WO2011069280A1 - Antimicrobial pexiganan analogue and preparation process thereof - Google Patents
Antimicrobial pexiganan analogue and preparation process thereof Download PDFInfo
- Publication number
- WO2011069280A1 WO2011069280A1 PCT/CN2009/001423 CN2009001423W WO2011069280A1 WO 2011069280 A1 WO2011069280 A1 WO 2011069280A1 CN 2009001423 W CN2009001423 W CN 2009001423W WO 2011069280 A1 WO2011069280 A1 WO 2011069280A1
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- Prior art keywords
- pexiganan
- lys
- analogue
- analog
- ttt
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- KGZGFSNZWHMDGZ-KAYYGGFYSA-N pexiganan Chemical class C([C@H](NC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(N)=O)C1=CC=CC=C1 KGZGFSNZWHMDGZ-KAYYGGFYSA-N 0.000 title claims abstract description 93
- 230000000845 anti-microbial effect Effects 0.000 title abstract description 4
- 238000002360 preparation method Methods 0.000 title abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 11
- 238000003786 synthesis reaction Methods 0.000 claims description 16
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- 238000000746 purification Methods 0.000 claims description 13
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- 230000000844 anti-bacterial effect Effects 0.000 claims description 10
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- 238000000034 method Methods 0.000 claims description 9
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- 239000003814 drug Substances 0.000 claims description 6
- 229940079593 drug Drugs 0.000 claims description 6
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- 108010062940 pexiganan Proteins 0.000 claims description 6
- 229950001731 pexiganan Drugs 0.000 claims description 6
- 229920001184 polypeptide Polymers 0.000 claims description 6
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- -1 Fmoc amino acids Chemical class 0.000 claims description 2
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- 210000004899 c-terminal region Anatomy 0.000 claims 1
- 230000002194 synthesizing effect Effects 0.000 claims 1
- 239000004599 antimicrobial Substances 0.000 abstract description 2
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- 229960000723 ampicillin Drugs 0.000 description 9
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- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 3
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- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 description 2
- UGNIYGNGCNXHTR-SFHVURJKSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UGNIYGNGCNXHTR-SFHVURJKSA-N 0.000 description 1
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- CEOOTQDKDICVJW-QNGWXLTQSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-(tritylamino)hexanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)CCCNC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 CEOOTQDKDICVJW-QNGWXLTQSA-N 0.000 description 1
- QTWZCODKTSUZJN-LJAQVGFWSA-N (2s)-5-[[amino-[(2,2,5,7,8-pentamethyl-3,4-dihydrochromen-6-yl)sulfonylamino]methylidene]amino]-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C12=CC=CC=C2C2=CC=CC=C2C1COC(=O)N[C@H](C(O)=O)CCCN=C(N)NS(=O)(=O)C(C(C)=C1C)=C(C)C2=C1OC(C)(C)CC2 QTWZCODKTSUZJN-LJAQVGFWSA-N 0.000 description 1
- QXVFEIPAZSXRGM-DJJJIMSYSA-N (2s,3s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H]([C@@H](C)CC)C(O)=O)C3=CC=CC=C3C2=C1 QXVFEIPAZSXRGM-DJJJIMSYSA-N 0.000 description 1
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- XUUXCWCKKCZEAW-YFKPBYRVSA-N Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](N)CCCN=C(N)N XUUXCWCKKCZEAW-YFKPBYRVSA-N 0.000 description 1
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- VBOBNHSVQKKTOT-YUMQZZPRSA-N Gly-Lys-Ala Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O VBOBNHSVQKKTOT-YUMQZZPRSA-N 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 108060003100 Magainin Proteins 0.000 description 1
- QXOHLNCNYLGICT-YFKPBYRVSA-N Met-Gly Chemical compound CSCC[C@H](N)C(=O)NCC(O)=O QXOHLNCNYLGICT-YFKPBYRVSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- RGMLUHANLDVMPB-ULQDDVLXSA-N Phe-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N RGMLUHANLDVMPB-ULQDDVLXSA-N 0.000 description 1
- UYKREHOKELZSPB-JTQLQIEISA-N Trp-Gly Chemical compound C1=CC=C2C(C[C@H](N)C(=O)NCC(O)=O)=CNC2=C1 UYKREHOKELZSPB-JTQLQIEISA-N 0.000 description 1
- 108010011559 alanylphenylalanine Proteins 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
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- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
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- WHWZLSFABNNENI-UHFFFAOYSA-N epinastine Chemical compound C1C2=CC=CC=C2C2CN=C(N)N2C2=CC=CC=C21 WHWZLSFABNNENI-UHFFFAOYSA-N 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
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- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to a Pexiganan analog (1-23) which has an antibacterial action and can be used as an antibacterial polypeptide drug.
- the present invention also provides a method of producing the Pexiganan analog (1-23) by chemical synthesis or genetic engineering.
- Insects of organisms invading the outside microbes produce antibacterial peptides that protect the body from pathogens through natural immunity. More than 800 antimicrobial peptides have been isolated from various organisms in the past 20 years, and more than 200 antimicrobial peptides have been isolated from various frog skins. This peptide, which has a relatively low molecular weight (20-60 amino acids), kills bacteria, fungi, protozoa and certain viruses. It is effective against some resistant microorganisms and is a new broad-spectrum antibacterial drug. "New antibiotics”. The antibacterial peptide itself is not toxic, and is amino acid after metabolism, which has been widely noted and studied at home and abroad.
- the high price of chemical synthetic products limits its wide application.
- the difficulty in preparing antimicrobial peptides by genetic engineering techniques is that the antibacterial peptides expressed by the fermentation will kill the engineered bacteria, and the fermentation is difficult to carry out.
- Fermented with Pichia Pastori s yeast the small peptide secreted into the fermentation broth has a small molecular weight, a small amount, and is difficult to recover.
- it is expressed by fusion protein, which forms insoluble inclusion bodies and avoids damage to engineering bacteria.
- the fusion protein carrier has a small molecular weight and a high expression level to increase the yield of the polypeptide of interest.
- the object of the present invention is to overcome the defects of the prior art and to successfully produce by using genetic engineering technology.
- Pexiganan analogues (1-23) can reduce costs by up to 500 mg per liter of fermentation broth.
- the Pexiganan analog (1-23) of the present invention is characterized in that the amino acid sequence of the Pexiganan analog (1-23) consists of 23 amino acids, and the Pexiganan analog (1-23) has the structural formula:
- the 22 amino acid sequence is Pexiganan
- the C terminus is Arg
- the C terminus Arg is -OH or NH2.
- the present invention also provides a method of producing the Pexiganan analog (1-23) by genetic engineering or chemical synthesis.
- the Pexiganan analog (1-23) of the present invention is an analog of the antibacterial peptide Magainin, which has a strong antibacterial action and can directly kill bacteria, fungi, protozoa and certain viruses without causing drug resistance.
- the use of the Pexiganan analog (1-23) of the present invention is useful as an antimicrobial polypeptide drug.
- antibacterial peptides such as Pexiganan used so far are chemically synthesized products, which are expensive and hinder their wide application. Genetic engineering using Pichia Pastoris yeast to secrete the expression of antimicrobial peptides to fermentation In the liquid, since the molecular weight is small and the amount of expression is also low, it is very difficult to recover from the fermentation broth. Antibacterial peptide
- Pexigarmn analogues (1-23) are expressed in the form of fusion proteins, which are simple to prepare and have industrial production significance.
