CN101880327A - Scorpion arialgesic anti-tumoral peptide fusion and acquisition method thereof - Google Patents
Scorpion arialgesic anti-tumoral peptide fusion and acquisition method thereof Download PDFInfo
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Abstract
The invention, belonging to the field of biotechnology, relates to a scorpion arialgesic anti-tumoral peptide fusion as well as derivative or analog or active fragment thereof and an acquisition method thereof, specifically to the fact that: by means of a simple short-peptide linker, interstitial cell stimulating hormone taken as the parts of scorpion arialgesic anti-tumoral peptide and cancel cell homing peptides releases chimeric protein composed of hormone or partial fragments inside insuline-like growth factor II or Octreotide analog or the above analog, the cancel cell homing peptides are respectively fused at N-end and C-end of the scorpion arialgesic anti-tumoral peptide. According to the invention, a series of carriers of fusion proteins of the scorpion arialgesic anti-tumoral peptide with different linkers and cancel cell homing peptides are constructed by means of DNA recombination technology, and the chimeric protein is acquired and then verified whether double functions, namely in vivo analgesic activity and in vitro tumor cell antigrowth activity, are contained therein, and that targeted anti-tumor activity against specific tumor cells can be realized.
Description
Technical field
The present invention relates to the biological medicine technology field, relate to scorpion arialgesic anti-tumoral peptide fusion and preparation method thereof, specifically, the present invention relates to serial scorpion arialgesic anti-tumoral peptide fusion or part fragment that should the series syzygy or should series syzygy derivative, the structure of analogue and preparation method thereof, and the application in field of medicaments as analgesic and antitumor drug.
Background technology
Scorpion is traditional rare Chinese medicine of China as the medicinal history in existing more than 2000 year of traditional Chinese medicine, has bigger exploitation and is worth.It has been found that multiple scorpion venom single component polypeptide in recent years with pharmacological actions such as antitumor, anti-inflammatory, analgesia, anti-epileptics.Natural scorpion venom output is extremely low, therefore, adopts gene engineering method, and the method that has the active Buthotoxin polypeptide of pharmaceutical use with simple protokaryon or the production of eukaryote expressive host seems particularly important.
A kind of method that improves the tumour cell specific selectivity of antitumor drug is preferentially to the tumor cell surface delivering drugs.Can be by several method with the drug targeting tumour cell.Wherein one of method is to rely on the specific receptors molecule that is present on the tumor cell surface.The molecule that is called as " directed agents " can be discerned these cell surface receptors and specificity combination with it.Said " directed agents " can comprise antibody, somatomedin or hormone etc." directed agents " of identification specificity cell surface receptor can have drug targeting the cell of these surface receptors.
The functional protein syzygy is the biotherapy novel method with wide application prospect that development in recent years is got up, and mainly is by targeting part that medicine is special and be gathered in lesions position efficiently, thereby realizes the purpose of target killing sick cell.The many parts of adding between targeting part and protein drug of generalized case flexibly connect the thing sequence, can keep the proteic configuration separately in two ends effectively, improve the space flexibility of molecule, with combining and the therapeutic action of protein drug performance inherent of favourable syzygy albumen targeting part and corresponding acceptor.
Based on this, there is the people once to utilize mouse interleukin-13 (mIL-3) to combine with diphtheria toxin, and between two parts, inserts a small peptide joint sequence Gly as targeting part
4Ser obtains fused protein DAB389-Gly
4Ser-mIL-3, experimental result shows in the external and body, compares with the fusion rotein DAB389-mIL-3 of belt lacing sequence not, and the former improves about 5-10 times (referring to Domunique.et al to the specific cell toxic action of tumour cell, FEBS Letters 406:157-161,1997).Reported first such as Amotz Nechushtan are also used GnRH 10 peptides and are made up chimeric toxin (GnRH-PE66) as targeted peptide, through the inside and outside Related Experimental Study and inquired into its target killing activity at adenocarcinoma cell, thereby be the broader applications of luteinizing hormone-releasing hormone open thinking (Amotz Nechushtan, Shai Yarkoni, IrinaMarianovsky etal.Adenocarcinoma Cel ls Are Targeted by the New GnRH-PE66Chimeric Toxin through Specific Gonadotropin-releasing Hormone BindingSites.THE JOURNAL OF BIOLOGICAL CHEMISTRY, 272:11597-11063,1997).Also reported a kind of based on joint sequence recently, the chimeric protein (GnRH-L-PE66 or GnRH-L-PE40) that has Pseudomonas exotoxin (PE) and gonadoliberin (GnRH) to form.The interior experimental result of external and body shows that GnRH-L-PE has cytotoxic activity and tumor growth to suppress active to the kinds of tumor cells cording, and the active GnRH-PE that does not have joint sequence that is significantly higher than.Therefore, have the chimeric toxin that flexibly connects thing and will become a kind of potential anti-tumor medicine.
Patent name is that (patent of invention number: invention ZL01128235.5) provides a kind of have better analgesic effect and antineoplastic scorpion arialgesic antitumoral peptide and preparation method for scorpion arialgesic antitumoral peptide and preparation method, and to obtain having analgesia and anti-tumor biological, simultaneously again to tumour cell realize the serial syzygy of target killing function or part fragment that should the series syzygy or should series syzygy derivative, analogue and preparation method thereof but await further research.
The selected five kinds of small molecules cancer cells targeted peptides that merge with scorpion arialgesic antitumoral peptide of the present invention, its specific recognition acceptor of bibliographical information is at liver cancer cell surface high expression level and normal cell is expressed hardly or very low (the Schally AV of expression amount, Srkalovic G, etal.Antitumor effects of analogsof LHRH and somatostatin:experimental and clinical studies.J steroidBiochem Mol Biol, 1990,37:1061-1067; Raderer M, Hejna MH, Muller C, etal.Treatment of hepatocellular cancer with the long actingsomatostatin analog lanreotide in vitro and in vivo.Int J oncol, 2000,16 (6): 1197-1201; Zhou Xiaomei, Chen Yuanqing.Human insulin-like growth factor acceptor and somatomedin thereof be the while overexpression in primary hepatocarcinoma.Tumour, 1991,11:241), these researchs just provide theoretical basis for selectively targeted killing hepatoma cell.There is the specificity overexpression acceptor of the selected cancer cells targeted peptide of report the present invention not only to be confined to the liver cancer cell surface simultaneously, is present in equally on the cytolemma as other cancerous tissues such as lung cancer, prostate cancers.
