WO2017101089A1 - 一种α促黑素细胞激素的融合蛋白及其制备方法和应用 - Google Patents
一种α促黑素细胞激素的融合蛋白及其制备方法和应用 Download PDFInfo
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- WO2017101089A1 WO2017101089A1 PCT/CN2015/097783 CN2015097783W WO2017101089A1 WO 2017101089 A1 WO2017101089 A1 WO 2017101089A1 CN 2015097783 W CN2015097783 W CN 2015097783W WO 2017101089 A1 WO2017101089 A1 WO 2017101089A1
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- fusion protein
- amino acid
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- stimulating hormone
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Classifications
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- 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
- C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
- C07K14/68—Melanocyte-stimulating hormone [MSH]
- C07K14/685—Alpha-melanotropin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
- A61K38/34—Melanocyte stimulating hormone [MSH], e.g. alpha- or beta-melanotropin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- 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
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
Definitions
- the invention belongs to the field of genetic engineering and pharmacy, and particularly relates to a fusion protein of ⁇ melanocyte stimulating hormone and a preparation method and application thereof.
- ⁇ -MSH immunoregulatory peptide ⁇ -Melanocyte stimulating hormone
- ⁇ -MSH The immunomodulatory effects of ⁇ -MSH are produced by: (1) ⁇ -MSH receptors acting directly on immune cells such as peripheral macrophages, mononuclear and neutrophils; and (2) acting on intracerebral nerves.
- the ⁇ -MSH receptor on the element which in turn initiates the downstream anti-inflammatory neural pathway; (3) local inflammation of the central nervous system is inhibited by the local production of ⁇ -MSH by acting on central microglia and astrocytes, ⁇ -MSH, which can be produced by peripheral cells, acts on the center through cerebrospinal fluid circulation.
- ⁇ -MSH which can be produced by peripheral cells, acts on the center through cerebrospinal fluid circulation.
- the anti-inflammatory activity of ⁇ -MSH has been confirmed in various animal models. These animal models have allergic dermatitis, atopic dermatitis, vasculitis, arthritis, ocular inflammation, gastroenteritis, brain inflammation, and allergic inflammation.
- ⁇ -MSH In the central nervous system inflammation, the molecular mechanism of ⁇ -MSH involved in the neuroimmune regulatory pathway is as follows: ⁇ -MSH is mediated by its receptor, which reduces the transcriptional activity mediated by NF-excellentB, the largest regulator molecule in the inflammatory response. Prevent the release of pro-inflammatory factors, inhibit the expression of adhesion molecules, thereby reducing brain tissue damage, and play a role in nutrition, protection, and repair of damaged brain tissue.
- ⁇ -MSH is mainly produced by the hypothalamus, pituitary and various peripheral tissues. It consists of 13 amino acids. The half-life of intravenous injection in rats is several minutes. For such protein drugs, glomerular filtration must be considered. Therefore, it is necessary to extend the half-life of the ⁇ -MSH by structural modification and modification or other methods.
- HSA is a human endogenous protein with a molecular weight of 66kDa and a half-life of 19 days in humans. It has non-immunogenic properties, good human compatibility, no enzyme activity, and is endogenous metabolite and exogenous. The carrier of the drug. These properties make HSA an ideal carrier for the development of long-acting protein/polypeptide drugs.
- the present invention utilizes albumin fusion technology to fuse ⁇ -MSH with HSA, and a flexible molecular linker (linking peptide) having a special structure is added between the two, and the technical solution disclosed by the present invention is not only guaranteed.
- the stability and biological activity of the fusion protein are increased, and the molecular weight is increased.
- the present invention adds a protein transduction domain (PTD) to the amino acid sequence of the fusion protein, and the PTD is in recent years.
- PTD protein transduction domain
- the ideal trans-BBB transporter has been found to have a powerful carrying function, capable of transporting macromolecules 100 times higher than its own molecular weight, and transporting exogenous proteins, DNA, RNA, chemical molecules, magnetic beads, and liposomes. Through the cell membrane, and this process is not limited by the size and type of the molecule.
- the inventors Based on the above status quo, the inventors have disclosed a fusion protein of ⁇ -melanocyte cytokine, which has a unique amino acid sequence which ensures high-level stable expression in the host, while retaining the original function of ⁇ -MSH. At the same time, the half-life of the body is significantly prolonged, and at the same time, it can cross the blood-brain barrier and treat brain inflammation and related diseases.
- the object of the present invention is to provide a fusion protein of ⁇ -melanocyte stimulating hormone, which can stably express at a high level in a host, has a long half-life, and can effectively cross the blood-brain barrier to treat brain inflammation and related diseases.
- Another object of the present invention is to provide a method for producing a fusion protein of ⁇ melanocyte stimulating hormone.
- Another object of the present invention is to provide a recombinant expression vector.
- Another object of the invention is to provide a host expression system.
- Another object of the present invention is to provide a fusion protein application of alpha melanocyte stimulating hormone.
- the ⁇ -melanocyte stimulating hormone fusion protein of the present invention comprises a protein transduction domian (PTD), a human serum albumin (HSA) and an alpha melanocyte stimulating hormone.
