CN101469031A - High cell toxicity targeting fusion protein - Google Patents
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- CN101469031A CN101469031A CNA2007103013164A CN200710301316A CN101469031A CN 101469031 A CN101469031 A CN 101469031A CN A2007103013164 A CNA2007103013164 A CN A2007103013164A CN 200710301316 A CN200710301316 A CN 200710301316A CN 101469031 A CN101469031 A CN 101469031A
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Abstract
The invention relates to a high-toxicity targeted fused protein, which is a fused protein GnRH-PE39KDELconsisting of GnRH and PE derivatives. Compared with the prior targeted medicine of the same type, the high-toxicity targeted fused protein has higher targeted cytotoxic activity, so as to have potential application value in the aspect of treating tumor.
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 cell during treatment.
Targeted therapy is a kind of new treatment means that is expected to replace chemotherapy, and many pieces of documents and patent (Chinese patent: the method for cancer diagnosis using chimeric toxin, 99806580.3 is arranged; Chinese patent: the genetically engineered recombinant protein of the special kill tumor cell of a kind of energy, 03137587.1; Chinese patent: the series function albumen of high-efficiency low-toxicity, 200410033621.6) reported about end user's luetinizing hormone releasing factor (GnRH) and derivative and false pseudomonas bacillus exotoxin A (PE) structure targent fused protein, can be used for the targeted therapy of tumour; Other has document (Mol Microbiol.1999 Mar; 31 (5): 1385-93.Adeletion within the translocation domin of Pseudomonas exotoxin A enhancestranslocation efficiency and cytotoxicity concomitantly) reported that the activity of PE toxin can increase substantially with after the 359-365 amino acids of the PE toxin disappearance; After also having document (US Patent 5602095, Recombinant pseudomonas exotoxin with increased activity) report that terminal 5 the amino acid REDLK of PE are sported KDEL, the activity of PE also can increase substantially.Based on this and conjugated protein three dimensional analysis, the present invention has carried out the following structure that changes in order to obtain active higher targent fused protein at targent fused protein GnRH-PE40 (SEQ ID NO:1):
1 has carried out lacking (being PE39) with the 359-365 amino acids of PE40.
25 amino acid REDLK of end with PE39 sport KDEL (being PE39KDEL).
Finally we have obtained highly active targent fused protein GnRH-PE39KDEL, and very high using value is arranged aspect oncotherapy.
Two. background technology
Treatment for cancer remains a difficult problem at present, and the main treatment means that people use remains operation, radiation and chemotherapy, and traditional therapeutics is when bringing curative effect, and side effect is also often very big, and more limited for the oncotherapy effect of recurrence.The shortcoming of traditional therapy is that mainly it has also killed normal cell in large quantities in the kill tumor cell, the appearance of targeted therapy makes us realize targeted therapy to tumour cell, be that the medicine molecule can guide to arrive tumour cell and play a role, reach the effect of targeted therapy.Immunotoxin or targeted toxin are important developing direction that reaches targeted therapy at present, be connected with toxin by specific antibody or cytokine, hormone 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.Be targeting part with the GnRH or derivatives thereof at present, the target therapeutic agent that is toxin moiety with the clipped form PE40 and the PE40 derivative of PE toxin has had report (Chinese patent: the method for cancer diagnosis using chimeric toxin, 99806580.3; Chinese patent: the genetically engineered recombinant protein of the special kill tumor cell of a kind of energy, 03137587.1; Chinese patent: the series function albumen of high-efficiency low-toxicity, 200410033621.6), the targeting type of these fusion roteins is extensively verified, identifying purpose cell and it is killed specifically, but in application, also there is a very big problem at present, that is exactly because the structure problem of solid tumor, the PE derivative is little for eugonic solid tumor onset in the animal model in the present target toxic protein, so how intensifier target PE cytotoxicity partly in toxic protein has just become a problem that urgency is to be solved.