Summary of the invention
An object of the present invention is to provide by scorpion arialgesic antitumoral peptide and different carcinoma cell targeted peptide syzygy albumen dimerous, and between has inserted suitable joint sequence.
Another object of the present invention is the proteic carrier of syzygy that makes up scorpion arialgesic antitumoral peptide and different carcinoma cell targeted peptide, proteic expression and purification method of this series syzygy and inside and outside bioactivity research.Different carcinoma cell targeted peptide is respectively targeting part, and after different flexible peptide linker connected, the N end or the C end that merge at scorpion arialgesic antitumoral peptide by flexible peptide linker constituted corresponding carrier.
Another object of the present invention provides the method for producing the chimeric protein that is defined as above, and this method comprises:
(1) provides the recombinant expression vector of the coding DNA that carries different carcinoma cell targeted peptide, different flexible peptide linker, scorpion arialgesic antitumoral peptide;
(2) recombinant vectors with step (1) transforms appropriate host cell (protokaryon or eukaryotic cell);
(3) be suitable for expressing culturing step (2) under the condition of scorpion arialgesic anti-tumoral peptide fusion by transformed host cells;
(4) results and the resulting scorpion arialgesic anti-tumoral peptide fusion protein of purifying.
A further object of the present invention provides and contains the syzygy protein that is defined as above and the pharmaceutical composition of one or more pharmaceutically acceptable carriers or vehicle.
A further object of the present invention provides the application of fused protein in producing analgesia and antitumor drug that is defined as above.
Serial scorpion arialgesic anti-tumoral peptide fusion albumen provided by the invention or part fragment that should the series syzygy or should series syzygy derivative, analogue all from genetically engineered host cell separation and purification obtain.Can use methods such as salt precipitation, ultrafiltration, ion exchange chromatography, hydrophobic interaction chromatography and gel-filtration from the lysate of cell and nutrient solution, to separate and the required protein expression product of purifying.In the separation and purge process of product, can use sodium dodecyl sulfate-polyacrylamide gel electrophoresis method (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA) or detected by Western blot (WESTERN) to detect the existence and the corresponding molecular size of product.
The present invention also provide serial scorpion arialgesic anti-tumoral peptide fusion albumen or part fragment that should the series syzygy or should series syzygy derivative, analogue is in the application of field of biological pharmacy, specifically comprises analgesia and anti-tumor biological.
The present invention utilizes different cancer cells targeted peptides to merge with scorpion arialgesic antitumoral peptide first and between is introduced the flexible peptide linker sequence that length is different and amino acid composition is different.
According to a preferred embodiment of the invention, wherein the connection peptides sequence is (Gly
4Ser)
n, n=1.
According to a preferred embodiment of the invention, wherein the connection peptides sequence is (Gly
4Ser)
n, n=2.
According to a preferred embodiment of the invention, wherein the connection peptides sequence is (Gly
4Ser)
n, n=3.
According to a preferred embodiment of the invention, wherein the connection peptides sequence is (Gly
4Ser)
n, n=4.
According to a preferred embodiment of the invention, wherein the connection peptides sequence is (Gly
4Ser)
n, n=5.
The present invention carry out first serial scorpion arialgesic anti-tumoral peptide fusion albumen or part fragment that should the series syzygy or should series syzygy derivative, analgesic activities and the experiment of extracorporeal anti-tumor activity of cell biology in the mouse body of analogue.
The invention further relates to the pharmaceutical composition that contains above-mentioned syzygy albumen and at least a pharmaceutically acceptable inert support or vehicle.Can be suitable for the pharmaceutical composition (as referring to Remington ' s PharmaceuticalScience, 15th., Mack Publishing Company, 1980) of the outer administration of gi tract according to the preparation of the known fundamental principle in pharmaceutical industry field and method.Can by in various route of administration, particularly intravenously, intramuscular, intraarticular, intraperitoneal, the nose, intracutaneous, the outer approach of gi tract such as the subcutaneous pharmaceutical composition of the present invention that comes into operation.
Can use syzygy albumen of the present invention or contain the proteinic pharmaceutical composition of this syzygy, be used for the treatment of the various diseases of particular type human body cell as therapeutical agent.A kind of preferred purposes is to be used for the treatment of the cell associated diseases that overexpression luteinizing hormone-releasing hormone acceptor, insulin-like growth factor II acceptor or somatostatin receptor are gone up in its surface.Syzygy protein of the present invention or the treatment effective dose that contains the proteinic pharmaceutical composition of this syzygy generally should be according to the character of disease, severity and to the responsive adaptability of medicine, and route of administration etc. is all is multifactorly determined according to principle of individuation by the clinician.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell, and the example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell etc.
In the present invention, english abbreviation " BmkAGAP " is meant scorpion arialgesic antitumoral peptide.X is meant the cancer cells targeted peptide, and X1 is QHWSYGLRPG, and X2 is QHWSYGWRPG, and X3 is QHWSYGFRPG, and X4 is EPFRSPDLALETYG, and X5 is PFCFWKTCT.J is meant connection peptides, and J1 is SSHHHHHHSSGLVPRGSHM, and J2 is GGGSGGGS, and J3 is SGGSGGSGGGSGGGSGGGGSGGGGS, and J4 is GSGGGGSGGGGS and (G
4S)
n(n=1 or 2 or 3 or 4 or 5).
Can use the various detection methods in the document that proteinic character of syzygy of the present invention and biologic activity are identified.Specifically comprise: analgesic activities experiment in (1) mouse body: this method is partly to have tangible analgesic activities according to the scorpion arialgesic antitumoral peptide in the syzygy protein, detects it to mouse acetic acid twisting analgesic inhibiting rate; (2) MTT cell survival rate experiment: this method is according to viable cell MTT to be changed into the influence of the ability detection of blue purple first a ceremonial jade-ladle, used in libation xln to tumour cell; (3) receptors bind experiment: this method is that competitive displacement combines with the target cell serous coat according to syzygy protein
125The ability of I-GnRH detects its specific receptors in conjunction with activity; (4) transplanted tumor in nude mice growth-inhibiting experiment: this method is to detect its target killing activity to tumour cell according to the inhibition ability that syzygy protein is grown to transplanted tumor in nude mice.