- PTD protein transduction domian
- HSA human serum albumin
- ⁇ -MSH alpha melanocyte stimulating hormone
- the fusion protein package further comprises a linker peptide L, and the HSA is linked to the ⁇ -MSH via the linker peptide L.
- the DNA sequence of L is The amino acid sequence is
- the PTD is located at the N-terminus of the fusion protein
- the ⁇ -MSH is located at the C-terminus of the fusion protein
- the fusion protein is represented by the structural formula as PTD-HSA-L- ⁇ -MSH.
- the PTD has the amino acid sequence shown in SEQ ID NO: 2, and the DNA sequence encoding the amino acid sequence of the PTD is as shown in SEQ ID NO: 1; or the amino acid residue is substituted, deleted or inserted in the amino acid sequence.
- the HSA has the amino acid sequence shown in SEQ ID NO: 4, and the DNA sequence encoding the amino acid sequence of the HSA is shown in SEQ ID NO: 3; or the amino acid residue is substituted, deleted or inserted in the amino acid sequence.
- SEQ ID NO: 4 the DNA sequence encoding the amino acid sequence of the HSA is shown in SEQ ID NO: 3; or the amino acid residue is substituted, deleted or inserted in the amino acid sequence.
- the ⁇ -MSH has the amino acid sequence shown in SEQ ID NO: 6, and the DNA sequence encoding the amino acid sequence of the ⁇ -MSH is as shown in SEQ ID NO: 5; or the amino acid sequence is substituted, deleted or inserted in the amino acid sequence.
- amino acid sequence of the fusion protein is set forth in SEQ ID NO: 8
- DNA sequence encoding the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 7.
- the above fusion protein was prepared by yeast cell expression, and the yeast was Pichia pastoris.
- the method for preparing a fusion protein of ⁇ -melanocyte stimulating hormone according to the present invention comprises the following steps:
- the recombinant yeast expression vector described in step 3 is transformed into competent E. coli TOP10 cells, and the recombinant expression vector plasmid is extracted and sequenced, and the correctly sequenced plasmid is transformed into a host expression system for expression, thereby obtaining the fusion protein.
- the host expression system is Pichia methanolophilus.
- a recombinant expression vector comprising a DNA sequence encoding an amino acid sequence of a fusion protein of alpha melanocyte stimulating hormone.
- a host expression system comprising the recombinant expression vector described above.
- the present invention utilizes human serum albumin fusion technology to fuse ⁇ -MSH with HSA, and a molecular linker (linking peptide) having a special structure and a certain flexibility is added between the two to ensure the stability and biological activity of the fusion protein, and is effective.
- the molecular weight is increased to achieve stable and high-efficiency expression of ⁇ -MSH, and the purpose of prolonging its half-life is achieved.
- the invention adds PTD to the amino acid sequence of the fusion protein, and can effectively cross the blood-brain barrier and treat brain inflammation and related diseases while retaining the original function of ⁇ -MSH.
- the fusion protein of ⁇ -melanocyte stimulating hormone of the present invention can be used in the preparation of a medicament for inhibiting or treating inflammation of the central nervous system, and is suitable for large-scale promotion in this field.
- Vector pPink ⁇ -HC vector pcDNA3.1-HSA, Pichia strain, Infusion kit (Invitrogen, USA)
- yeast extract 5 g of yeast extract, 10 g of peptone, 10 g of NaCl, dissolved in 1000 ml of deionized water, and adjusted to pH 7.0 with 1 mol/L NaOH, autoclaved.
- Yeast extract 10g peptone 20g, amino acid yeast nitrogen source 13.4g, glycerol 10g, potassium phosphate 26.631g, autoclaved in 1000ml double distilled water, cooled to room temperature, adjusted to pH 6.0, stored at 4 ° C for use.
- agarose a small amount of ethidium bromide (EB) when it is not hot. Mix it and pour it into it. Put the comb in the glue tank beforehand, cool it until it is completely solidified, and then pull out the comb to use.
- EB ethidium bromide
- Example 1 Fusion protein yeast of pPINK ⁇ -HC/PTD-HSA-L- ⁇ -MSH and pPINK ⁇ -HC/HSA-L- ⁇ -MSH Construction and expression of expression vector
- PCR amplification was carried out using pcDNA3.1-HSA plasmid DNA as a template and NFT2 and R2 as upper and lower primers, respectively.
- the reaction conditions are as follows: 1 denaturation: 94 ° C, 5 min; 2 denaturation: 94 ° C, 1 min; 3 renaturation: 55 ° C, 30 S; 4 extension: 72 ° C, 2 min; 5 return to step "2", 35 cycles; : 72 ° C, 5 min, the total number of cycles is 30 times.
- the PCR product was subjected to 1% agarose gel electrophoresis, and it was revealed that a partial PTD-HSA-L- ⁇ -MSH band of about 1.8 kb in size was amplified.
- the second round of PCR amplification was carried out using the products of the first round of PCR amplification as a template and NFT1 and R1 as the upstream and downstream primers, respectively.
