CN101433713B (en) | 2007-11-15 | 2007-11-15 | GnRH-PE mutant fusion protein and uses thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101433713B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102250252A (en) * | 2010-05-17 | 2011-11-23 | 中国人民解放军军事医学科学院军事兽医研究所 | Gastrin-releasing peptide (GRP) guided fusion protein |
| CN102321178A (en) * | 2011-05-10 | 2012-01-18 | 北京易德新奥生物科技有限公司 | Novel gonadotropin releasing hormone oriented fusion protein mutant |
| 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 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1294149C (en) * | 2003-06-18 | 2007-01-10 | 北京诺思兰德生物技术有限责任公司 | Genet engineering recombinant protein capable of specifically killing tumor cell |
| CN1840546A (en) * | 2005-04-01 | 2006-10-04 | 北京诺思兰德生物技术有限责任公司 | Recombinant fusion protein with targeted cell for killing tumor |
-
2007
- 2007-11-15 CN CN2007101879158A patent/CN101433713B/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102250252A (en) * | 2010-05-17 | 2011-11-23 | 中国人民解放军军事医学科学院军事兽医研究所 | Gastrin-releasing peptide (GRP) guided fusion protein |
| CN102321178A (en) * | 2011-05-10 | 2012-01-18 | 北京易德新奥生物科技有限公司 | Novel gonadotropin releasing hormone oriented fusion protein mutant |
| CN102321178B (en) * | 2011-05-10 | 2013-07-31 | 北京易德新奥生物科技有限公司 | Novel gonadotropin releasing hormone oriented fusion protein mutant |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101433713B (en) | 2011-10-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110845603B (en) | Human collagen 17-type polypeptide, production method and use thereof | |
| CN106632682A (en) | Fusion protein IFN-ELP and application thereof | |
| CN101433713B (en) | GnRH-PE mutant fusion protein and uses thereof | |
| US11746134B2 (en) | Human FGF21 mutant with improved effectiveness and stability and pharmaceutical composition thereof | |
| CN101914561B (en) | Fusion protein with antibacterial and repairing function and production method and application thereof | |
| CN101225110B (en) | Human interleukins-22 mutant as well as construction method and use thereof | |
| JP2023527093A (en) | rhFGF21 fusion proteins, polynucleotides encoding rhFGF21 fusion proteins, compositions containing rhFGF21 fusion proteins, and uses of rhFGF21 fusion proteins | |
| CN104293821A (en) | Gene cloning, expression and application of recombinant human fibroblast growth factor-20 | |
| CN112851791B (en) | Novel FGF analogue for resisting metabolic disorder and application thereof | |
| CN103145851B (en) | Recombinant protein PACAP38-NtA, and coding gene and application thereof | |
| CN100457778C (en) | Mutant of ciliary nerves trophic factor (CNTF), producing method and usage | |
| CN102516392B (en) | Cancer-targeted super antigen fusion protein, and preparation method and application thereof | |
| CN103360497A (en) | Novel antitumor fusion protein vaccine, and preparation method and application thereof | |
| CN113292656A (en) | Fusion protein of mesencephalon astrocyte-derived neurotrophic factor for preventing and treating obesity | |
| CN106749561B (en) | A kind of stenotrophomonas maltophilia outer membrane protein and its application | |
| CN101580846A (en) | Human cytoglobin for preventing and curing cirrhosis and preparation method thereof | |
| CN101875699A (en) | Fusion protein of human epidermal growth factor and metallothionein and preparation method and application thereof | |
| CN112341523B (en) | Small peptide encoded by DLEU2 and application thereof in preparation of immunomodulatory drugs | |
| CN101897953B (en) | Non-invasive high-penetrability epidermal growth factor and application thereof | |
| KR20140094837A (en) | Novel Polypeptides Binding with Interleukin-6 and Uses Thereof | |
| CN103601805B (en) | A kind of light chain of people FGF21 monoclonal antibody and variable region of heavy chain | |
| CN1904041B (en) | Human recombination phospholipase D2, its preparation method and application in preparation medicine | |
| CN101089181A (en) | Production method of recombination human interleukin-4 | |
| CN106674352B (en) | Derivative of antineoplastic protein endostatin and application thereof | |
| CN101812135B (en) | Fusion protein with high-efficient platelet-increasing role and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20090520 Assignee: Beijing North Rand Medical Technology Co., Ltd. Assignor: Beijing Northland Biotechnology Co., Ltd. Contract record no.: 2012990000805 Denomination of invention: GnRH-PE mutant fusion protein and uses thereof Granted publication date: 20111012 License type: Exclusive License Record date: 20121031 |
|
| LICC | Enforcement, change and cancellation of record of contracts on the licence for exploitation of a patent or utility model | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: GnRH-PE mutant fusion protein and uses thereof Effective date of registration: 20200628 Granted publication date: 20111012 Pledgee: Beijing technology intellectual property financing Company limited by guarantee Pledgor: BEIJING NORTHLAND BIOTECH Co.,Ltd. Registration number: Y2020990000671 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20210118 Granted publication date: 20111012 Pledgee: Beijing technology intellectual property financing Company limited by guarantee Pledgor: BEIJING NORTHLAND BIOTECH Co.,Ltd. Registration number: Y2020990000671 |