With five kinds of cancer cells targeted peptides be respectively the serial scorpion arialgesic anti-tumoral peptide fusion of guiding or part fragment that should the series syzygy or should series syzygy derivative, analogue can guidance quality ground selective action in primary tumors and metastasis, toxic side effect is little simultaneously, and serial syzygy albumen of the present invention also can play analgesic activity preferably, can also alleviate patient's slight illness in the time of the treatment disease, so its application prospect will be very wide.
Embodiment
The following examples can make those skilled in the art more fully understand the present invention, rather than limit the scope that the present invention gives special approval to claim by any way.The experimental technique of unreceipted actual conditions among the embodiment is usually according to normal condition, as the molecular cloning laboratory manual.
Embodiment 1:
The acquisition of X1-J1-BmkAGAP syzygy and derivative thereof, analogue
1.X1-J1-BmkAGAP the structure of syzygy gene
Present embodiment is enumerated and is described construction strategy and the basic skills be used to express X1-J1-BmkAGAP syzygy of the present invention and derivative thereof, analogue gene.
Aminoacid sequence according to cancer cells targeted peptide-X1 (QHWSYGLRPG) and scorpion arialgesic antitumoral peptide (BmkAGAP) designs corresponding oligonucleotide upstream primer and downstream primer, with the pET28a-Tag that builds
(His)-BmkAGAP plasmid is that template is carried out pcr amplification, and agarose gel electrophoresis detects the gel that product also carries out nucleic acid fragment and reclaims, through behind restriction endonuclease NcoI and the BamHI double digestion with carry out the plasmid pSYPU of double digestion at T equally
4Connect under the effect of DNA ligase, thermal transition competent escherichia coli cell DH 5 α, process bacterium colony PCR and restriction endonuclease enzyme cut the checking screening and obtain to submit to biotechnology service company to carry out the DNA determining nucleic acid sequence behind the positive transformant.The result shows, successfully make up and obtain recombinant plasmid pSYPU-X1-J1-BmkAGAP by engineered method, obtain the fusion gene sequence of cancer cells targeted peptide-QHWSYGLRPG, connection peptides-SSHHHHHHSSGLVPRGSHM and scorpion arialgesic antitumoral peptide.
2.X1-J1-BmkAGAP the acquisition of syzygy and derivative thereof, analogue
Utilize genetic engineering technique, make up X1-J1-BmkAGAP syzygy protein expressing plasmid, extract plasmid and check order.
The constitutional features of this recombinant expression plasmid coding expression product is: MGQHWSYGLRPGSSHHHHHHSSGLVPRGSHMVRDGYIADDKNCAYFCGRNAYCDDE CKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCNGG.
The expression product of fermentation expression and separating Escherichia coli host cell according to a conventional method after order-checking is confirmed.At first with positive recombinant plasmid thermal transition e. coli bl21 (λ DE3), be seeded to 3ml LB (containing ammonia benzyl microbiotic, 60 μ g/ml) behind the picking list bacterium colony from this LB solid plate then, in 37 ℃, the 200r/min shaken overnight is cultivated.According to 1% inoculum size overnight culture is seeded in the triangular flask that 400ml contains corresponding antibiotic fresh LB substratum, in 37 ℃, 200r/min shaking culture to OD value is 0.6-0.8, adding final concentration is inductor isopropyl ss-D-thiogalactoside (IPTG) of 0.166mmol/L, cultivates 4h.Finish fermentation, 4 ℃ of centrifugal collection thalline.With 40ml lysis buffer (0.1M PBS, 0.15M NaCl, the 50mM imidazoles) resuspended thalline, carry out ultrasonication, ultrasonic end back is in 12,000g, 4 ℃ of centrifugal 20min, get supernatant liquor, the gained precipitation repeats fragmentation once according to above-mentioned steps, merge supernatant liquor twice, directly be splined on the good metal ion-chelant chromatography column of 0.1M PBS (pH 8.0) pre-balance, behind 5 column volumes of pH damping fluid difference thorough washing through two different stepss, use 0.5M imidazoles (pH 9.0) to carry out wash-out and gather in the crops this elutriant.Products therefrom is verified its purity through 15%SDS-PAGE.By column chromatography chromatogram figure and the explanation of SDS-PAGE collection of illustrative plates, reorganization syzygy protein obtains to efficiently express, and through obtaining the single purpose protein band behind the conventional column chromatography of two steps, reaches electrophoresis purity.
According to above-mentioned strategy and basic skills, the constitutional features of X1-J1-BmkAGAP syzygy derivative, analogue is: MGQHWSYGLRPGSSHHHHHHSSGLVPRGSHVRDGYIADDKNCAYFCGRNAYCDDEC KKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCNGG.Utilize chemical method at M place fracture removal M, thereby obtain X1-J1-BmkAGAP syzygy derivative, analogue, constitutional features is: GQHWSYGLRPGSSHHHHHHSSGLVPRGSHVRDGYIADDKNCAYFCGRNAYCDDECK KNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCNGG.
3.X1-J1-BmkAGAP expression and the purifying thereof of syzygy albumen in yeast
To encode X1-J1-BmkAGAP or X1-J1 (M)-BmkAGAP syzygy protein gene increases, PCR reacts the encoding sequence that employed 5 ' Oligonucleolide primers sequence contains the N-terminal zone aminoacid sequence of the restriction enzyme site of EcoRV restriction enzyme and scorpion arialgesic antitumoral peptide.3 ' end primer sequence contains the encoding sequence of the restriction enzyme site of EcoR I restriction enzyme, translation stop codon and scorpion arialgesic antitumoral peptide scorpion pain-stopping anti-tumor bioactive peptide C-terminal zone aminoacid sequence.
The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the Yeast expression carrier pPIC9K (wherein the restriction endonuclease otch of EcoRV and SnaB I for flat terminal), this plasmid vector coding antibiotics resistance (Amp
rAnd Kan
r), a replicon from 2 μ plasmids, an AOX1 promotor can be efficiently expressed by methanol induction in pichia spp, the signal peptide sequence of a α-factor, a transcription termination signal, HIS4 selected marker and integration sequence.