Three. summary of the invention:
The present invention relates to a kind of GnRH-PE derivative, in order further to improve the biological activity of targent fused protein, strengthen its lethality to eugonic tumour cell, thereby better targent fused protein is applied to the treatment field, the inventor is by carrying out the tertiary protein structure analysis to known GnRH-PE fusion rotein, in conjunction with document, adopt gene engineering method that the toxin moiety of described fusion rotein is lacked and suddenly change, by a large amount of screenings, obtained obviously to be better than aspect active the new GnRH-PE derivative of existing GnRH-PE derivative.
GnRH-PE derivative among the present invention comprises the 359-365 amino acids of PE40 has been carried out lacking (being PE39), and 5 amino acid REDLK of end of PE40 are sported KDEL, finally we have obtained highly active targent fused protein GnRH-PE39KDEL.
The targent fused protein that obtains by foregoing invention has advantage of high activity, and tumour cell is had very strong specific killing effect, is the potential anti-tumor medicine.
One aspect of the present invention relates to a kind of GnRH-PE derivative GnRH-PE39KDEL, is the aminoacid sequence shown in the SEQ ID NO:2.
Those of ordinary skills know, and described above-mentioned fusion rotein can be by the method for dna recombinant expression or the method preparation of chemosynthesis.In the present invention, described GnRH-PE39KDEL obtains by the recombinant expressed method of genetically engineered.
In one embodiment of the invention, obtain GnRH-PE39KDEL by the following method, comprise the steps:
1) structure of expression vector
According to known amino acid sequence (SEQ ID NO:1), select the full gene synthetic gene sequence of intestinal bacteria preference codon for use, add restriction enzyme site at N-terminal and C-terminal simultaneously corresponding to coded aminoacid sequence.
Using the method for PCR operates the gene order of SEQ ID NO:1, reach the 359-365 amino acids of PE40 has been carried out lacking (being PE39), and 5 amino acid REDLK of end of PE40 are sported the purpose of KDEL, the nucleotide sequence of the fusion rotein of the SEQ ID NO:2 that obtains encoding.
To handle well with restriction enzyme as the GnRH-PE derivative gene order and the screening plasmid pBSK of above-mentioned preparation, press certain mol proportion as calculated behind each sample concentration and add the T4 linked system.Through the recon that transforms, screening is successful.
Downcut antigen-4 fusion protein gene with Restriction Enzyme, again this gene is connected with the plasmid pET21b of NOVAGEN company that handles well with Restriction Enzyme, obtain recon correctly through screening.
2) expression of recombination targent fused protein and separation and purification
To be transformed into the expressive host bacterium as above-mentioned gained recon, go out the cance high-expression gene engineering bacteria,, obtain the engineering bacteria of target protein high expression level then through fermentation through expression screening.Use the broken bacterium of osmotic pressure method, pass through centrifugal, clarifying step then, by a series of chromatography methods commonly used, as ion-exchange, gel-filtration and hydrophobic chromatography etc., purifying finally obtain purity greater than 95% target protein.
3) the bioactive mensuration of target protein: use mtt assay, at first the Hela cell is digested and collect, cell is diluted to 6-8 * 10 with the RPMI1640 nutrient solution
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 the IC50 value with microplate reader 570nm with mtt assay dyeing.
Another aspect of the present invention relates to a kind of pharmaceutical composition, and it contains GnRH-PE39KDEL of the present invention, and optional pharmaceutically acceptable carrier.
Know that in the present invention term " pharmaceutically acceptable " means the animal that can be used for that pharmacy field generally acknowledges, more particularly can be used for the people's.Term " carrier " refers to thinner, adjuvant (for example (fully or not exclusively) freund's adjuvant), vehicle or be used to holds or the medium of administering therapeutic agent.