- Figure 1 is an HPLC detection map of Pexiganan analog (1-23)
- Figure 2 is a diagram showing the bactericidal effect of Pexiganan analogues (1-23);
- Figure 3 is a diagram showing the synthesis process of a Pexiganan analogue (1-23) synthesized by solid phase synthesis. detailed description
- Embodiment 1 is a diagrammatic representation of Embodiment 1:
- the solid phase resin is Wang Resin, which performs solid phase synthesis. After the synthesis, the obtained peptide resin with side chain protecting group is cleaved, that is, the connection between the polypeptide and the resin is cut off, and the deprotection step is completed in one step.
- the C-terminus is a free carboxyl group.
- the reagents used were of analytical grade or peptide synthesis grade: N-methylpyrrolidone, chloroform, hexahydrobipyridyl, 2.0 M DIEA NMP, DMAP/DMF, HBTU 100 mmole/0.5M HOBT in F, and the like.
- Electromagnetic stirring at a constant temperature of 30 ° C for 6 hours removing the resin by filtration, collecting the supernatant, and washing the resin once with a small amount of trifluoroacetic acid, combining the collected liquid, adding diethyl ether (about 20-30 ml) to precipitate, and filtering with diethyl ether. The precipitate was washed once and immediately placed in a desiccator.
- the crude Pexiganan analog (1-23) was dissolved in 0.5% acetic acid at a concentration of 5 mg/ml, and subjected to HPLC.
- the main peak of Pexiganan analog (1-23) was purified by preparative HPLC using Dalian elestat.
- the separation and purification conditions were as follows: Gradient separation was carried out by using 0.1% trifluoroacetic acid and 80% acetonitrile containing 0.1% trifluoroacetic acid, and the obtained Pexiganan analog (1-23) was concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain a product.
- the fusion protein is cleaved by Clostripain.
- GCA CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG CCG TTT CGC AAG CAA
- Lys lie Leu Lys Lys Arg
- the Arg-containing codon was amplified by a conventional PCR method, cloned into a Lac-containing promoter plasmid to form a fusion protein gene, and an expression plasmid was constructed and transfected into Escherichia coli JM109.
- Pexiganan analogue (1-23) genetically engineered bacteria were inoculated into 300 ml X 2 bottles of LB medium, added with ampicillin (Ampici ll in ) to a final concentration of 50 ⁇ g/ml, 37 ° C, and shaken overnight. 150rpm). On the second day, transfer to 20L New Brunswik fermenter.
- the culture medium is composed of M9 medium, with glucose concentration of 1%, ampicillin (Ampici ll in ) concentration of 50 ⁇ g/ml, aeration of 20L/min, dissolved oxygen. Maintained at more than 20%, the antifoaming agent is domestically produced, the pH is maintained at 7 ⁇ 8, and it is adjusted with ammonia water.
- adding of IPTG at a concentration of 0. 5mM, continue to ferment 4 ⁇ 6 hours, bacterial cells were collected by centrifugation wet weight 30 ⁇ 50g / L o
- the collected cells were homogenized with pH 8.0 Tri s buffer 20 mM, then lysozyme lg/L was added, incubated at 37 ° C for 1 hour, frozen at 20 ° C overnight, sonicated 3 times, each time 1 minute or so.
- the precipitate was collected by centrifugation, washed 2 times with 2M Urea, and washed once with water. Separation and purification of inclusion bodies: The precipitate is dissolved in 8 M urea. Sephadex G-100 was chromatographed at A280 nm, and the collected solution was dialyzed overnight, lysed with Clostripain protease, and traced by HPLC.
- the Met-containing codon was amplified by a conventional PCR method, cloned into a Lac-containing promoter plasmid to form a fusion protein gene, and an expression plasmid was constructed and transfected into Escherichia coli JM109.
- Pexiganan analogue (1-23) genetically engineered bacteria were inoculated into 300 ml X 2 bottles of LB medium, and added with ampicillin (Ampici ll in) to a final concentration of 50 ⁇ g/ml, 37 ° C, and shaken overnight. 150rpm). On the second day, transfer to 20L New Brunswik fermenter.
- the culture medium is composed of M9 medium, with glucose concentration of 1%, ampicillin (Ampici ll in ) concentration of 50 ⁇ g/ml, aeration of 20L/min, dissolved oxygen. Maintained at more than 20%, the antifoaming agent is domestically produced, the pH is maintained at 7 ⁇ 8, and it is adjusted with ammonia water.
- Lys lie Leu Lys Lys Arg
- the codons containing Asn-Gly were amplified by conventional PCR and cloned into the Lac-containing promoter plasmid to form a fusion protein gene, and the expression plasmid was constructed and transfected into Escherichia coli JM109.
- Pexiganan analog (1-23) genetically engineered bacteria were inoculated into 300 ml X 2 bottles of LB medium, added with ampicillin (Ampici ll in ) to a final concentration of 50 ⁇ g/ml, and 37 shaker overnight (150 rpm). On the second day, transfer to 20L New Brunswik fermenter.
- the culture medium is composed of M9 medium and added with glucose. Degree 1%, ampicillin (Ampici llin) concentration of 50 ⁇ g / ml, ventilation capacity of 20L / min, dissolved oxygen maintained above 20%, antifoaming agent is domestic foaming enemy, pH is maintained at 7 ⁇ 8, with Ammonia adjustment.
- IPTG was added, and the concentration was 0.5 mM.
- the fermentation was continued for 4 to 6 hours, and the cells were collected by centrifugation to a wet weight of 30 to 50 g/L.
- the fusion protein of Pexiganan analogue (1-23) was lysed with 2M NH20H/6M guanidine Guanidin ⁇ HC1 at pH 9. 0, 45 °C for 8 hours, followed by HPLC, cleavage and ultrafiltration with molecular weight of 1000
- the salt was purified by HPLC to give the Pexiganan analog (1-23) product.
- Trp Gly lie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val 4 ⁇ TGG GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC GGC AAA GCG TTC GTT
- Lys lie Leu Lys Lys Arg
- Trp-containing codon was amplified by a conventional PCR method, cloned into a Lac-containing promoter plasmid to form a fusion protein gene, and an expression plasmid was constructed and transfected into Escherichia coli JM109.
- Pexiganan analogue (1-23) genetically engineered bacteria were inoculated into 300 ml X 2 bottles of LB medium, and added with ampicillin (Ampici ll in ) to a final concentration of 50 ⁇ g/ml, 37 ° C, and shaken overnight. 150rpm). On the second day, transfer to 20L New Brunswik fermenter.
- the culture medium is composed of M9 medium, with glucose concentration of 1%, ampicillin (Ampici ll in ) concentration of 50 ⁇ g/ml, aeration of 20L/min, dissolved oxygen. Maintained at more than 20%, the antifoaming agent is domestically produced, the pH is maintained at 7 ⁇ 8, and it is adjusted with ammonia water.
- IPTG IPTG
- the concentration is 0.5 mM
- reaction was carried out for 48 hours (or 5 hours at 37 ° C), and the reaction was stopped by adding a 10-fold amount of a reducing agent (such as mercaptoethanol) under nitrogen in the dark.