With SnaB I and EcoR I digestion pPIC9K carrier, insert fragment with EcoR V and EcoR I digestion, to insert fragment subsequently and be connected into the pPIC9K carrier, connect product thermal transition E.coli DH 5 α bacterial strains, screen transformant containing on the LB culture dish of Amp, incubated overnight contains the clone of required construction in the LB liquid nutrient medium that contains Amp (100 μ g/ml).The extracting plasmid, sequence verification is inserted fragment and is correctly inserted.
After getting plasmid linearization, electricity is transformed into yeast cell, and linearizing recombinant vectors produces stable yeast conversion body by the homologous sequence generation homologous recombination with host genome.Utilize G418 to filter out the integration transformant of high copy.The inoculation that screening obtains is shaken in the bottle in the 250ml that 25ml MGY, BMG or BMGY substratum are housed, be cultured to OD in 28～30 ℃/250～300 rev/mins
600=2～6 (16～18h); The following 3000 rev/mins of centrifugal 5min of room temperature collect thalline, with MM, BMM or the resuspended thalline of BMMY (about 10～20ml) of 1/5 to 1/10 former volume of culture; The bacterium liquid of step 2 gained is placed the bottle that shakes of 100ml, seal, be positioned over continued growth on 28～30 ℃/250～300 rev/mins the shaking table with double gauze or cheese cloth; Every 24h adds 100% methyl alcohol to final concentration in substratum be 0.5～1.0%; The supernatant liquor of sample separation is that pigment is removed in the ultra-filtration membrane ultrafiltration of 3000Da with molecular weight cut-off, detects and identifies Recombinant Protein Expression with SDS-PAGE, Western-Blot and activity experiment.
The expression product constitutional features that relates to is:
1.GQHWSYGLRPGSSHHHHHHSSGLVPRGSHMVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVP?I?RVPGKCNGG。
2.QHWSYGLRPGSSHHHHHHSSGLVPRGSHMVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCNGG。
3.GQHWSYGLRPGSSHHHHHHSSGLVPRGSHVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCNGG。
4.QHWSYGLRPGSSHHHHHHSSGLVPRGSHVRDGYIADDKNCAYFCGRNAYCDDECKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCNGG。
Embodiment 2:
The acquisition of X2-J1-BmkAGAP syzygy and derivative thereof, analogue
The strategy of present embodiment, basic skills and primary process are with embodiment 1.
The constitutional features of X2-J1-BmkAGAP syzygy and derivative thereof, analogue is:
1.QHWSYGWRPGSSHHHHHHSSGLVPRGSHM-BmkAGAP。
2.QHWSYGWRPGSSHHHHHHSSGLVPRGSH-BmkAGAP。
3.MQHWSYGWRPGSSHHHHHHSSGLVPRGSHM-BmkAGAP。
4.MQHWSYGWRPGSSHHHHHHSSGLVPRGSH-BmkAGAP。
Embodiment 3:
The acquisition of X3-J1-BmkAGAP syzygy and derivative thereof, analogue
The strategy of present embodiment, basic skills and primary process are with embodiment 1.
The constitutional features of X3-J1-BmkAGAP syzygy and derivative thereof, analogue is:
1.QHWSYGFRPGSSHHHHHHSSGLVPRGSHM-BmkAGAP。
2.QHWSYGFRPGSSHHHHHHSSGLVPRGSH-BmkAGAP。
3.MQHWSYGFRPGSSHHHHHHSSGLVPRGSHM-BmkAGAP。
4.MQHWSYGFRPGSSHHHHHHSSGLVPRGSH-BmkAGAP。
Embodiment 4:
The acquisition of X4-J1-BmkAGAP syzygy and derivative thereof, analogue
The strategy of present embodiment, basic skills and primary process are with embodiment 1.
The constitutional features of X4-J1-BmkAGAP syzygy and derivative thereof, analogue is:
1.MEPFRSPDLALETYGSSHHHHHHSSGLVPRGSHM-BmkAGAP。
2.MEPFRSPDLALETYGSSHHHHHHSSGLVPRGSH-BmkAGAP。
3.EPFRSPDLALETYGSSHHHHHHSSGLVPRGSHM-BmkAGAP。
4.EPFRSPDLALETYGSSHHHHHHSSGLVPRGSH-BmkAGAP。
Embodiment 5:
The acquisition of X5-J1-BmkAGAP syzygy and derivative thereof, analogue
The strategy of present embodiment, basic skills and primary process are with embodiment 1.
The constitutional features of X5-J1-BmkAGAP syzygy and derivative thereof, analogue is:
1.MPFCFWKTCTSSHHHHHHSSGLVPRGSHM-BmkAGAP。
2.MPFCFWKTCTSSHHHHHHSSGLVPRGSH-BmkAGAP。
3.PFCFWKTCTSSHHHHHHSSGLVPRGSHM-BmkAGAP。
4.PFCFWKTCTSSHHHHHHSSGLVPRGSH-BmkAGAP。
Embodiment 6:
It with J2 the acquisition of the BmkAGAP syzygy of connection peptides and derivative thereof, analogue
The strategy of present embodiment, basic skills and primary process are with embodiment 1.In this embodiment, be that connection peptides forms BmkAGAP syzygy and derivative thereof, analogue with J2 (GGGSGGGS), its constitutional features is:
1.MQHWSYGLRPGGGGSGGGS-BmkAGAP，2.QHWSYGLRPGGGGSGGGS-BmkAGAP，3.M-BmkAGAP-GGGSGGGSQHWSYGLRPG，4.BmkAGAP-GGGSGGGSQHWSYGLRPG，5.MQHWSYGWRPGGGGSGGGS-BmkAGAP，6.QHWSYGWRPGGGGSGGGS-BmkAGAP，7.M-BmkAGAP-GGGSGGGSQHWSYGWRPG，8.BmkAGAP-GGGSGGGSQHWSYGWRPG，9.MQHWSYGFRPGGGGSGGGS-BmkAGAP，10.QHWSYGFRPGGGGSGGGS-BmkAGAP，11.M-BmkAGAP-GGGSGGGSQHWSYGFRPG，12.BmkAGAP-GGGSGGGSQHWSYGFRPG，13.MEPFRSPDLALETYGGGGSGGGS-BmkAGAP，14.EPFRSPDLALETYGGGGSGGGS-BmkAGAP，15.M-BmkAGAP-GGGSGGGSEPFRSPDLALETYG，16.BmkAGAP-GGGSGGGSEPFRSPDLALETYG，17.MPFCFWKTCTGGGSGGGS-BmkAGAP，18.PFCFWKTCTGGGSGGGS-BmkAGAP，19.M-BmkAGAP-GGGSGGGSPFCFWKTCT，20.BmkAGAP-GGGSGGGSPFCFWKTCT。
Embodiment 7:
It with J3 the acquisition of the BmkAGAP syzygy of connection peptides and derivative thereof, analogue
The strategy of present embodiment, basic skills and primary process are with embodiment 1.In this embodiment, be that connection peptides forms BmkAGAP syzygy and derivative thereof, analogue with J3 (SGGSGGSGGGSGGGSGGGGSGGGGS), its constitutional features is:
1.MQHWSYGLRPG-J3-BmkAGAP，2.QHWSYGLRPG-J3-BmkAGAP，3.M-BmkAGAP-J3-QHWSYGLRPG，4.BmkAGAP-J3-QHWSYGLRPG，5.MQHWSYGWRPG-J3-BmkAGAP，6.QHWSYGWRPG-J3-BmkAGAP，7.M-BmkAGAP-J3-QHWSYGWRPG，8.BmkAGAP-J3-QHWSYGWRPG，9.MQHWSYGFRPG-J3-BmkAGAP，10.QHWSYGFRPG-J3-BmkAGAP，11.M-BmkAGAP-J3-QHWSYGFRPG，12.BmkAGAP-J3-QHWSYGFRPG，13.MEPFRSPDLALETYG-J3-BmkAGAP，14.EPFRSPDLALETYG-J3-BmkAGAP，15.M-BmkAGAP-J3-EPFRSPDLALETYG，16.BmkAGAP-J3-EPFRSPDLALETYG，17.MPFCFWKTCT-J3-BmkAGAP，18.PFCFWKTCT-J3-BmkAGAP，19.M-BmkAGAP-J3-PFCFWKTCT，20.BmkAGAP-J3-PFCFWKTCT。
Embodiment 8:
It with J4 the acquisition of the BmkAGAP syzygy of connection peptides and derivative thereof, analogue
The strategy of present embodiment, basic skills and primary process are with embodiment 1.In this embodiment, be that connection peptides forms BmkAGAP syzygy and derivative thereof, analogue with J4 (GSGGGGSGGGGS), its constitutional features is:
1.MQHWSYGLRPG-J4-BmkAGAP，2.QHWSYGLRPG-J4-BmkAGAP，3.M-BmkAGAP-J4-QHWSYGLRPG，4.BmkAGAP-J4-QHWSYGLRPG，5.MQHWSYGWRPG-J4-BmkAGAP，6.QHWSYGWRPG-J4-BmkAGAP，7.M-BmkAGAP-J4-QHWSYGWRPG，8.BmkAGAP-J4-QHWSYGWRPG，9.MQHWSYGFRPG-J4-BmkAGAP，10.QHWSYGFRPG-J4-BmkAGAP，11.M-BmkAGAP-J4-QHWSYGFRPG，12.BmkAGAP-J4-QHWSYGFRPG，13.MEPFRSPDLALETYG-J4-BmkAGAP，14.EPFRSPDLALETYG-J4-BmkAGAP，15.M-BmkAGAP-J4-EPFRSPDLALETYG，16.BmkAGAP-J4-EPFRSPDLALETYG，17.MPFCFWKTCT-J4-BmkAGAP，18.PFCFWKTCT-J4-BmkAGAP，19.M-BmkAGAP-J4-PFCFWKTCT，20.BmkAGAP-J4-PFCFWKTCT。
Embodiment 9:
With (G
4S)
1Acquisition for the BmkAGAP syzygy of connection peptides and derivative thereof, analogue
The strategy of present embodiment, basic skills and primary process are with embodiment 1.In this embodiment, with (G
4S)
1For connection peptides forms BmkAGAP syzygy and derivative thereof, analogue, its constitutional features is:
1.MQHWSYGLRPG-(G
4S)
1-BmkAGAP，2.QHWSYGLRPG-(G
4S)
1-BmkAGAP，3.M-BmkAGAP-(G
4S)
1-QHWSYGLRPG，4.BmkAGAP-(G
4S)
1-QHWSYGLRPG，5.MQHWSYGWRPG-(G
4S)
1-BmkAGAP，6.QHWSYGWRPG-(G
4S)
1-BmkAGAP，7.M-BmkAGAP-(G
4S)
1-QHWSYGWRPG，8.BmkAGAP-(G
4S)
1-QHWSYGWRPG，9.MQHWSYGFRPG-(G
4S)
1-BmkAGAP，10.QHWSYGFRPG-(G
4S)
1-BmkAGAP，11.M-BmkAGAP-(G
4S)
1-QHWSYGFRPG，12.BmkAGAP-(G
4S)
1-QHWSYGFRPG，13.MEPFRSPDLALETYG-(G
4S)
1-BmkAGAP，14.EPFRSPDLALETYG-(G
4S)
1-BmkAGAP，15.M-BmkAGAP-(G
4S)
1-EPFRSPDLALETYG，16.BmkAGAP-(G
4S)
1-EPFRSPDLALETYG，17.MPFCFWKTCT-(G
4S)
1-BmkAGAP，18.PFCFWKTCT-(G
4S)
1-BmkAGAP，19.M-BmkAGAP-(G
4S)
1-PFCFWKTCT，20.BmkAGAP-(G
4S)
1-PFCFWKTCT。
Embodiment 10:
With (G
4S)
5Acquisition for the BmkAGAP syzygy of connection peptides and derivative thereof, analogue
The strategy of present embodiment, basic skills and primary process are with embodiment 1.In this embodiment, with (G
4S)
5For connection peptides forms BmkAGAP syzygy and derivative thereof, analogue, its constitutional features is:
1.MQHWSYGLRPG-(G
4S)
5-BmkAGAP，2.QHWSYGLRPG-(G
4S)
5-BmkAGAP，3.M-BmkAGAP-(G
4S)
5-QHWSYGLRPG，4.BmkAGAP-(G
4S)
5-QHWSYGLRPG，5.MQHWSYGWRPG-(G
4S)
5-BmkAGAP，6.QHWSYGWRPG-(G
4S)
5-BmkAGAP，7.M-BmkAGAP-(G
4S)
5-QHWSYGWRPG，8.BmkAGAP-(G
4S)
5-QHWSYGWRPG，9.MQHWSYGFRPG-(G
4S)
5-BmkAGAP，10.QHWSYGFRPG-(G
4S)
5-BmkAGAP，11.M-BmkAGAP-(G
4S)
5-QHWSYGFRPG，12.BmkAGAP-(G
4S)
5-QHWSYGFRPG，13.MEPFRSPDLALETYG-(G
4S)
5-BmkAGAP，14.EPFRSPDLALETYG-(G
4S)
5-BmkAGAP，15.M-BmkAGAP-(G
4S)
5-EPFRSPDLALETYG，16.BmkAGAP-(G
4S)
5-EPFRSPDLALETYG，17.MPFCFWKTCT-(G
4S)
5-BmkAGAP，18.PFCFWKTCT-(G
4S)
5-BmkAGAP，19.M-BmkAGAP-(G
4S)
5-PFCFWKTCT，20.BmkAGAP-(G
4S)
5-PFCFWKTCT。
Embodiment 11:
Analgesic activities in series BmkAGAP syzygy and derivative thereof, the similar object-mouse acetic acid twisting method
Present embodiment is intended to verify the analgesic activities of BmkAGAP syzygy and derivative thereof, analogue by analgesic model in the mouse body.