- the reaction conditions are as follows: 1 denaturation: 94 ° C, 5 min; 2 denaturation: 94 ° C, 1 min; 3 renaturation: 55 ° C, 30 S; 4 extension: 72 ° C, 2 min; 5 return to step "2", 35 cycles; : 72 ° C, 5 min, the total number of cycles is 30 times.
- the PCR product was subjected to 1% agarose gel electrophoresis, and it was revealed that a complete PTD-HSA-L- ⁇ -MSH DNA band of about 1.8 kb in size was amplified, and the above PCR product was subjected to gel recovery.
- the first round of PCR amplification PCR amplification was carried out using pcDNA3.1-HSA plasmid DNA as a template and NF and R2 as upper and lower primers, respectively.
- the reaction conditions are as follows: 1 denaturation: 94 ° C, 5 min; 2 denaturation: 94 ° C, 1 min; 3 renaturation: 55 ° C, 30 S; 4 extension: 72 ° C, 2 min; 5 return to step "2", 35 cycles; 6 extension: 72 ° C, 5 min, the total number of cycles is 30 times.
- the PCR product was subjected to 1% agarose gel electrophoresis, and it was revealed that a partial HSA-L- ⁇ -MSH band of about 1.8 kb in size was amplified.
- the second round of PCR amplification was performed using the products of the first round of PCR amplification as a template and NF and R1 as the upstream and downstream primers, respectively.
- the reaction conditions are as follows: 1 denaturation: 94 ° C, 5 min; 2 denaturation: 94 ° C, 1 min; 3 renaturation: 55 ° C, 30 S; 4 extension: 72 ° C, 2 min; 5 return to step "2", 35 cycles; : 72 ° C, 5 min, the total number of cycles is 30 times.
- the PCR product was subjected to 1% agarose gel electrophoresis, and it was revealed that a complete HSA-L- ⁇ -MSH DNA band of about 1.8 kb in size was amplified, and the above PCR product was subjected to gel recovery.
- the correctly sequenced pPINK ⁇ -HC/PTD-HSA-L- ⁇ -MSH plasmid DNA and pPINK ⁇ -HC/HSA-L- ⁇ -MSH plasmid DNA were digested with AflII to obtain linearized pPINK ⁇ -HC/PTD-HSA.
- -L- ⁇ -MSH and pPINK ⁇ -HC/HSA-L- ⁇ -MSH fragments were transformed into Pichia methanolica, respectively, and then the transformation broth was inoculated into PAD plates, cultured at 30 ° C for 3-4 days, and positive clones were picked. .
- the positive clones were inoculated into BMGY liquid medium, cultured at 30 ° C for 48 hours, then transferred to BMMY medium to induce expression. After 96 hours, centrifuge at 1500 rpm for 15 minutes, the supernatant was taken, and protein expression was detected by SDS-PAGE electrophoresis. happening.
- the fusion protein PTD-HSA-L- ⁇ -MSH has a molecular weight of about 70 kDa, and the amino acid sequence of PTD-HSA-L- ⁇ -MSH is shown in SEQ ID NO: 8, and the DNA encoding PTD-HSA-L- ⁇ -MSH The sequence is shown in SEQ ID NO: 7; the fusion protein HSA-L- ⁇ -MSH has a molecular weight of about 69 kDa, and the amino acid sequence of HSA-L- ⁇ -MSH is shown in SEQ ID NO: 10, encoding HSA-L- ⁇ .
- the DNA sequence of MSH is shown as SEQ ID NO: 9.
- mice Grouping and administration of mice:
- mice Ten Kunming mice were randomly divided into two groups, weighing about 18-22 g: 1-control group (injected HSA-L- ⁇ -MSH), 2-alpha melanocyte stimulating hormone fusion protein group (injected PTD-HSA) -L- ⁇ -MSH).
- 1-control group injected HSA-L- ⁇ -MSH
- 2-alpha melanocyte stimulating hormone fusion protein group injected PTD-HSA
- the control group was injected with tail vein (150 uL, 1 ⁇ m/kg) HSA-L- ⁇ -MSH.
- the fusion protein group of ⁇ -melanocyte cytokines was injected into the tail vein (150 uL, 1 ⁇ m/kg) PTD-HSA-L- ⁇ -MSH.
- the mice were anesthetized, and hippocampus tissues were taken out and homogenized.
- the level of PTD-HSA-L- ⁇ -MSH in hippocampal tissue homogenate was determined by ELISA.
- mice Grouping and administration of mice:
- Lipopolysaccharides are the cell wall components of Gram-negative bacteria. LPS injection into the lateral ventricle of mice can cause neurodegenerative diseases and inflammatory reactions in the brain.
- the tumor necrosis factor a (TNF-a) in the hippocampus was used as an indicator to test the efficacy of the ⁇ -MSH fusion protein of the present invention in a brain-induced inflammation model induced by LPS in mice.
- mice Twenty Kunming mice were divided into four groups and weighed about 18-22 g: 1-control group, 2-LPS group, 3-PTD-HSA-L- ⁇ -MSH+LPS group, 4-a-MSH+LPS group. .