These pharmaceutical carriers can be sterile liquids, such as water and oil, comprise being derived from oil, animal, plant or synthetic oil, such as peanut oil, soybean oil, mineral oil, sesame oil, like that.When intravenously was used medicinal compositions, water was preferred carrier.Salt brine solution and aqueous dextrose and glycerine solution also can be used as liquid carrier, especially for Injectable solution.Suitable pharmaceutical excipient comprises starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talcum, sodium-chlor, milk powder, glycerine, propylene, ethylene glycol, water, ethanol, like that.If desired, composition can also comprise the wetting or emulsifying agent such as the hyaluronate sodium of lesser amt, or the pH buffer reagent.These compositions can be taked solution, suspension, milk sap, tablet, pill, capsule powder, slowly-releasing prescription, suchlike form.
In one embodiment of the invention, described pharmaceutical composition is the freeze dried injection form, wherein contains the GnRH-PE39KDEL of 0.01%-0.2%, and 5% N.F,USP MANNITOL, and pharmaceutically acceptable carrier.
Medicinal compositions of the present invention is mixed with to use the path compatible with its purpose.The example of using the path includes but not limited to parenteral, for example (for example sucking) in intravenously, intracutaneous, subcutaneous, oral cavity, the nose, through skin (for example local), through mucous membrane and rectal administration.In a specific embodiments, be mixed with composition in suitable intravenously, subcutaneous, intramuscular, oral cavity, the nose or be locally applied to human medicinal compositions according to old process.Usually, being used for the composition that intravenously uses is the solution of sterile isotonic water-based damping fluid.If desired, composition can also comprise solubilizing agent and local anesthetic such as Ergotamine to alleviate the pain of injection site.
If want topical application composition of the present invention, then composition can be mixed with form or well-known other form of those skilled in the art of ointment, emulsifiable paste, skin subsides, lotion, gel, shampoo, sprays, aerosol, solution, emulsion.Consult for example " Lei Mingdunshi pharmaceutical science and pharmaceutical dosage form introduction " (Remington ' s Pharmaceutical Sciencesand Introduction to Pharmaceutical Dosage Forms), the 19th edition, Mack publishing company, Easton, the Pennsylvania, nineteen ninety-five.
For non-sprayable local dose form, usually adopt the viscosity that comprises the carrier compatible or one or more vehicle and have the dynamic viscosity that is preferably greater than water to semisolid or solid form with topical application.Suitable prescription includes but not limited to solution, suspension, milk sap, emulsifiable paste, ointment, pulvis, liniment, ointment, like that, if desired, be aseptic or mixed to change various characteristics, such as for example osmotic pressure with auxiliary reagent (for example sanitas, stablizer, wetting agent buffer reagent or salt).Other suitable local dose form comprises sprayable aerosol formulation, and the activeconstituents of wherein preferably uniting solid phase or liquid phase inert support is packaged in the mixture that contains supercharging volatile matter (for example pressurized gas, such as freonll-11) or squeezes in the bottle.If desired, can also in pharmaceutical composition and dosage form, add moistening agent or wetting agent.The example of these extra compositions is well known in the art.
In another embodiment of the present invention, described pharmaceutical composition is an ointment, wherein contains GnRH-PE39KDEL and pharmaceutically acceptable vehicle such as the Vaseline of 0.01%-0.2%.
If method of the present invention comprises the intranasal administration of composition, then composition can be mixed with the form of aerosol, sprays, mist agent or drops.Particularly, the preventative or therapeutic agent of using according to the present invention can use suitable propelling agent (for example Refrigerant 12, the single fluoromethane of trichlorine, dichloro tetrafluoro ethane, carbonic acid gas or other suitable other) to be delivered easily by supercharging packing or atomizer with the form that aerosol spray presents.In the situation of pressurized aerosol, can determine dose unit by valve is provided, to deliver metered amounts.Employed capsule and cartridge case in sucker or the insufflator (being made of for example gelatin) can be mixed with the powdered mixture that contains compound and suitable powder matrix such as lactose or starch.
Method of the present invention comprises that also preparation is used for the using of composition by injection (for example by injecting or transfusion continuously) parenteral administration.The prescription that is used for injecting can exist with the unit dosage form (for example at ampoule or in multi-dose container) that contains extra sanitas.Composition can be taked such as forms such as the suspension in oiliness or the aqueous medium, solution or milk sap, and can contain preparaton, such as suspension agent, stablizer and/or dispersion agent.Perhaps, activeconstituents can be a powder type, uses suitable media (for example aseptic, apirogen water) dissolving before use.