Mouse acetic acid twisting method analgesic model: Glacial acetic acid is injected the Kunming mouse intraperitoneal as chemical irritant, then cause the deep, big area and more persistent pain stimulation, cause mouse to produce " turning round body " reaction (belly indent, trunk and back leg extension, hips up).
The 18-22g Kunming mouse, male and female half and half, random packet, every group 8, abdominal injection BmkAGAP syzygy and derivative thereof, analogue sample cause Encelialgia by 0.2ml/20g abdominal injection 0.6% (v/v) acetic acid behind the 20min, turn round the body number of times behind the 5min in the record mouse 10min.With the positive contrast of morphine, physiological saline is blank, calculates the writhing response inhibiting rate of each administration group according to the following equation.
The analgesia biological activity test is the result show:
1. the blank group of physiological saline laboratory animal is turned round the body number of times (Mean ± SEM) is 46.75 ± 3.65; The positive controls laboratory animal is turned round the body number of times, and (Mean ± SEM) is 29.37 ± 1.85, and turning round the body inhibiting rate is 37.17%.
The laboratory animal of scorpion arialgesic antitumoral peptide group to turn round the body inhibiting rate be 55.6%.
3.BmkAGAP syzygy and derivative thereof, analogue group
(1) X1-J1-BmkAGAP syzygy and derivative thereof, analogue group
Sample: it is 54.1% that the laboratory animal of MGQHWSYGLRPGSSHHHHHHSSGLVPRGSHMVRDGYIADDKNCAYFCGRNAYCDDE CKKNGAESGYCQWAGVYGNACWCYKLPDKVPIRVPGKCNGG is turned round the body inhibiting rate.The above-mentioned sample of this group selection carries out the analgesic activities experiment, be because this structures of samples contains the structure of X1-J1-BmkAGAP syzygy and derivative thereof, analogue group, therefore, its result is enough to represent the analgesic activities of X1-J1-BmkAGAP syzygy and derivative thereof, analogue group.
(2) X2-J1-BmkAGAP syzygy and derivative thereof, analogue group
It is 55.1% that the laboratory animal of sample MQHWSYGWRPGSSHHHHHHSSGLVPRGSHM-BmkAGAP is turned round the body inhibiting rate.The above-mentioned sample of this group selection carries out the analgesic activities experiment, be because this structures of samples contains the structure of X2-J1-BmkAGAP syzygy and derivative thereof, analogue group, therefore, its result is enough to represent the analgesic activities of X2-J1-BmkAGAP syzygy and derivative thereof, analogue group.
(3) X3-J1-BmkAGAP syzygy and derivative thereof, analogue group
It is 53.8% that the laboratory animal of sample MQHWSYGFRPGSSHHHHHHSSGLVPRGSHM-BmkAGAP is turned round the body inhibiting rate.The above-mentioned sample of this group selection carries out the analgesic activities experiment, be because this structures of samples contains the structure of X3-J1-BmkAGAP syzygy and derivative thereof, analogue group, therefore, its result is enough to represent the analgesic activities of X3-J1-BmkAGAP syzygy and derivative thereof, analogue group.
(4) X4-J1-BmkAGAP syzygy and derivative thereof, analogue group
It is 55.8% that the laboratory animal of sample MEPFRSPDLALETYGSSHHHHHHSSGLVPRGSHM-BmkAGAP is turned round the body inhibiting rate.The above-mentioned sample of this group selection carries out the analgesic activities experiment, be because this structures of samples contains the structure of X4-J1-BmkAGAP syzygy and derivative thereof, analogue group, therefore, its result is enough to represent the analgesic activities of X4-J1-BmkAGAP syzygy and derivative thereof, analogue group.
(5) X5-J1-BmkAGAP syzygy and derivative thereof, analogue group
It is 56.5% that the laboratory animal of sample MPFCFWKTCTSSHHHHHHSSGLVPRGSHM-BmkAGAP is turned round the body inhibiting rate.The above-mentioned sample of this group selection carries out the analgesic activities experiment, be because this structures of samples contains the structure of X5-J1-BmkAGAP syzygy and derivative thereof, analogue group, therefore, its result is enough to represent the analgesic activities of X5-J1-BmkAGAP syzygy and derivative thereof, analogue group.
(6) with J2 be the BmkAGAP syzygy of connection peptides and derivative thereof, analogue group
Sample: MQHWSYGLRPGGGGSGGGS-BmkAGAP, M-BmkAGAP-GGGSGGGSQHWSYGLRPG, M-BmkAGAP-GGGSGGGSQHWSYGWRPG, M-BmkAGAP-GGGSGGGSQHWSYGFRPG, M-BmkAGAP-GGGSGGGSEPFRSPDLALETYG, the laboratory animal of M-BmkAGAP-GGGSGGGSPFCFWKTCT is turned round the body inhibiting rate all more than 52.5%.The above-mentioned sample of this group selection carries out the analgesic activities experiment, be because above-mentioned structures of samples contains with J2 is the structure of the BmkAGAP syzygy of connection peptides and derivative thereof, analogue group, therefore, to be enough to represent with J2 be the analgesic activities of the BmkAGAP syzygy of connection peptides and derivative thereof, analogue group to its result.