- the control group was injected with 300 ⁇ L of normal saline twice, each injection was 150 ⁇ L; the LPS group was injected with 150 ⁇ L LPS (5 mg/kg), followed by 150 ⁇ L of normal saline; the 3-PTD-HSA-L- ⁇ -MSH+LPS group was first.
- the level of TNF- ⁇ in hippocampal tissue homogenate was determined by ELISA.
- TNF- ⁇ was significantly increased in the hippocampus of the LPS group, indicating that the model was successfully established; compared with the ⁇ -MSH+LPS group, the hippocampus of the mouse in the PTD-HSA-L- ⁇ -MSH group
- the TNF- ⁇ is significantly decreased, indicating that the PTD-HSA-L- ⁇ -MSH of the present invention can significantly reduce the inflammation of the central nervous system, and more strongly prove that PTD-HSA-L- ⁇ -MSH can cross the blood-brain barrier.
- the original effect of ⁇ -MSH is maintained to treat brain inflammation and related diseases.
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Abstract
公开了一种α促黑素细胞激素的融合蛋白,包含1个蛋白转导结构域(Protein transduction domian,PTD),1个人血清白蛋白(Albumin Human,HSA)和一个α促黑素细胞激素(α-Melanocyte stimulating hormone,α-MSH)。还公开了该融合蛋白的制备方法及其在抑制或治疗中枢神经系统炎症中的应用。
Description
本发明属于基因工程制药领域,具体涉及一种α促黑素细胞激素的融合蛋白及其制备方法和应用。
血管损伤,炎症以及外伤引起的脑组织损伤会导致机体的认知功能障碍,这类疾病严重影响着人们的生活质量和工作效率。目前针对上述疾病的临床药物大多关注脑神经组织的修复和保护,事实上,除了神经细胞以外,星型胶质细胞、寡树突胶质细胞、内皮细胞以及小胶质细胞同样需要损伤后的修复。内源性免疫神经调节肽α促黑素细胞激素(α-Melanocyte stimulating hormone,α-MSH)具有潜在的抗炎,神经营养,抗凋亡效应。对上述疾病具有显著的治疗潜力,而且相对于传统的抗代谢免疫抑制剂副作用较小,在基础研究与临床治疗中都具有重要价值,有望成为新型的神经保护剂,是一种前景广阔的临床候选药物。
α-MSH的免疫调节效应经下述途径产生:(1)直接作用于外周巨噬、单核和嗜中性粒细胞等免疫细胞上的α-MSH受体;(2)作用于脑内神经元上的α-MSH受体,进而启动下游抗炎神经通路;(3)中枢神经系统的局部炎症由局部产生的α-MSH通过作用于中枢小胶质细胞和星形细胞而被抑制,也可由外周细胞产生的α-MSH通过脑脊液循环作用于中枢。此外α-MSH的抗炎活性已经在多种动物模型中被证实。这些动物模型有刺激过敏性皮炎、接触过敏性皮炎、血管炎、关节炎、眼部炎症、胃肠炎、脑部炎症以及过敏性炎症。
在中枢神经系统炎症中,α-MSH参与神经免疫调节通路的分子机制如下:α-MSH由其受体介导,降低炎症反应中最大的调节器分子NF-кB的介导的转录活性,进而阻止前炎因子的释放,抑制粘着分子的表达,从而降低脑组织损伤,起到营养、保护、修复受损脑组织的作用。
α-MSH主要由下丘脑,垂体和多种外周组织细胞产生,由13个氨基酸组成,大鼠静脉注射体内半衰期为几分钟,对于此类蛋白质药物来说,必须考虑肾小球滤过问题,因此,就要通过对α-MSH进行结构的修饰和改造或者其他方法来延长其半衰期。
HSA是人体内源性蛋白,分子量达66kDa,在人体内的半衰期达到19天,具有无免疫原性、人体相容性好、无酶活等特性,是许多内源性代谢物质和外源性药物的运输载体。上述特性使HSA成为长效蛋白/多肽类药物开发的一个理想载体。