Can be used for the suitable media of parenteral dosage form of the present invention is provided is well-known to those skilled in the art.In certain embodiments, the medium that is suitable for the parenteral dosage form includes but not limited to water for injection USP; Aqueous medium includes but not limited to sodium chloride injection, RingerShi injection liquid, glucose injection, dextrose ﹠ sodium chloride injection and lactic acid RingerShi injection liquid; Water easily mixes medium and includes but not limited to ethanol, polyoxyethylene glycol and polypropylene glycol; And non-aqueous media includes but not limited to Semen Maydis oil, Oleum Gossypii semen, peanut oil, sesame oil, ethyl oleate, Isopropyl myristate and phenylamino benzoic acid methyl esters.
One side more of the present invention relates to the purposes that GnRH-PE39KDEL is used to prepare the cancer patients's who treats tumor cell surface expression GnRH acceptor medicine.
Prove by experiment, by the animal pattern experimental study to the body of tumour in target kill and wound active aspect, GnRH-PE39KDEL of the present invention has apparently higher than present known GnRH-PE40KDEL (SEQ ID NO:3) and the active characteristics of GnRH-PE39 (SEQ ID NO:4).
The inventor has studied the effect that GnRH-PE39KDEL kills and wounds in-vivo tumour.With A549 lung carcinoma cell on 120 nude inoculations, thereby obtain lung cancer nude mouse model.Inject GnRH-PE39KDEL every other day totally 10 times nude mice tumor mass reduce the most obviously, tumour inhibiting rate is the highest, result of treatment obviously is better than GnRH-PE39 and present known GnRH-PE40KDEL.
The above results shows, GnRH-PE39KDEL of the present invention aspect the treatment tumour activity apparently higher than GnRH-PE39 and present known GnRH-PE40KDEL.
Those of ordinary skills know, and the mode of using, frequency and dosage will be according to the illness of being treated, the patient's condition with individual different and different.In general, can use modes such as (for example eye drops) by injection (for example intracutaneous, intramuscular, intravenously or subcutaneous), topical application (for example epidermis is used) or dropping uses.Also can be according to rational route of administration of the different choice of individual patients and application program.The amount of proper dosage for after using above-mentioned pharmaceutical composition, can effectively treating the cancer patients of tumor cell surface expression GnRH acceptor.
In general, for the pharmaceutical composition that contains the GnRH-PE39KDEL of containing of the present invention, the amount that is present in each dosage is approximately 100 μ g-5mg.What of proper dosage will be different because of patient's illness and administering mode, but general about scope is 0.5mg-2mg, preferred 1mg.
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.
Example one.
According to the principle of intestinal bacteria preference codon, synthesize the pairing gene order of SEQ IDNO:1 by full gene synthetic method in conjunction with the method for PCR, the method by PCR sudden change disappearance obtains the pairing gene order of SEQ ID NO:2-4 then.Through after the sequence verification, goal gene is connected into expression vector, the commentaries on classics of recombinant plasmid after the checking is imported in the host bacterium, obtain the purpose thalline through expressing, fermenting, then by behind the broken bacterium of osmometry, centrifuging and taking supernatant, the method purifying by hydrophobic chromatography and ion exchange chromatography obtain purity greater than 4 kinds of pure product of targent fused protein of 95%, carry out the interior pharmacodynamics test of cytoactive test and body then respectively simultaneously.
Example two.