(7) with J3 be the BmkAGAP syzygy of connection peptides and derivative thereof, analogue group
Sample: MQHWSYGLRPG-J3-BmkAGAP, M-BmkAGAP-J3-QHWSYGLRPG, MEPFRSPDLALETYG-J3-BmkAGAP, M-BmkAGAP-J3-EPFRSPDLALETYG, MPFCFWKTCT-J3-BmkAGAP, the laboratory animal of M-BmkAGAP-J3-PFCFWKTCT is turned round the body inhibiting rate all more than 57.5%.The above-mentioned sample of this group selection carries out the analgesic activities experiment, be because above-mentioned structures of samples contains with J3 is the structure of the BmkAGAP syzygy of connection peptides and derivative thereof, analogue group, in addition, in conjunction with the experimental data of (1)-(6) among the analgesia biological activity test result 3.It is the analgesic activities of the BmkAGAP syzygy of connection peptides and derivative thereof, analogue group that this group experimental result is enough to represent with J3.
(8) with J4 be the BmkAGAP syzygy of connection peptides and derivative thereof, analogue group
Sample: MQHWSYGLRPG-J4-BmkAGAP, M-BmkAGAP-J4-QHWSYGLRPG, laboratory animal turn round the body inhibiting rate all more than 54.7%.In conjunction with the experimental data of (1)-(7) among the analgesia biological activity test result 3, it is the analgesic activities of the BmkAGAP syzygy of connection peptides and derivative thereof, analogue group that this group experimental result is enough to represent with J4.
(9) with (G
4S)
nBe the BmkAGAP syzygy of connection peptides and derivative thereof, analogue group
Sample: MQHWSYGLRPG-(G
4S)
1-BmkAGAP, M-BmkAGAP-(G
4S)
1-QHWSYGLRPG, MQHWSYGLRPG-(G
4S)
5-BmkAGAP, M-BmkAGAP-(G
4S)
5-QHWSYGLRPG, MEPFRSPDLALETYG-(G
4S)
1-BmkAGAP, M-BmkAGAP-(G
4S)
1-EPFRSPDLALETYG, MEPFRSPDLALETYG-(G
4S)
5-BmkAGAP, M-BmkAGAP-(G
4S)
5-EPFRSPDLALETYG, MPFCFWKTCT-(G
4S)
1-BmkAGAP, M-BmkAGAP-(G
4S)
1-PFCFWKTCT, MPFCFWKTCT-(G
4S)
5-BmkAGAP, M-BmkAGAP-(G
4S)
5The laboratory animal of-PFCFWKTCT is turned round the body inhibiting rate all more than 50.6%.In conjunction with the experimental data of (1)-(8) among the analgesia biological activity test result 3, this group experimental result is enough to representative with (G
4S)
N (n is 1 or 2 or 3 or 4 or 5)Analgesic activities for the BmkAGAP syzygy of connection peptides and derivative thereof, analogue group.
Embodiment 12:
Series BmkAGAP syzygy and derivative thereof, the outer antitumor cell biological activity-mtt assay of similar object
Present embodiment detects serial BmkAGAP syzygy that the present invention obtained and derivative thereof, the analogue cytotoxic activity to cultured tumor cells in vitro by mtt assay.
With the cell dissociation of logarithmic phase, centrifugal, make cell suspension, blow even and density is transferred to 6 * 10
5Individual/ml, add in the 96 hole microtest plates with 100 μ l/ holes, constantly blow even and fine born of the same parents therebetween, the cell count that guarantees every hole is about equally.In 37 ℃, CO
2Concentration is to hatch observation of cell growing state under inverted microscope 24 hours in 5% the cell culture incubator.Growing state is good, can add the sample of different weaker concns.Sample is with the dissolving of RPMI 1640 substratum, and the long-pending dilution method dilution of diploid is five concentration gradients, and each concentration is provided with three multiple holes.Negative control hole adds the nutrient solution of equal volume.Other establishes only to add and contains corresponding nutrient solution (acellular) and be blank zeroing hole.In 37 ℃, CO
2Concentration is to continue in 5% the cell culture incubator to cultivate after 48 hours to discard nutrient solution, every hole adds 100 μ l MTT (being diluted to final concentration is 0.5mg/ml), cultivate after 4 hours, supernatant is abandoned in suction, every hole adds 150 μ l dimethyl sulfoxide (DMSO), room temperature was put on the shaking table low-speed oscillation 10 minutes, fully the absorbance in each hole, 490nm place is measured in the dissolving back on microplate reader, calculate the growth inhibition ratio of every group of sample pair cell according to the absorbance of each sample, utilize the half-inhibition concentration IC50 value (promptly the survival cells amount reduces the required drug level of a half after medication) of SPSS 13.0 statistical software calculation samples.The method of calculation of inhibitory rate of cell growth are carried out with reference to following formula:
Series BmkAGAP syzygy and derivative thereof, similar object is outer antitumor cell biological activity test result show: compare X1-J1-BmkAGAP syzygy and derivative thereof and analogue with the scorpion arialgesic antitumoral peptide group, X2-J1-BmkAGAP syzygy and derivative thereof and analogue, X3-J1-BmkAGAP syzygy and derivative thereof and analogue, X4-J1-BmkAGAP syzygy and derivative thereof and analogue, X5-J1-BmkAGAP syzygy and derivative thereof and analogue, with J2 is BmkAGAP syzygy and the derivative and the analogue of connection peptides, with J3 is BmkAGAP syzygy and the derivative and the analogue of connection peptides, with J4 the BmkAGAP syzygy of connection peptides and derivative thereof and analogue and with (G
4S)
nFor the extracorporeal anti-tumor cell bio-activity of the BmkAGAP syzygy of connection peptides and derivative and analogue group does not obviously reduce, the BmkAGAP syzygy that has and the extracorporeal anti-tumor cell bio-activity of derivative and analogue thereof then have raising.