- a reducing agent such as mercaptoethanol
- the reaction solution was extracted with ethyl acetate to remove BNPS-Skatole, and the aqueous phase was stored in lyophilized, dissolved and then purified by HPLC to obtain Pexiganan analog (1-23) product.
- a reducing agent such as mercaptoethanol
- the Pexiganan analog (1-23) of the present invention has an antibacterial action as an antibacterial polypeptide drug, and can directly kill bacteria, fungi, protozoa and certain viruses without causing drug resistance.
- the left dish is a Pexiganan analogue (1-23) and the E.Coli resistant bacteria are pre-mixed in a saline solution for half an hour after plating, and the right dish is the premixed physiology of E. coli resistant bacteria.
- the plate was incubated for half an hour in a saline liquid.
- the plate was incubated overnight at 37 ° C.
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Abstract
The present invention provides an antimicrobial Pexiganan analogue, the amino acid sequence of which is GIGKFLKKAKKFGKAFVKILKKR. Moreover, the present invention also provides a preparation process of the analogue.
Description
说 明 书 . 抗微生物的 PEXIGANAN类似物及其制备方法 Description. Antimicrobial PEXIGANAN analogue and preparation method thereof
技术领域 Technical field
本发明涉及一种 Pexiganan类似物 (1-23 ) , 该 Pexiganan类似物 (1-23 ) 具 有抗菌作用, 可用作为抗菌多肽药物。 本发明还提供了通过化学合成或基因工程生 产该 Pexiganan类似物 (1-23 ) 的方法。 The present invention relates to a Pexiganan analog (1-23) which has an antibacterial action and can be used as an antibacterial polypeptide drug. The present invention also provides a method of producing the Pexiganan analog (1-23) by chemical synthesis or genetic engineering.
背景技术 Background technique
生物体(动物、 植物、 昆虫……)对外界微生物的入侵本能的反应产生抗菌肽, 通过天然免疫从而保护机体不受病菌的侵害。 迄今 20余年来已从各种生物体内分 离出 800多种抗菌多肽, 仅从各种蛙皮中就分离出 200多种抗菌肽。 这种多肽, 分 子量比较小 (20- 60个氨基酸) , 却能杀死细菌、 真菌、 原虫和某些病毒, 对一些 耐药型微生物有效, 是一种新型广谱抗菌药物, 又被称为 "新型抗生素" 。 抗菌肽 本身没有毒性, 代谢后为氨基酸, 受到国内外广泛注意和研究。 随着各种细菌对抗 生素的耐药性增加, 对抗菌肽的研究愈来愈多, 因为抗菌肽对那些抗药性的菌类也 能遏制。 化学合成品 Pexiganan由 22个氨基酸组成, 临床试验局部用药获得很好 的治疗效果。 Insects of organisms (animals, plants, insects...) invading the outside microbes produce antibacterial peptides that protect the body from pathogens through natural immunity. More than 800 antimicrobial peptides have been isolated from various organisms in the past 20 years, and more than 200 antimicrobial peptides have been isolated from various frog skins. This peptide, which has a relatively low molecular weight (20-60 amino acids), kills bacteria, fungi, protozoa and certain viruses. It is effective against some resistant microorganisms and is a new broad-spectrum antibacterial drug. "New antibiotics". The antibacterial peptide itself is not toxic, and is amino acid after metabolism, which has been widely noted and studied at home and abroad. As the resistance of various bacterial antibiotics increases, more and more research on antimicrobial peptides has been made, because antimicrobial peptides can also be suppressed against those resistant bacteria. Chemically synthesized Pexiganan consists of 22 amino acids, which are used in clinical trials to achieve good therapeutic results.
化学合成品的高价制约了其广泛应用。 基因工程技术制备抗菌肽的困难在于发 酵表达的抗菌肽会将工程菌杀死, 发酵很难进行。 用 Pichia Pastori s酵母发酵, 分泌到发酵液中的小肽, 分子量小, 量少, 回收困难。 目前多以融合蛋白方式表达, 形成不溶性包涵体, 避免了对工程菌的伤害, 但要求融合蛋白载体分子量要小, 表 达水平要高, 以增加目的多肽产量。 参考文献: The high price of chemical synthetic products limits its wide application. The difficulty in preparing antimicrobial peptides by genetic engineering techniques is that the antibacterial peptides expressed by the fermentation will kill the engineered bacteria, and the fermentation is difficult to carry out. Fermented with Pichia Pastori s yeast, the small peptide secreted into the fermentation broth has a small molecular weight, a small amount, and is difficult to recover. At present, it is expressed by fusion protein, which forms insoluble inclusion bodies and avoids damage to engineering bacteria. However, it is required that the fusion protein carrier has a small molecular weight and a high expression level to increase the yield of the polypeptide of interest. references:
US patene : 7332800 US patene : 7332800
Pexiganan: Antimicrob Agents Chemither(1999)43,782-788 Pexiganan: Antimicrob Agents Chemither (1999) 43, 782-788
Diagn. Microbiol Infect. Dis.(1999)35,45-53 Diagn. Microbiol Infect. Dis. (1999) 35, 45-53
Drugs.(1998)56, 1047-1052 Drugs. (1998) 56, 1047-1052
Rev. in Medical Microbiology(2004) 15, 17-25 Rev. in Medical Microbiology (2004) 15, 17-25
Drug Communication.(2008) 确认本
Pexiganan基因工程制备: Drug Communication. (2008) Confirmation Pexiganan genetic engineering preparation:
Biotechnology and applied Biochemistry(2004)39,339-345. Biotechnology and applied Biochemistry (2004) 39, 339-345.
Protein Expression and purification( 1998) 12,53-60. Protein Expression and purification (1998) 12, 53-60.
Biochemical and Biophysical. Res.comm(2000)277,575-579. Biochemical and Biophysical. Res.comm (2000) 277, 575-579.
US patent(2001)6183992 US patent (2001) 6138992
生物工程学报 (2008 ) 24, 21-26. 发明内容 Journal of Bioengineering (2008) 24, 21-26.
本发明的目的在于克服已有技术的缺陷, 利用基因工程技术成功的生产 The object of the present invention is to overcome the defects of the prior art and to successfully produce by using genetic engineering technology.
Pexiganan类似物 (1-23 ) , 每立升发酵液可达 500mg, 能降低成本。 Pexiganan analogues (1-23) can reduce costs by up to 500 mg per liter of fermentation broth.