因此,本发明利用白蛋白融合技术(Albumin Fusion technology)将α-MSH与HSA融合,两者之间加入具有特殊结构的柔韧性分子接头(连接肽),采用本发明公开的技术方案,不仅保证了融合蛋白的稳定性和生物活性,而且增加了分子量。
虽然现有技术中公开了通过融合蛋白的表达的策略可以延长目的蛋白的半衰期,但是融合蛋白的设计本身是一个程序复杂、影响因素众多的过程,仅通过序列的简单加和是无法实现α-MSH的稳定高效表达、延长其半衰期的目的的,这是本领域技术人员所公知的。
同时,为了使上述融合蛋白能够有效地跨过血脑屏障,治疗脑部炎症及相关疾病,本发明在融合蛋白氨基酸序列中加入了蛋白转导结构域(Protein transduction domian,PTD),PTD是近年来发现的较理想的跨BBB转运载体,具有强大的运载功能,能够转运高于自身分子量100倍的大分子,可将外源性蛋白质、DNA、RNA、化学分子、磁珠、脂质体转运通过细胞膜,且此过程不受分子大小和种类的限制。
基于上述现状,发明人公开了一种α促黑素细胞激素的融合蛋白,该融合蛋白所具有的独特的氨基酸序列可以保证其在宿主体内高水平稳定表达,在保留α-MSH原有功能的同时,体内半衰期显著延长,同时能够跨过血脑屏障,治疗脑部炎症及相关疾病。
发明内容
本发明的目的在于提供一种α促黑素细胞激素的融合蛋白,该融合蛋白能在宿主体内高水平稳定表达,半衰期长,能够有效地跨过血脑屏障,治疗脑部炎症及相关疾病。
本发明的另一个目的在于提供一种α促黑素细胞激素的融合蛋白的制备方法。
本发明的另一个目的在于提供一种重组表达载体。
本发明的另一个目的在于提供一种宿主表达系统。
本发明的另一个目的在于提供一种α促黑素细胞激素的融合蛋白应用。
本发明所述的α促黑素细胞激素的融合蛋白,包含1个蛋白转导结构域(Protein transduction domian,PTD),1个人血清白蛋白(Albumin Human,HSA)和一个α促黑素细胞激素(α-Melanocyte stimulating hormone,α-MSH)。
其中,所述融合蛋白包还含有连接肽L,HSA通过连接肽L与α-MSH连接。
所述PTD位于融合蛋白的N-末端,α-MSH位于融合蛋白的C-末端,融合蛋白用结构式表示为PTD-HSA-L-α-MSH。
所述PTD具有SEQ ID NO:2所示的氨基酸序列,编码所述PTD的氨基酸序列的DNA序列如SEQ ID NO:1所示;或在该氨基酸序列中取代、缺失或插入氨基酸残基所得到的具有所述PTD的活性的氨基酸序列,及编码所述的氨基酸序列的DNA序列。
所述HSA具有SEQ ID NO:4所示的氨基酸序列,编码所述HSA的氨基酸序列的DNA序列如SEQ ID NO:3所示;或在该氨基酸序列中取代、缺失或插入氨基酸残基所得到的具
有所述HSA的活性的氨基酸序列,及编码所述的氨基酸序列的DNA序列。
所述α-MSH具有SEQ ID NO:6所示的氨基酸序列,编码所述α-MSH的氨基酸序列的DNA序列如SEQ ID NO:5所示;或在该氨基酸序列中取代、缺失或插入氨基酸残基所得到的具有所述α-MSH的活性的氨基酸序列,及编码所述的氨基酸序列的DNA序列。
所述融合蛋白的氨基酸序列如SEQ ID NO:8所示,编码所述融合蛋白的氨基酸序列的DNA序列如SEQ ID NO:7所示。
上述融合蛋白采用酵母细胞表达制备,所述酵母为嗜甲醇毕赤酵母(Pichia pastoris)。
本发明所述的α促黑素细胞激素的融合蛋白的制备方法,包含以下步骤:
①全基因合成α-MSH序列;
②通过PCR扩增获取PTD-HSA序列;
③使用融合延长PCR技术连接①中α-MSH与②中PTD-HSA,通过In-fusion技术连接目的片段与载体,获得含编码所述α促黑素细胞激素的融合蛋白的DNA序列的重组酵母表达载体;其中,所述载体为pPinkα-HC。
④将步骤③所述的重组酵母表达载体转化到感受态大肠杆菌TOP10细胞中,提取重组表达载体质粒后测序,将测序正确的质粒转化到宿主表达系统进行表达,即得所述融合蛋白。其中,所述宿主表达系统为嗜甲醇毕赤酵母。
一种含有编码α促黑素细胞激素的融合蛋白氨基酸序列的DNA序列的重组表达载体。
一种含有上述的重组表达载体的宿主表达系统。
一种α促黑素细胞激素的融合蛋白在制备抑制或治疗中枢神经系统炎症的药物中的应用。本发明的有益效果:
1.本发明利用人血清白蛋白融合技术将α-MSH与HSA融合,两者之间加入具有特殊结构及一定柔韧性的分子接头(连接肽),保证融合蛋白的稳定性和生物活性,有效地增加了分子量,实现α-MSH的稳定高效表达,达到延长其半衰期的目的。
2.本发明在融合蛋白氨基酸序列中加入了PTD,在保留α-MSH原有功能的同时,能够有效地跨过血脑屏障,治疗脑部炎症及相关疾病。
3.本发明所述的α促黑素细胞激素的融合蛋白可在制备抑制或治疗中枢神经系统炎症的药物中应用,适合在此领域大规模推广。