Targent fused protein and the three kinds of controlled trial fusion roteins got among the present invention carry out the cytoactive test: use mtt assay, at first the Hela cell is digested and collect, 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.11 kinds of samples are diluted to 100ul/mL with the RPMI RPMI-1640 earlier, as starter hole, press 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, 37 degree were cultivated 24 hours, took out, with mtt assay dyeing, measure and calculate the IC50 value with microplate reader 570nm.Determination of activity the results are shown in following table,
Example three:
The intravital test of pesticide effectiveness: A549 lung carcinoma cell in 120 male BALB/C nude mices (SPF level) inoculation, thus obtain lung cancer nude mouse model.Be divided into 12 groups at random, every group of 10 nude mices are respectively the physiological saline group, GnRH-PE derivative SEQID NO:1-4 group is inoculated back 7 days and is risen, every one day intravenous injection 75ug/kg, administration is 10 times altogether, surveys major diameter (a), the minor axis (b) of knurl piece in per 4 days, according to formula V=ab
2/ 2 calculate gross tumor volume (mm
3), inoculate back 27 days and put to death animal, dissect and get the knurl piece, claim knurl heavy, calculate tumour inhibiting rate.
Test-results:
| Fusion rotein | Tumour inhibiting rate |
| GnRH-PE40(SEQ?ID?NO：1) | 30.5％ |
| GnRH-PE40KDEL(SEQ?ID?NO：3) | 44.7％ |
| GnRH-PE39(SEQ?ID?NO：4) | 56.8％ |
| GnRH-PE39KDEL(SEQ?ID?NO：2) | 81.8％ |
Conclusion: as can be seen from the above results, the cell in vitro activity of GnRH-PE39KDEL, the interior antitumor activity of body all are significantly higher than present known GnRH-PE derivative GnRH-PE40KDEL (SEQ ID NO:3) and GnRH-PE39 among the present invention, illustrate that the present invention has strengthened tumor-inhibiting action in its biological activity and the body by disappearance and sudden change to targent fused protein.
Sequence table
SEQ?ID?NO：1
SEQ?ID?NO：2
SEQ?ID?NO：3
SEQ?ID?NO：4
Claims (4)
1. fusion rotein GnRH-PE39KDEL who forms by human luteinizing hormone's releasing hormone derivative GnRH and false pseudomonas bacillus exotoxin A PE derivative, and be the tumor disease that the main component of medicine is used for the treatment of cell surface overexpression GnRH acceptor with this fusion rotein.
2. according to claim 1, the aminoacid sequence of said fusion rotein 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-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 2, can give expression to all nucleotide sequences of aminoacid sequence in 2.
4. the fusion rotein in the claim 1 uses intestinal bacteria, yeast or eukaryotic cell to be the host bacterium, and the method for using gene engineering is expressed, purifying obtains.
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| CNA2007103013164A CN101469031A (en) | 2007-12-27 | 2007-12-27 | High cell toxicity targeting fusion protein |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102603897A (en) * | 2011-12-08 | 2012-07-25 | 山东省科学院生物研究所 | Fusion protein containing guide peptide and GnRH-PE39KDEL as well as nucleic acid and application thereof |
| CN103509119A (en) * | 2012-06-29 | 2014-01-15 | 山东省科学院生物研究所 | Recombinant fusion protein of short peptide hormone and recombinant pseudomonas exotoxin A and application thereof |
Citations (1)
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| 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 |
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- 2007-12-27 CN CNA2007103013164A patent/CN101469031A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| MARIE-PIERRE TAUPIAC ET AL: "A deletion within the translocation domain of Pseudomonas exotoxin A enhances translocation efficiency and cytotoxicity concomitantly", 《MOLECULAR MICROBIOLOGY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102603897A (en) * | 2011-12-08 | 2012-07-25 | 山东省科学院生物研究所 | Fusion protein containing guide peptide and GnRH-PE39KDEL as well as nucleic acid and application thereof |
| CN102603897B (en) * | 2011-12-08 | 2014-10-08 | 山东省科学院生物研究所 | Fusion protein containing guide peptide and GnRH-PE39KDEL as well as nucleic acid and application thereof |
| CN103509119A (en) * | 2012-06-29 | 2014-01-15 | 山东省科学院生物研究所 | Recombinant fusion protein of short peptide hormone and recombinant pseudomonas exotoxin A and application thereof |
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Application publication date: 20090701 |