本发明的 Pexiganan 类似物(1-23), 其特征在于: Pexiganan 类似物(1-23) 的氨 基酸序列由 23个氨基酸组成, Pexiganan 类似物 (1-23) 其结构式为: The Pexiganan analog (1-23) of the present invention is characterized in that the amino acid sequence of the Pexiganan analog (1-23) consists of 23 amino acids, and the Pexiganan analog (1-23) has the structural formula:
Gly-I le-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys Gly-I le-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys
-I le-Leu-Lys-Lys-Arg -I le-Leu-Lys-Lys-Arg
其中, 22个氨基酸序列为 Pexiganan, C末端为 Arg, C末端 Arg为 -OH或 NH2。 根据 Pexiganan类似物氨基酸序列及相应 DNA序列, 合成 DNA片段 Among them, the 22 amino acid sequence is Pexiganan, the C terminus is Arg, and the C terminus Arg is -OH or NH2. Synthesis of DNA fragments based on the amino acid sequence of the Pexiganan analog and the corresponding DNA sequence
BamH I X Gly l ie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe < > GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC BamH I X Gly l ie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe < > GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC
CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG
Gly Lys Ala Phe Val Lys lie Leu Lys Lys Arg Gly Lys Ala Phe Val Lys lie Leu Lys Lys Arg
GGC AAA GCG TTC GTT AAA ATC CTG AAA AAA CGT TAG GGC AAA GCG TTC GTT AAA ATC CTG AAA AAA CGT TAG
<==> <==>
CCG TTT CGC AAG CAA TTT TAG GAC TTT TTT GCA ATC CCG TTT CGC AAG CAA TTT TAG GAC TTT TTT GCA ATC
Sal I Sal I
其中 X=Met ¾=Asp或 =Trp或 Arg。 Where X=Met 3⁄4=Asp or =Trp or Arg.
另一方面, 本发明还提供了该 Pexiganan 类似物 (1-23 )利用基因工程或化学 合成法生产的方法。 In another aspect, the present invention also provides a method of producing the Pexiganan analog (1-23) by genetic engineering or chemical synthesis.
本发明的 Pexiganan 类似物 (1-23 ) 是抗菌肽 Magainin的类似物, 具有很强 的抗菌作用, 能直接杀死细菌、 真菌、 原虫和某些病毒, 不产生抗药性。 本发明的 Pexiganan 类似物 (1-23 ) 的用途可作为抗菌多肽药物。 The Pexiganan analog (1-23) of the present invention is an analog of the antibacterial peptide Magainin, which has a strong antibacterial action and can directly kill bacteria, fungi, protozoa and certain viruses without causing drug resistance. The use of the Pexiganan analog (1-23) of the present invention is useful as an antimicrobial polypeptide drug.
迄今所用的 Pexiganan等抗菌肽都是化学合成品, 价格昂贵, 阻碍了其广泛的 应用。 基因工程利用 Pichia Pastoris酵母以甲醇诱导表达的抗菌肽分泌到发酵
液中, 由于分子量小, 表达量也低, 从发酵液中回收十分困难。 本发明将抗菌肽The antibacterial peptides such as Pexiganan used so far are chemically synthesized products, which are expensive and hinder their wide application. Genetic engineering using Pichia Pastoris yeast to secrete the expression of antimicrobial peptides to fermentation In the liquid, since the molecular weight is small and the amount of expression is also low, it is very difficult to recover from the fermentation broth. Antibacterial peptide
Pexigarmn类似物 (1-23) 以融合蛋白形式表达, 制备, 工艺简单, 具有工业量产 意义。 附图说明 Pexigarmn analogues (1-23) are expressed in the form of fusion proteins, which are simple to prepare and have industrial production significance. DRAWINGS
图 1是 Pexiganan 类似物 (1-23) 的 HPLC检测图谱; Figure 1 is an HPLC detection map of Pexiganan analog (1-23);
图 2是 Pexiganan类似物 (1-23) 杀菌效果图; Figure 2 is a diagram showing the bactericidal effect of Pexiganan analogues (1-23);
图 3是固相合成法合成 Pexiganan类似物 (1-23) 合成过程图。 具体实施方式 Figure 3 is a diagram showing the synthesis process of a Pexiganan analogue (1-23) synthesized by solid phase synthesis. detailed description
实施例一: Embodiment 1:
固相合成法合成 Pexiganan类似物 (1-23) 。 Solid phase synthesis of Pexiganan analogues (1-23).
(1) 化学结构 (1) Chemical structure
Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys-Ile- Leu-Lys-Lys-Arg Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys-Ile- Leu-Lys-Lys-Arg
(2) 固相树脂是王树脂 (Wang Resin) , 进行固相合成, 合成结束后将得到 的带侧链保护基的多肽树脂进行裂解, 即切断多肽与树脂的联结同时脱保护基一步 完成, C端为自由羧基。 (2) The solid phase resin is Wang Resin, which performs solid phase synthesis. After the synthesis, the obtained peptide resin with side chain protecting group is cleaved, that is, the connection between the polypeptide and the resin is cut off, and the deprotection step is completed in one step. The C-terminus is a free carboxyl group.
(3) 合成步骤 (3) Synthesis step
8种带保护基 Fmoc氨基酸 ~~► 固相合成一"► 切断树脂, 同时脱保护基一 HPLC纯化——►冷冻干燥, 得到 Pexiganan类似物 (1-23) 。 8 kinds of protected groups Fmoc amino acids ~~► Solid phase synthesis - "► Cut the resin, while deprotecting the substrate - HPLC purification - ► freeze-drying to obtain Pexiganan analogues (1-23).
带保护基 Fmoc的氨基酸- Fmoc-L- Ala-OH; Fmoc-L-Lys(Trt)-OH; Amino acid with a protecting group Fmoc - Fmoc-L-Ala-OH; Fmoc-L-Lys(Trt)-OH;
Fmoc-L-Arg(pmc)-OH; Fmoc-L-Val-OH; Fmoc-L-Arg(pmc)-OH; Fmoc-L-Val-OH;
Fmoc-L-Ile-OH; Fmoc-Gly-OH; Fmoc-L-Ile-OH; Fmoc-Gly-OH;
Fmoc-L-Phe-OH; Fmoc-L-Leu-OH Fmoc-L-Phe-OH; Fmoc-L-Leu-OH
(4) 合成过程参见图 3, 从 C端幵始将第一个氨基酸 Arg联在树脂 (R) 上, 然后从 C端一步完成一个氨基酸的联结, 向 N端延伸直至最后完成, 共 进行 24步。
( 5 ) 固相多肽合成仪 (Applied Biosystem433A型) (4) The synthesis process is shown in Figure 3. The first amino acid Arg is linked to the resin (R) from the C-terminus, and then an amino acid linkage is completed from the C-terminus, extending to the N-terminus until the final completion. step. (5) Solid phase peptide synthesizer (Applied Biosystem 433A)
所用试剂皆系分析纯或多肽合成级: N-甲基吡咯烷酮, 二氯甲垸, 六氢 B比啶, 2.0M DIEA NMP, DMAP/DMF, HBTU100mmole/0.5M HOBT in F等。 The reagents used were of analytical grade or peptide synthesis grade: N-methylpyrrolidone, chloroform, hexahydrobipyridyl, 2.0 M DIEA NMP, DMAP/DMF, HBTU 100 mmole/0.5M HOBT in F, and the like.
( 6) 操作 (6) Operation
以 0.25mmole规模, 称树脂 0.5g置入多肽合成仪的反应器中, 将 8种带 保护基 Fmoc的氨基酸各秤 lmmole装瓶按 Pexiganan类似物 ( 1-23 ) 的 氨基酸序列从 C端向 N端排列在合成仪中, 于 25'C室温条件下由电脑 程序 (Synth Assiat) 控制自动进行, 脱 Fmoc保护基, 活化, 联结完成 后进入下一个循环步骤。 On the scale of 0.25 mmole, 0.5 g of resin was placed in the reactor of the peptide synthesizer, and 8 kinds of amino acids with protective group Fmoc were bottled in 1 mmole according to the amino acid sequence of Pexiganan analogue (1-23) from C to N. The ends are arranged in a synthesizer, automatically controlled by a computer program (Synth Assiat) at room temperature of 25 ° C, the Fmoc protecting group is removed, activated, and the coupling is completed and then proceeds to the next cycle step.