图1载体pPinkα-HC
图2载体pPINKα-HC/PTD-HSA-L-α-MSH
主要实验仪器:
移液枪、超净工作台(安泰)、磁力搅拌器、微波炉、高温蒸汽灭菌锅、-80℃低温冰箱(Forma)、超纯水仪(Millipore)、制冰机、离心机(Hitachi)、HDB-PLUS型恒温金属浴、HZQ-F16OA型恒温振荡培养箱(上海一恒)、PCR仪(Applied Biosystems)、台式冷冻离心机(Thermo)、DYY-8B型电泳仪(伯乐)、Image Quant 300型凝胶成像仪(GE)等。
主要实验材料:
1.限制性核酸内切酶StuI、KpnI、XhoI、AflII(NEB公司产品,美国)
2.小提质粒试剂盒、PCR纯化试剂盒、DNA胶回收试剂盒(生工生物,中国)
3.T4DNA连接酶试剂盒(Takara公司产品,中国大连)
4.载体pPinkα-HC、载体pcDNA3.1-HSA、毕赤酵母菌株、Infusion试剂盒(Invitrogen公司产品,美国)
5.大肠杆菌TOP10(天根生化科技(北京)有限公司)
6.酵母提取物、蛋白胨(Oxford公司产品,美国)
7.LB培养基
酵母提取物5g,蛋白胨10g,NaCl 10g,溶于1000ml去离子水中,并用1mol/L的NaOH调节pH值至7.0,高压蒸气灭菌。
8.YPD培养基
酵母提取物10g,胰蛋白胨20g,Agar 20g,溶于900ml去离子水中,高压灭菌,冷却后加入100ml经滤器除菌后的20%的右旋糖。
9.YPDS培养基
酵母提取物10g,蛋白胨20g,山梨糖醇182.2g,溶于900ml去离子水中,高压灭菌,冷却后加入100ml经滤器除菌后的20%的右旋糖。
10.BMGY液体培养基
酵母提取物10g,蛋白胨20g,无氨基酸酵母氮源13.4g,甘油10g,磷酸钾26.631g,溶于1000ml双蒸水中高压灭菌,冷至室温,调节pH至6.0,4℃保存备用。
11.1%琼脂糖凝胶的配置
根据用量,每100ml的TAE缓冲液,加入1g琼脂糖,使用微波炉加热煮沸,使琼脂糖完全融解,室温冷却至不烫手时滴加少量溴化乙锭(EB),混匀后将其倒入事先摆放好梳子的胶槽中,待到室温冷却至完全凝固后拔去梳子即可使用。
实施例1pPINKα-HC/PTD-HSA-L-α-MSH和pPINKα-HC/HSA-L-α-MSH的融合蛋白酵母
表达载体的构建与表达
一、pPINKα-HC/PTD-HSA-L-α-MSH载体的构建
1.设计PCR引物:
2.第一轮PCR扩增:以pcDNA3.1-HSA质粒DNA为模版,以NFT2和R2分别作为上、下游引物,进行PCR扩增。反应条件如下:①变性:94℃,5min;②变性:94℃,1min;③复性:55℃,30S;④延伸:72℃,2min;⑤返回步骤“②”,进行35循环;⑥延伸:72℃,5min,总循环次数为30次。将PCR产物进行1%琼脂糖凝胶电泳,结果显示扩增出约1.8kb大小的部分PTD-HSA-L-α-MSH条带。
3.第二轮PCR扩增:以第一轮PCR扩增的产物为模板,以NFT1和R1分别作为上、下游引物,进行PCR扩增。反应条件如下:①变性:94℃,5min;②变性:94℃,1min;③复性:55℃,30S;④延伸:72℃,2min;⑤返回步骤“②”,进行35循环;⑥延伸:72℃,5min,总循环次数为30次。将PCR产物进行1%琼脂糖凝胶电泳,结果显示扩增出约1.8kb大小的完整PTD-HSA-L-α-MSH DNA条带,将以上PCR产物进行胶回收。
4.KpnI和StuI双酶切pPINKα-HC(Invitrogen公司产品)质粒DNA,胶回收获得pPINKα-HC(KpnI/StuI)载体片段,利用Infusion试剂盒将上述胶回收的pPINKα-HC(KpnI/StuI)载体片段和PTD-HSA-L-α-MSH DNA目的基因片段进行重组反应,反应产物转化大肠杆菌感受态TOP10,涂于氨苄抗性LB板37℃培养过夜,筛选阳性克隆。所得克隆送Invitrogen公司测序,序列正确的克隆命名为pPINKα-HC/PTD-HSA-L-α-MSH。
二、pPINKα-HC/HSA-L-α-MSH载体的构建
1.设计PCR引物:
2.第一轮PCR扩增:以pcDNA3.1-HSA质粒DNA为模版,以NF和R2分别作为上、下游引物,进行PCR扩增。反应条件如下:①变性:94℃,5min;②变性:94℃,1min;
③复性:55℃,30S;④延伸:72℃,2min;⑤返回步骤“②”,进行35循环;⑥延伸:72℃,5min,总循环次数为30次。将PCR产物进行1%琼脂糖凝胶电泳,结果显示扩增出约1.8kb大小的部分HSA-L-α-MSH条带。