Embodiment 13:
The targeting specific test of series BmkAGAP syzygy and derivative thereof, analogue
Present embodiment detects the cell receptor binding ability of serial BmkAGAP syzygy of the present invention and derivative thereof, analogue in conjunction with test by cell-membrane receptor.According to Qaycum, the described method of people such as A. (Br.J.Cancer 62:96-99,1990) detects serial BmkAGAP syzygy of the present invention and derivative thereof, analogue competition replacement and human breast cancer cell bonded
125The ability of I-GnRH.
The human breast carcinoma Mcf-7 cell monolayer of cultivating is separated in the damping fluid that contains 10mM Tris-HCl (pH7.5), 1mMDTT and 1mg/ml bovine serum albumin with the preparation cell homogenates.After low-speed centrifugal removed big cell debris, again with 25,000g obtained the cytoplasm membrane portions with centrifugal 30 minutes of 4 ℃ of supernatant parts.The final volume of serous coat suspension by every hole 100 μ l is added in the 24 well culture plate holes, and then in each hole, adds 5 μ M
125I-GnRH also bathed 1 hour in 37 ℃ of temperature.In every hole, add unlabelled GnRH or scorpion arialgesic antitumoral peptide control sample or the present invention's series BmkAGAP syzygy and derivative thereof, analogue sample (0.1-100 μ M) after temperature is bathed again, and continue insulation 24 hours.The insulation back is washed sample and is detected the bonded part with the γ calculating instrument with above-mentioned damping fluid.After adding the sample of cumulative concentration, can replace and Mcf-7 cytolemma bonded gradually along with the increase of concentration
125I-GnRH.
Series BmkAGAP syzygy and derivative thereof, the targeting specific test-results of analogue shows: compare X1-J1-BmkAGAP syzygy and derivative thereof and analogue with the scorpion arialgesic antitumoral peptide sample sets, X2-J1-BmkAGAP syzygy and derivative thereof and analogue, X3-J1-BmkAGAP syzygy and derivative thereof and analogue, X4-J1-BmkAGAP syzygy and derivative thereof and analogue, X5-J1-BmkAGAP syzygy and derivative thereof and analogue, with J2 is BmkAGAP syzygy and the derivative and the analogue of connection peptides, with J3 is BmkAGAP syzygy and the derivative and the analogue of connection peptides, with J4 is BmkAGAP syzygy and the derivative and the analogue of connection peptides, with (G
4S)
nBmkAGAP syzygy and derivative and analogue group for connection peptides all are improved with the receptor in target cell binding ability, and this combination degree increases with the increase of sample concentration.
Claims (8)
1. scorpion arialgesic anti-tumoral peptide fusion, it is characterized in that, scorpion arialgesic antitumoral peptide forms serial scorpion arialgesic anti-tumoral peptide fusion by connection peptides and cancer cells targeted peptide, syzygy had both had the original biological activity of scorpion arialgesic antitumoral peptide, also had the biological activity of cancer cells targeted peptide simultaneously.
2. scorpion arialgesic anti-tumoral peptide fusion according to claim 1 is characterized in that, by the N end or the C end of scorpion arialgesic antitumoral peptide, forms serial scorpion arialgesic anti-tumoral peptide fusion by connection peptides and cancer cells targeted peptide respectively.
3. scorpion arialgesic anti-tumoral peptide fusion according to claim 1 is characterized in that, described cancer cells targeted peptide comprises: QHWSYGLRPG, QHWSYGWRPG, QHWSYGFRPG, EPFRSPDLALETYG, PFCFWKTCT.
4. scorpion arialgesic anti-tumoral peptide fusion according to claim 1 is characterized in that, described connection peptides comprises: SSHHHHHHSSGLVPRGSHM, GGGSGGGS, SGGSGGSGGGSGGGSGGGGSGGGGS, GSGGGGSGGGGS, GGGGS, (G
4S)
2, (G
4S)
3, (G
4S)
4And (G
4S)
5
5. the derivative of scorpion arialgesic anti-tumoral peptide fusion or analogue or active fragments is characterized in that, it comprises claim 1-4 any one described aminoacid sequence total order or fragment.
6. scorpion arialgesic anti-tumoral peptide fusion according to claim 5 and derivative thereof or analogue or active fragments can utilize genetic engineering technique to express acquisition, it is characterized in that, expression host cell is prokaryotic cell prokaryocyte or eukaryotic cell.
7. scorpion arialgesic anti-tumoral peptide fusion and derivative thereof or analogue or the active fragments application in the preparation analgesic.
8. application according to claim 7 is characterized in that: described scorpion arialgesic anti-tumoral peptide fusion and derivative thereof or analogue or active fragments can be mixed with acceptable injection, oral preparations, transdermal absorption formulation, mucosal absorption preparation clinically with pharmaceutically acceptable carrier.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690342A (en) * | 2011-03-22 | 2012-09-26 | 沈阳药科大学 | Anti-cancer analgesic peptide VKVR, its preparation method and application |
| CN103102403A (en) * | 2011-11-10 | 2013-05-15 | 沈阳药科大学 | Two mutants of analgesic bioactive peptide GRR, and preparation and application thereof |
| CN110357968A (en) * | 2018-04-08 | 2019-10-22 | 颜浩为 | Antineoplastic amalgamation protein and its preparation method and application |
-
2010
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690342A (en) * | 2011-03-22 | 2012-09-26 | 沈阳药科大学 | Anti-cancer analgesic peptide VKVR, its preparation method and application |
| CN102690342B (en) * | 2011-03-22 | 2014-10-29 | 沈阳药科大学 | Anti-cancer analgesic peptide VKVR, its preparation method and application |
| CN103102403A (en) * | 2011-11-10 | 2013-05-15 | 沈阳药科大学 | Two mutants of analgesic bioactive peptide GRR, and preparation and application thereof |
| CN103102403B (en) * | 2011-11-10 | 2014-04-23 | 沈阳药科大学 | Two mutants of analgesic bioactive peptide GRR, and preparation and application thereof |
| CN110357968A (en) * | 2018-04-08 | 2019-10-22 | 颜浩为 | Antineoplastic amalgamation protein and its preparation method and application |
| CN110357968B (en) * | 2018-04-08 | 2023-08-25 | 吉林省汇融生物科技有限公司 | Anti-tumor fusion protein and preparation method and application thereof |
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