( 7) 裂解与脱保护基 (7) Pyrolysis and deprotection
将带保护基的 Pexiganan类似物 (1-23 ) - 放入带塞的三角瓶中, 放入 裂解试剂如下: Place the Pexiganan analogue (1-23) with a protecting group in a stoppered flask and place the lysis reagent as follows:
试剂 体积 (ml) Reagent volume (ml)
水 0.50 Water 0.50
苯甲醚 0.50 Anisole 0.50
苯酚 0.75 Phenol 0.75
巯基乙酸 0.20 Thioglycolic acid 0.20
三氟乙酸 10.0 Trifluoroacetic acid 10.0
恒温 30°C条件下, 电磁搅拌 6小时, 过滤除去树脂, 收集清液, 再用少 量三氟乙酸将树脂洗涤一次, 合并收集液, 加入乙醚 (约 20-30ml) 产 生沉淀, 过滤, 用乙醚将沉淀洗一次, 立即放入干燥器中。 Electromagnetic stirring at a constant temperature of 30 ° C for 6 hours, removing the resin by filtration, collecting the supernatant, and washing the resin once with a small amount of trifluoroacetic acid, combining the collected liquid, adding diethyl ether (about 20-30 ml) to precipitate, and filtering with diethyl ether. The precipitate was washed once and immediately placed in a desiccator.
用 0.5%乙酸将上述 Pexiganan类似物( 1-23 )粗品溶解,浓度为 5mg/ml, 进行 HPLC, 以大连依列特制备型 HPLC纯化分离 Pexiganan类似物 ( 1-23 )主峰, 分离纯化条件: 0.1%三氟乙酸及含 0.1%三氟乙酸的 80% 的乙腈进行梯度分离, 将分离所得的 Pexiganan类似物 (1-23 ) 减压浓 缩除去乙腈后进行冷冻干燥得到产品。 The crude Pexiganan analog (1-23) was dissolved in 0.5% acetic acid at a concentration of 5 mg/ml, and subjected to HPLC. The main peak of Pexiganan analog (1-23) was purified by preparative HPLC using Dalian elestat. The separation and purification conditions were as follows: Gradient separation was carried out by using 0.1% trifluoroacetic acid and 80% acetonitrile containing 0.1% trifluoroacetic acid, and the obtained Pexiganan analog (1-23) was concentrated under reduced pressure to remove acetonitrile, followed by lyophilization to obtain a product.
Pexiganan 类似物 (1-23 ) 的 HPLC检测参见图 1。
实施例二: See Figure 1 for HPLC detection of Pexiganan analog (1-23). Embodiment 2:
融合蛋白的 Clostripain切断。 The fusion protein is cleaved by Clostripain.
BamH I Arg Gly lie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val ^ ^ CGT GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC GGC AAA GCG TTC GTT BamH I Arg Gly lie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val ^ ^ CGT GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC GGC AAA GCG TTC GTT
GCA CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG CCG TTT CGC AAG CAA GCA CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG CCG TTT CGC AAG CAA
Lys lie Leu Lys Lys Arg Lys lie Leu Lys Lys Arg
AAA ATC CTG AAA AAA CGT TAG ^ ► AAA ATC CTG AAA AAA CGT TAG ^ ►
TTT TAG GAC TTT TTT GCA ATC TTT TAG GAC TTT TTT GCA ATC
Sal I Sal I
基因合成及克隆: Gene synthesis and cloning:
根据 Pexiganan类似物 (1-23)氨基酸序列及相应 DNA序列, 合成 DNA片段 Synthesis of DNA fragments based on the amino acid sequence of Pexiganan analogue (1-23) and the corresponding DNA sequence
1 BamH I CGTGGCATCGGCAAATTCCTGAAAAAAGCGAAAAAATT 2 1 BamH I CGTGGCATCGGCAAATTCCTGAAAAAAGCGAAAAAATT 2
4 Sal I^ CTAGGATCCACGTTTTTTCA 4 Sal I^ CTAGGATCCACGTTTTTTCA
5 TGCCGATGCCACG ^amH 5 TGCCGATGCCACG ^amH
以常规 PCR方法扩增联接构成含 Arg的密码子, 克隆到含 Lac启动子质粒中, 形成融合蛋白基因, 构建表达质粒, 转染大肠杆菌 JM109。 The Arg-containing codon was amplified by a conventional PCR method, cloned into a Lac-containing promoter plasmid to form a fusion protein gene, and an expression plasmid was constructed and transfected into Escherichia coli JM109.
发酵: Fermentation:
Pexiganan类似物 (1-23 ) 基因工程菌接种于 300ml X 2瓶 LB培养基中, 加氨苄西 林 (Ampici l l in ) 使其最后浓度为 50 μ g/ml, 37 °C , 摇床培养过夜 (150rpm) 。 第 二日转至 20L New Brunswik 发酵罐培养, 培养液组成为 M9培养液, 加葡萄糖浓 度 1%, 氨苄西林 (Ampici l l in )浓度为 50 μ g/ml , 通气量为 20L/min, 溶氧维持在 20%以上, 防泡剂为国产泡敌, pH维持在 7〜8, 用氨水调节。 当菌体浓度达 log曲 线中线, 加 IPTG, 浓度为 0. 5mM, 继续发酵 4〜6小时, 离心收集菌体为湿重 30〜 50g/Lo Pexiganan analogue (1-23) genetically engineered bacteria were inoculated into 300 ml X 2 bottles of LB medium, added with ampicillin (Ampici ll in ) to a final concentration of 50 μg/ml, 37 ° C, and shaken overnight. 150rpm). On the second day, transfer to 20L New Brunswik fermenter. The culture medium is composed of M9 medium, with glucose concentration of 1%, ampicillin (Ampici ll in ) concentration of 50 μg/ml, aeration of 20L/min, dissolved oxygen. Maintained at more than 20%, the antifoaming agent is domestically produced, the pH is maintained at 7~8, and it is adjusted with ammonia water. When the cell concentration reached log curve line, adding of IPTG, at a concentration of 0. 5mM, continue to ferment 4 ~ 6 hours, bacterial cells were collected by centrifugation wet weight 30~ 50g / L o
下游工艺: Downstream process:
收集的菌体用 pH 7. 0 Tri s 缓冲液 20mM匀浆, 之后加溶菌酶 lg/L , 37°C保温 1 小时, 冷冻 一 20°C 过夜, 超声破碎 3次, 每次 1分钟 左右, 离心收集沉淀, 2M Urea 打碎洗涤 1次, 再打碎水洗 1次。 分离、 纯化包涵体: 沉淀溶于 8M尿素,
Sephadex G- 100层析 A280nm检测, 将收集液透析过夜, 用 Clostripain蛋白酶 裂解, HPLC追踪。 完成酶解反应后以 30000分子量超滤, 再经 HPLC纯化, 得到 Pexiganan 类似物 (1-23 ) 。 制备型 HPLC纯化 (参见图 1 ) 。 冷冻干燥获得产品。 实施例三: The collected cells were homogenized with pH 8.0 Tri s buffer 20 mM, then lysozyme lg/L was added, incubated at 37 ° C for 1 hour, frozen at 20 ° C overnight, sonicated 3 times, each time 1 minute or so. The precipitate was collected by centrifugation, washed 2 times with 2M Urea, and washed once with water. Separation and purification of inclusion bodies: The precipitate is dissolved in 8 M urea. Sephadex G-100 was chromatographed at A280 nm, and the collected solution was dialyzed overnight, lysed with Clostripain protease, and traced by HPLC. After the enzymatic hydrolysis reaction was completed, ultrafiltration was carried out at a molecular weight of 30,000, followed by purification by HPLC to obtain a Pexiganan analog (1-23). Preparative HPLC purification (see Figure 1). Freeze drying to obtain the product. Embodiment 3:
融合蛋白的 BrCN切断。 BrCN cleavage of the fusion protein.