3.第二轮PCR扩增:以第一轮PCR扩增的产物为模板,以NF和R1分别作为上、下游引物,进行PCR扩增。反应条件如下:①变性:94℃,5min;②变性:94℃,1min;③复性:55℃,30S;④延伸:72℃,2min;⑤返回步骤“②”,进行35循环;⑥延伸:72℃,5min,总循环次数为30次。将PCR产物进行1%琼脂糖凝胶电泳,结果显示扩增出约1.8kb大小的完整HSA-L-α-MSH DNA条带,将以上PCR产物进行胶回收。
4.KpnI和StuI双酶切pPINKα-HC(Invitrogen公司产品)质粒DNA,胶回收获得pPINKα-HC(KpnI/StuI)载体片段,利用Infusion试剂盒将上述胶回收的pPINKα-HC(KpnI/StuI)载体片段和HSA-L-α-MSH DNA目的基因片段进行重组反应,反应产物转化大肠杆菌感受态TOP10,涂于氨苄抗性LB板37℃培养过夜,筛选阳性克隆。所得克隆送Invitrogen公司测序,序列正确的克隆命名为pPINKα-HC/HSA-L-α-MSH。
三、PTD-HSA-L-α-MSH及HSA-L-α-MSH融合蛋白在酵母中的表达
将测序正确的pPINKα-HC/PTD-HSA-L-α-MSH质粒DNA和pPINKα-HC/HSA-L-α-MSH质粒DNA用AflII酶切回收后得到线性化的pPINKα-HC/PTD-HSA-L-α-MSH及pPINKα-HC/HSA-L-α-MSH片段,分别转化嗜甲醇毕赤酵母,然后将转化菌液接种于PAD平板,30℃培养3-4天,挑取阳性克隆。将得到阳性克隆分别接种BMGY液体培养基,30℃培养48小时,然后转接至BMMY培养基中诱导表达,持续96小时后,1500rpm低温离心15分钟,取上清,SDS-PAGE电泳检测蛋白表达情况。融合蛋白PTD-HSA-L-α-MSH的分子量约为70kDa,PTD-HSA-L-α-MSH的氨基酸序列如SEQ ID NO:8所示,编码PTD-HSA-L-α-MSH的DNA序列如SEQ ID NO:7所示;融合蛋白HSA-L-α-MSH的分子量约为69kDa,HSA-L-α-MSH的氨基酸序列如SEQ ID NO:10所示,编码HSA-L-α-MSH的DNA序列如SEQ ID NO:9所示。
实施例2PTD-HSA-L-α-MSH跨血脑屏障功能验证
实验材料
1.实验仪器
注射器、移液枪、离心机(Hitachi)、超纯水仪(Millipore)、超声仪、恒温培
养箱(上海一恒)、酶标仪(Thermo)等。HSA Elisa试剂盒(Cygnus Technologies)
2.实验动物
10只标准体重昆明小鼠,购于兰州大学实验动物中心。
3.实验方法
小鼠的分组及给药方式:
将10只昆明小鼠随机分为两组,体重18~22g左右:1-对照组(注射HSA-L-α-MSH),2-α促黑素细胞激素的融合蛋白组(注射PTD-HSA-L-α-MSH)。
对照组尾静脉注射(150uL,1μm/kg)HSA-L-α-MSH。α促黑素细胞激素的融合蛋白组尾静脉注射(150uL,1μm/kg)PTD-HSA-L-α-MSH。6小时后,麻醉小鼠,取出海马组织,进行组织匀浆。ELISA法检测海马组织匀浆中的PTD-HSA-L-α-MSH水平。
实验结果分析:
表1
以上实验说明本发明提供PTD-HSA-L-α-MSH融合蛋白能有效地够跨过血脑屏障。
实施例3PTD-HSA-L-α-MSH生物活性研究
实验材料
1.实验仪器
注射器、移液枪、离心机(Hitachi)、超纯水仪(Millipore)、超声仪、恒温培
养箱(上海一恒)、酶标仪(Thermo)等。TNF-a Elisa试剂盒(达科为)
2.实验动物
20只标准体重昆明小鼠,购于兰州大学实验动物中心。
3.实验方法
小鼠的分组及给药方式:
脂多糖LPS(Lipopolysaccharides)即革兰氏阴性菌内毒素是革兰氏阴性细菌的细胞壁组成成分,小鼠侧脑室注射LPS能够引起大脑中的神经退行性病变及炎症反应,本实验以小鼠脑部海马组织中的肿瘤坏死因子(tumor necrosis factors a,TNF-a)为指标,检测本发明所述的α-MSH的融合蛋白在小鼠由LPS引起的脑补炎症模型中的药效。
将20只昆明小鼠分为四组,体重18~22g左右:1-对照组,2-LPS组,3-PTD-HSA-L-α-MSH+LPS组,4-a-MSH+LPS组。对照组分两次注射300μL生理盐水,每次注射量为150μL;LPS组先注射150μL LPS(5mg/kg),后注射150μL生理盐水;3-PTD-HSA-L-α-MSH+LPS组先注射150μL LPS(5mg/kg),后注射150μL PTD-HSA-L-α-MSH(1μM/kg);a-MSH+LPS组先
注射150μL LPS(5mg/kg),后注射150μL a-MSH。2小时后,处死小鼠,迅速取出海马组织,进行组织匀浆。本实验所述注射方式均为腹腔注射。
ELISA法检测海马组织匀浆中的TNF-α水平。
实验结果分析:
分组 | TNF-α(pg/mg) |
对照组 | 14.8±4.2 |