BamH I Met Gly lie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val ►ATG GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC GGC AAA GCG TTC GTTBamH I Met Gly lie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val ►ATG GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC GGC AAA GCG TTC GTT
TAC CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG CCG TTT CGC AAG CAA TAC CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG CCG TTT CGC AAG CAA
Lys He Leu Lys Lys Arg Lys He Leu Lys Lys Arg
AAA ATC CTG AAA AAA CGT TAG ~► AAA ATC CTG AAA AAA CGT TAG ~►
TTT TAG GAC TTT TTT GCA ATC TTT TAG GAC TTT TTT GCA ATC
Sal I Sal I
基因合成及克隆- 根据 Pexiganan类似物(1-23)氨基酸序列及相应 DNA序列, 合成 DNA片段 Gene synthesis and cloning - synthesis of DNA fragments based on the amino acid sequence of the Pexiganan analogue (1-23) and the corresponding DNA sequence
1 BamH I ATGGGCATCGGCAAATTCCTGAAAAAAGCGAAAAAATT 2 1 BamH I ATGGGCATCGGCAAATTCCTGAAAAAAGCGAAAAAATT 2
CGGCAAAGCGTTCGTTAAAATCCTGAAAAAACGTGGATCCTAGSal l CGGCAAAGCGTTCGTTAAAATCCTGAAAAAACGTGGATCCTAGSal l
► ►
4 Sal CTAGGATCCACGTTTTTTCA 4 Sal CTAGGATCCACGTTTTTTCA
5 TGCCGATGCCCAT gamH 5 TGCCGATGCCCAT gamH
以常规 PCR方法扩增联接构成含 Met的密码子, 克隆到含 Lac启动子质粒中, 形成融合蛋白基因, 构建表达质粒, 转染大肠杆菌 JM109。 The Met-containing codon was amplified by a conventional PCR method, cloned into a Lac-containing promoter plasmid to form a fusion protein gene, and an expression plasmid was constructed and transfected into Escherichia coli JM109.
发酵: Fermentation:
Pexiganan类似物 (1-23 ) 基因工程菌接种于 300ml X 2瓶 LB培养基中, 加氨苄西 林 (Ampici l l in) 使其最后浓度为 50 μ g/ml, 37 °C , 摇床培养过夜 (150rpm) 。 第 二日转至 20L New Brunswik 发酵罐培养, 培养液组成为 M9培养液, 加葡萄糖浓 度 1%, 氨苄西林 (Ampici l l in )浓度为 50 μ g/ml, 通气量为 20L/min, 溶氧维持在 20%以上, 防泡剂为国产泡敌, pH维持在 7〜8, 用氨水调节。 当菌体浓度达 log曲 线中线, 加 IPTG, 浓度为 0. 5mM, 继续发酵 4〜6小时, 离心收集菌体为湿重 30〜 50g/L。
下游工艺- 收集的菌体用 pH 7. 0 Tri s 缓冲液 20mM匀浆, 之后加溶菌酶 lg/L , 37°C保温 1 小时, 冷冻 一 20°C 过夜, 超声破碎 3次, 每次 1分钟 左右, 离心收集沉淀, 2M Urea 打碎洗涤 1次, 再打碎水洗 1次。 分离、 纯化包涵体: 沉淀溶于 8M尿素, Sephadex G-100层析 A280nm检测, 将收集液透析过夜, 在 70%HC02H条件下经 CNBr切断, 加 Na2S03, 停止反应, 稀释后 1000分子量超滤, 再经 HPLC纯化, 得 到 Pexiganan类似物 (1-23 ) 。 制备型 HPLC纯化。 冷冻干燥获得产品。 实施例四: Pexiganan analogue (1-23) genetically engineered bacteria were inoculated into 300 ml X 2 bottles of LB medium, and added with ampicillin (Ampici ll in) to a final concentration of 50 μg/ml, 37 ° C, and shaken overnight. 150rpm). On the second day, transfer to 20L New Brunswik fermenter. The culture medium is composed of M9 medium, with glucose concentration of 1%, ampicillin (Ampici ll in ) concentration of 50 μg/ml, aeration of 20L/min, dissolved oxygen. Maintained at more than 20%, the antifoaming agent is domestically produced, the pH is maintained at 7~8, and it is adjusted with ammonia water. When the concentration of the bacteria reached the log line, IPTG was added, and the concentration was 0.5 mM. The fermentation was continued for 4 to 6 hours, and the cells were collected by centrifugation to a wet weight of 30 to 50 g/L. Downstream process - The collected cells were homogenized with pH 8.0 Tri s buffer 20 mM, then lysozyme lg/L was added, incubated at 37 ° C for 1 hour, frozen at 20 ° C overnight, sonicated 3 times, each time 1 In about minute, the precipitate was collected by centrifugation, washed 2 times with 2M Urea, and washed once with water. Isolation and purification of inclusion bodies: The precipitate was dissolved in 8 M urea, Sephadex G-100 chromatography was detected at A280 nm, the collected solution was dialyzed overnight, cut through CNBr under 70% HC02H conditions, Na2S03 was added, the reaction was stopped, and 1000 molecular weight ultrafiltration was diluted. Further purification by HPLC gave Pexiganan analog (1-23). Preparative HPLC purification. Freeze drying to obtain the product. Embodiment 4:
融合蛋白的羟胺裂解。 Hydroxylamine cleavage of the fusion protein.
BamH 】 Asn Gly lie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val i ► AAC GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC GGC AAA GCG TTC GTT BamH 】 Asn Gly lie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val i ► AAC GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC GGC AAA GCG TTC GTT
TTG CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG CCG TTT CGC AAG CAA TTG CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG CCG TTT CGC AAG CAA
Lys lie Leu Lys Lys Arg Lys lie Leu Lys Lys Arg
AAA ATC CTG AAA AAA CGT TAG ^ ~► AAA ATC CTG AAA AAA CGT TAG ^ ~►
TTT TAG GAC TTT TTT GCA ATC TTT TAG GAC TTT TTT GCA ATC
Sal I Sal I
基因合成及克隆: Gene synthesis and cloning:
根据 Pexiganan类似物 (1-23)氨基酸序列及相应 DNA序列, 合成 DNA片段 Synthesis of DNA fragments based on the amino acid sequence of Pexiganan analogue (1-23) and the corresponding DNA sequence
1 BamH I AACGGCATCGGCAAATTCCTGAAAAAAGCGAAAAAATT 2 1 BamH I AACGGCATCGGCAAATTCCTGAAAAAAGCGAAAAAATT 2
CGGCAAAGCGTTCGTTAAAATCCTGAAAAAACGTGGATCCTAGSal ICGGCAAAGCGTTCGTTAAAATCCTGAAAAAACGTGGATCCTAGSal I
4 ^Sal CTAGGATCCACGTTTTTTC A 4 ^Sal CTAGGATCCACGTTTTTTC A
5 TGCCGATGCCGTT ^amH I 5 TGCCGATGCCGTT ^amH I
以常规 PCR方法扩增联接构成含 Asn-Gly的密码子, 克隆到含 Lac启动子质粒 中, 形成融合蛋白基因, 构建表达质粒, 转染大肠杆菌 JM109。 The codons containing Asn-Gly were amplified by conventional PCR and cloned into the Lac-containing promoter plasmid to form a fusion protein gene, and the expression plasmid was constructed and transfected into Escherichia coli JM109.
发酵: Fermentation:
Pexiganan类似物 (1-23 )基因工程菌接种于 300ml X 2瓶 LB培养基中, 加氨苄西 林 (Ampici l l in ) 使其最后浓度为 50 μ g/ml, 37 摇床培养过夜 (150rpm) 。 第 二日转至 20L New Brunswik 发酵罐培养, 培养液组成为 M9培养液, 加葡萄糖浓
度 1%, 氨苄西林(Ampici l lin)浓度为 50 μ g/ml, 通气量为 20L/min, 溶氧维持在 20%以上, 防泡剂为国产泡敌, pH维持在 7〜8, 用氨水调节。 当菌体浓度达 log曲 线中线, 加 IPTG, 浓度为 0. 5mM, 继续发酵 4〜6小时, 离心收集菌体为湿重 30〜 50g/L。 Pexiganan analog (1-23) genetically engineered bacteria were inoculated into 300 ml X 2 bottles of LB medium, added with ampicillin (Ampici ll in ) to a final concentration of 50 μg/ml, and 37 shaker overnight (150 rpm). On the second day, transfer to 20L New Brunswik fermenter. The culture medium is composed of M9 medium and added with glucose. Degree 1%, ampicillin (Ampici llin) concentration of 50 μ g / ml, ventilation capacity of 20L / min, dissolved oxygen maintained above 20%, antifoaming agent is domestic foaming enemy, pH is maintained at 7~8, with Ammonia adjustment. When the concentration of the bacteria reached the log line, IPTG was added, and the concentration was 0.5 mM. The fermentation was continued for 4 to 6 hours, and the cells were collected by centrifugation to a wet weight of 30 to 50 g/L.
下游工艺- 收集的菌体用 pH 7. 0 Tri s 缓冲液 20mM匀浆, 之后加溶菌酶 lg/L , 37°C保温 1 小时, 冷冻 一 20°C 过夜, 超声破碎 3次, 每次 1分钟 左右, 离心收集沉淀, 2M Urea 打碎洗漆 1次, 再打碎水洗 1次。 分离、 纯化包涵体: 沉淀溶于 8M尿素, Sephadex G-100层析 A280nm检测,将收集液透析过夜。 Pexiganan类似物( 1-23 ) 的融合蛋白以 2M的 NH20H/6M的盐酸胍 Guanidin · HC1在 pH9. 0, 45 °C , 裂解 8小 时,过程中以 HPLC追踪,裂解后以分子量 1000超滤去盐, HPLC纯化,得 Pexiganan 类似物 (1-23 ) 产品。 制备型 HPLC纯化。 冷冻干燥获得产品。 实施例五: Downstream process - The collected cells were homogenized with pH 8.0 Tri s buffer 20 mM, then lysozyme lg/L was added, incubated at 37 ° C for 1 hour, frozen at 20 ° C overnight, sonicated 3 times, each time 1 In about a minute, the precipitate was collected by centrifugation, and 2M Urea was broken and washed once, and then washed once with water. Isolation and purification of inclusion bodies: The precipitate was dissolved in 8 M urea, Sephadex G-100 chromatography was detected at A280 nm, and the collected solution was dialyzed overnight. The fusion protein of Pexiganan analogue (1-23) was lysed with 2M NH20H/6M guanidine Guanidin·HC1 at pH 9. 0, 45 °C for 8 hours, followed by HPLC, cleavage and ultrafiltration with molecular weight of 1000 The salt was purified by HPLC to give the Pexiganan analog (1-23) product. Preparative HPLC purification. Freeze drying to obtain the product. Embodiment 5:
融合蛋白色氨酸残基的裂解。 Cleavage of the fusion egg white residue.
BamH I Trp Gly lie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val 4 ^ TGG GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC GGC AAA GCG TTC GTTBamH I Trp Gly lie Gly Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val 4 ^ TGG GGC ATC GGC AAA TTC CTG AAA AAA GCG AAA AAA TTC GGC AAA GCG TTC GTT
ACC CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG CCG TTT CGC AAG CAA ACC CCG TAG CCG TTT AAG GAC TTT TTT CGC TTT TTT AAG CCG TTT CGC AAG CAA
Lys lie Leu Lys Lys Arg Lys lie Leu Lys Lys Arg
AAA ATC CTG AAA AAA CGT TAG ~► AAA ATC CTG AAA AAA CGT TAG ~►
TTT TAG GAC TTT TTT GCA ATC TTT TAG GAC TTT TTT GCA ATC
Sal I Sal I
基因合成及克隆- 根据 Pexiganan类似物 (1-23)氨基酸序列及相应 DNA序列, 合成 DNA片段 Gene synthesis and cloning - synthesis of DNA fragments based on the amino acid sequence of the Pexiganan analogue (1-23) and the corresponding DNA sequence
3 3
CGGCAAAGCGTTCGTTAAAATCCTGAAAAAACGTGGATCCTAGSal I CGGCAAAGCGTTCGTTAAAATCCTGAAAAAACGTGGATCCTAGSal I
4 Sal I CTAGGATCCACGTTTTTTCA 4 Sal I CTAGGATCCACGTTTTTTCA
5 TGCCGATGCCCCA gamH^I
以常规 PCR方法扩增联接构成含 Trp的密码子, 克隆到含 Lac启动子质粒中, 形成 融合蛋白基因, 构建表达质粒, 转染大肠杆菌 JM109。 5 TGCCGATGCCCCA gamH^I The Trp-containing codon was amplified by a conventional PCR method, cloned into a Lac-containing promoter plasmid to form a fusion protein gene, and an expression plasmid was constructed and transfected into Escherichia coli JM109.