LPS组 | 191.9±9.1 |
LPS+PTD-HSA-L-α-MSH组 | 38.3±18.3 |
LPS+α-MSH组 | 155.8±10.3 |
与对照组相比,LPS组小鼠海马组织中TNF-α明显升高,说明模型建立成功;与α-MSH+LPS组相比,PTD-HSA-L-α-MSH组中小鼠海马组织中TNF-α显著降低,说明本发明所述PTD-HSA-L-α-MSH能够显著地降低中枢神经系统的炎症,更有力地证明PTD-HSA-L-α-MSH能够跨过血脑屏障,同时保持α-MSH的原有功效,用以治疗脑部炎症及相关疾病。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换,都应当视为属于本发明的保护范围。
Claims (14)
- 一种α促黑素细胞激素的融合蛋白,其特征在于,所述融合蛋白包含1个蛋白转导结构域(Protein transduction domian,PTD),1个人血清白蛋白(Albumin Human,HSA)和一个α促黑素细胞激素(α-Melanocyte stimulating hormone,α-MSH)。
- 根据权利要求1所述的一种α促黑素细胞激素的融合蛋白,其特征在于,所述融合蛋白还含有连接肽L,HSA通过连接肽L与α-MSH连接。
- 根据权利要求2所述的一种α促黑素细胞激素的融合蛋白,其特征在于,所述连接肽L的DNA序列为GGAGGTGGAGGTTCTGGAGGTGGATCTGGT,氨基酸序列为GGGGSGGGSG。
- 根据权利要求1所述的一种α促黑素细胞激素的融合蛋白,其特征在于,所述PTD位于融合蛋白的N-末端,α-MSH位于融合蛋白的C-末端,融合蛋白用结构式表示为PTD-HSA-L-α-MSH。
- 根据权利要求1所述的一种α促黑素细胞激素的融合蛋白,其特征在于,所述PTD具有SEQ ID NO:2所示的氨基酸序列,编码所述PTD的氨基酸序列的DNA序列如SEQ ID NO:1所示;或在该氨基酸序列中取代、缺失或插入氨基酸残基所得到的具有所述PTD的活性的氨基酸序列,及编码所述的氨基酸序列的DNA序列。
- 根据权利要求1所述的一种α促黑素细胞激素的融合蛋白,其特征在于,所述HSA具有SEQ ID NO:4所示的氨基酸序列,编码所述HSA的氨基酸序列的DNA序列如SEQ ID NO:3所示;或在该氨基酸序列中取代、缺失或插入氨基酸残基所得到的具有所述HSA的活性的氨基酸序列,及编码所述的氨基酸序列的DNA序列。
- 根据权利要求1所述的一种α促黑素细胞激素的融合蛋白,其特征在于,所述α-MSH具有SEQ ID NO:6所示的氨基酸序列,编码所述α-MSH的氨基酸序列的DNA序列如SEQ ID NO:5所示;或在该氨基酸序列中取代、缺失或插入氨基酸残基所得到的具有所述α-MSH的活性的氨基酸序列,及编码所述的氨基酸序列的DNA序列。
- 根据权利要求1-7中任意一项所述的一种α促黑素细胞激素的融合蛋白,其特征在于,所述融合蛋白的氨基酸序列如SEQ ID NO:8所示,编码所述融合蛋白的氨基酸序列的DNA序列如SEQ ID NO:7所示。
- 根据权利要求8所述的一种α促黑素细胞激素的融合蛋白,其特征在于,所述融合蛋白采用酵母细胞表达制备。
- 根据权利要求9所述的一种α促黑素细胞激素的融合蛋白,其特征在于,所述的酵母为嗜甲醇毕赤酵母(Pichia pastoris)。
- 一种如权利要求4所述的α促黑素细胞激素的融合蛋白的制备方法,其特征在于,所述方法包含以下步骤:①全基因合成α-MSH序列;②通过PCR扩增获取PTD-HSA序列;③使用融合延长PCR技术连接①中α-MSH与②中PTD-HSA,通过In-fusion技术连接目的片段与载体,获得含编码所述α促黑素细胞激素的融合蛋白的DNA序列的重组酵母表达载体;④将步骤③所述的重组酵母表达载体转化到感受态大肠杆菌TOP10细胞中,将所述质粒转化到宿主表达系统进行表达,即得所述融合蛋白,其中,所述宿主表达系统为嗜甲醇毕赤酵母。
- 一种含有编码如权利要求1-7、9、10中任意一项所述的α促黑素细胞激素的融合蛋白氨基酸序列的DNA序列的重组表达载体。
- 一种含有权利要求12所述的重组表达载体的宿主表达系统。
- 权利要求1-7、9、10中任意一项所述的α促黑素细胞激素的融合蛋白在制备抑制或治疗中枢神经系统炎症的药物中的应用。
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CN102066421A (zh) * | 2007-07-06 | 2011-05-18 | 瑟瑞技术公司 | α-促黑激素(α-MSH)和心房利尿钠蛋白(ANP)的双功能融合蛋白以及在高血压和急性肾损伤中的用途 |
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