发酵: Fermentation:
Pexiganan类似物 (1-23 ) 基因工程菌接种于 300ml X 2瓶 LB培养基中, 加氨苄西 林 (Ampici l l in ) 使其最后浓度为 50 μ g/ml , 37°C, 摇床培养过夜 (150rpm) 。 第 二日转至 20L New Brunswik 发酵罐培养, 培养液组成为 M9培养液, 加葡萄糖浓 度 1%, 氨苄西林 (Ampici l l in )浓度为 50 μ g/ml, 通气量为 20L/min, 溶氧维持在 20%以上, 防泡剂为国产泡敌, pH维持在 7〜8, 用氨水调节。 当菌体浓度达 log曲 线中线, 加 IPTG, 浓度为 0. 5mM, 继续发酵 4〜6小时, 离心收集菌体为湿重 30〜 50g/L o Pexiganan analogue (1-23) genetically engineered bacteria were inoculated into 300 ml X 2 bottles of LB medium, and added with ampicillin (Ampici ll in ) to a final concentration of 50 μg/ml, 37 ° C, and shaken overnight. 150rpm). On the second day, transfer to 20L New Brunswik fermenter. The culture medium is composed of M9 medium, with glucose concentration of 1%, ampicillin (Ampici ll in ) concentration of 50 μg/ml, aeration of 20L/min, dissolved oxygen. Maintained at more than 20%, the antifoaming agent is domestically produced, the pH is maintained at 7~8, and it is adjusted with ammonia water. When the concentration of the bacteria reaches the midline of the log curve, add IPTG, the concentration is 0.5 mM, continue the fermentation for 4 to 6 hours, and collect the cells by centrifugation to a wet weight of 30~50g/L.
下游工艺- 收集的菌体用 pH 7. 0 Tri s 缓冲液 20mM匀浆, 之后加溶菌酶 lg/L , 37°C保温 1 小时, 冷冻 一 20°C 过夜, 超声破碎 3次, 每次 1分钟 左右, 离心收集沉淀, 2M Urea 打碎洗涤 1次, 再打碎水洗 1次。 分离、 纯化包涵体: 沉淀溶于 8M尿素, Sephadex G- 100层析 A280nm检测, 将收集液透析过夜, 取 BNPS- Skatole溶于 70%醋酸 (含 0. 1%酚) , 加入融合蛋白, 室温反应 48小时 (或 37°C反应 5小时) , 暗处氮气条件下加 10倍量还原剂 (如巯基乙醇) 停止反应。 反应液经乙酸乙酯抽 提除去 BNPS- Skatole,水相冻干保存,溶解后 HPLC制备,得 Pexiganan类似物( 1-23 ) 产品。 实施例六: Downstream process - The collected cells were homogenized with pH 8.0 Tri s buffer 20 mM, then lysozyme lg/L was added, incubated at 37 ° C for 1 hour, frozen at 20 ° C overnight, sonicated 3 times, each time 1 In about minute, the precipitate was collected by centrifugation, washed 2 times with 2M Urea, and washed once with water. Isolation and purification of inclusion bodies: The precipitate was dissolved in 8 M urea, Sephadex G-100 chromatography was detected at A280 nm, and the collected solution was dialyzed overnight. BNPS-Skatole was dissolved in 70% acetic acid (containing 0.1% phenol), and the fusion protein was added at room temperature. The reaction was carried out for 48 hours (or 5 hours at 37 ° C), and the reaction was stopped by adding a 10-fold amount of a reducing agent (such as mercaptoethanol) under nitrogen in the dark. The reaction solution was extracted with ethyl acetate to remove BNPS-Skatole, and the aqueous phase was stored in lyophilized, dissolved and then purified by HPLC to obtain Pexiganan analog (1-23) product. Example 6:
本发明的 Pexiganan类似物 (1-23 ) 作为抗菌多肽药物, 具有很强的抗菌作 用, 能直接杀死细菌、 真菌、 原虫和某些病毒, 不产生抗药性。 The Pexiganan analog (1-23) of the present invention has an antibacterial action as an antibacterial polypeptide drug, and can directly kill bacteria, fungi, protozoa and certain viruses without causing drug resistance.
参见图 2, 图中: 左碟为 Pexiganan 类似物 (1-23 ) 与 E.Coli耐药菌预混 生理盐水液体中保温半小时后涂板, 右碟为 E.Coli耐药菌预混生理盐水液体 中保温半小时后涂板。 37°C保温过夜后摄片。
See Figure 2, in the figure: The left dish is a Pexiganan analogue (1-23) and the E.Coli resistant bacteria are pre-mixed in a saline solution for half an hour after plating, and the right dish is the premixed physiology of E. coli resistant bacteria. The plate was incubated for half an hour in a saline liquid. The plate was incubated overnight at 37 ° C.
Claims
1. 一种 Pexiganan 类似物(1-23), 其特征在于: Pexiganan 类似物 (1-23) 的氨基酸序 列由 23个氨基酸组成, Pexiganan 类似物 (1-23) 其结构式为: A Pexiganan analogue (1-23), characterized in that: the amino acid sequence of the Pexiganan analog (1-23) consists of 23 amino acids, and the Pexiganan analog (1-23) has the structural formula:
Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Qly-Lys-Ala-Phe-Val-Lys -Ile-Leu-Lys-Lys-Argo Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Qly-Lys-Ala-Phe-Val-Lys-Ile-Leu-Lys-Lys-Argo
2. 如权利要求 1所述的 Pexiganan 类似物 (1-23) , 其中, 1-22个氨基酸序列为 Pexiganan, C末端为 Arg。 2. The Pexiganan analog (1-23) according to claim 1, wherein the 1-22 amino acid sequence is Pexiganan and the C terminus is Arg.
3. 如权利要求 1或 2所述的 Pexiganan类似物 (1-23) , 其中, C末端 Arg为 -OH 或 NH2。 The Pexiganan analogue (1-23) according to claim 1 or 2, wherein the C-terminal Arg is -OH or NH2.
4. 如权利要求 1所述的 Pexiganan类似物(1-23)的生产方法, 为化学合成法生产, 包括如下步骤: 将 8种带保护基 Fmoc氨基酸经固相合成、 切断树脂, 同时脱保 护基、 HPLC纯化、 冷冻干燥, 得到 Pexiganan类似物 (1-23) The method for producing a Pexiganan analogue (1-23) according to claim 1, which is produced by a chemical synthesis method, comprising the steps of: synthesizing 8 kinds of protected Fmoc amino acids by solid phase, cutting the resin, and simultaneously deprotecting Purification by base chromatography, HPLC, and lyophilization to obtain Pexiganan analogues (1-23)
5. 如权利要求 1所述的 Pexiganan类似物(1-23)的生产方法, 为基因工程法生产。 The method for producing Pexiganan analog (1-23) according to claim 1, which is produced by genetic engineering.
6. 如权利要求 1所述的 Pexiganan类似物 (1-23) , 其用途为作为抗菌多肽药物。 6. The Pexiganan analogue (1-23) according to claim 1, which is used as an antibacterial polypeptide drug.
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CN104356221A (en) * | 2014-10-24 | 2015-02-18 | 杭州阿德莱诺泰制药技术有限公司 | Preparation method of pexiganan |
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US5424290A (en) * | 1992-10-26 | 1995-06-13 | Magainin Pharmaceuticals Inc. | Biologically active peptides and uses therefor |
CN1920020A (en) * | 2005-08-24 | 2007-02-28 | 天津天士力制药股份有限公司 | Gene engineering producing method of antibacterial peptide Pexiganan |
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US5424290A (en) * | 1992-10-26 | 1995-06-13 | Magainin Pharmaceuticals Inc. | Biologically active peptides and uses therefor |
CN1920020A (en) * | 2005-08-24 | 2007-02-28 | 天津天士力制药股份有限公司 | Gene engineering producing method of antibacterial peptide Pexiganan |
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CN104356221A (en) * | 2014-10-24 | 2015-02-18 | 杭州阿德莱诺泰制药技术有限公司 | Preparation method of pexiganan |
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