阅读说明：本技术 一种可产降铁除自由基治疗阿兹海默病的bs-5-YHEDA肽载体构建与生产方法 (Method for constructing and producing bs-5-YHEDA peptide vector capable of reducing iron and removing free radicals for treating Alzheimer's disease ) 是由 邹珍友 于 2020-07-02 设计创作，主要内容包括：本发明公开的属于临床化学药剂合成技术领域,具体为一种可产降铁除自由基治疗阿兹海默病的bs-5-YHEDA肽载体构建与生产方法,一种5’端为Ecor I限制性内切酶序列和3’端为Hind III限制性内切酶序列的含bs-5-YHEDA基因的DNA；该种可产降铁除自由基治疗阿兹海默病的bs-5-YHEDA肽载体构建与生产方法,通过缩合法开发制备了一种可跨越血脑屏障的bs-5-YHEDA肽,可进入AD脑组织降铁除自由基,将bs-5-YHEDA翻译成对应的DNA序列构建了人工基因,重组到分泌表达型载体pIN III ompA3,转化到大肠杆菌表达菌产bs-5-YHEDA肽,并借助载体表达的ompA导肽分泌到菌体外周,用盐析沉淀,透析和HPLC提纯,合成足够长度的多肽,节约生产成本,可实现量产,用于临床。(The invention discloses a method for constructing and producing a bs-5-YHEDA peptide vector capable of producing ferrum-reducing free radicals for treating Alzheimer disease, belonging to the technical field of synthesis of clinical chemical agents, in particular to a DNA containing a bs-5-YHEDA gene, wherein the 5 'end of the DNA is an Ecor I restriction endonuclease sequence and the 3' end of the DNA is a Hind III restriction endonuclease sequence; the bs-5-YHEDA peptide carrier capable of producing the iron-reducing free radical for treating the Alzheimer disease is constructed and produced by developing and preparing the bs-5-YHEDA peptide capable of crossing a blood brain barrier through a condensation method, entering AD brain tissues for reducing iron and removing free radicals, translating the bs-5-YHEDA into a corresponding DNA sequence to construct an artificial gene, recombining the artificial gene to a secretion expression carrier pIN III ompA3, transforming the artificial gene to escherichia coli expression bacteria to produce the bs-5-YHEDA peptide, secreting the cell periphery by virtue of ompA peptide expressed by the carrier, precipitating by salting out, dialyzing and purifying by HPLC, synthesizing a polypeptide with sufficient length, saving the production cost, realizing mass production and being used for clinic.)

1. A method for constructing a bs-5-YHEDA peptide vector capable of reducing iron and removing free radicals for treating Alzheimer's disease, which comprises the following steps: the DNA containing the bs-5-YHEDA gene, the 5 'end of which is an Ecor I restriction enzyme sequence and the 3' end of which is a Hind III restriction enzyme sequence, is cut by restriction enzymes Ecor I and Hind III and then inserted between polyclonal fragment cuts of pIN III ompA3 vector cut by restriction enzymes Ecor I and HindIII and after the ompA DNA of pIN III ompA3 vector encoding leader peptide, the pIN III ompA3-bs-5-YHEDA vector is formed;

pIN III ompA3-bs-5-YHEDA vector is transformed into JM101 competent Escherichia coli, after induction expression, a promoter in pINIII ompA3 vector can start bs-5-YHEDA gene transcription and translation, ompA DNA transcription and translation leader peptide can secrete bs-5-YHEDA into periplasm of thallus, and the leader peptide is automatically cut off.

2. The method for producing the bs-5-YHEDA peptide vector for producing iron-reducing and free radical-removing therapy for Alzheimer's disease according to claim 1, wherein: the production method for constructing the bs-5-YHEDA peptide vector capable of reducing iron and removing free radicals for treating the Alzheimer disease comprises the following steps:

s1: construction of bs-5-HAYED Artificial Gene: the DNA sequence encoding the bs-5-YHEDA peptide was 5'-catgcc tac cag gat cat gcc tac cag gat cat gcc tac cag gat cat gcc tac cag gatcat gcc tac cag gat cag tca gac atc gtc gca caa tcc tcc taa-3' according to the gene code;

adding EcoR I restriction enzyme recognition sequence ' G ^ AA TTC ' and HIND III restriction enzyme recognition sequence ' A ^ AG CTT ' at two ends respectively for facilitating splicing and insertion, and adding a stop codon taa at the 3' end;

s2: construction of secretion expression vector pIN III ompA 3-bs-5-YHEDA: entrusted company synthesizes the above DNA sequence, cuts the bs-5-HAYED artificial gene with EcoR I and Hind III restriction enzymes, extracts the secretory expression vector pIN III ompA3 plasmid stored in DH5 alpha E.coli, cuts the multiple cloning site of pIN III ompA3 plasmid with the above restriction enzymes, and attaches the cut bs-5-HAYED artificial gene into pIN III ompA3 cut with T4 DNA ligase, finally becomes pIN III ompA3-bs-5-YHEDA vector;

s3: transformation and positive colony screening: with CaCl₂Method for preparing JM101 competent cells, pINIII ompA3-bs-5-YHEDA vector plasmid was transformed into JM101 competent cells, plated on a plate containing ampicillin medium, and Amp was selected^RThe white colonies of (4) and identified by PCR and electrophoresis;

s4: selecting colony amplification and inducing expression: selecting a single colony, placing the single colony in a 10ml medium test tube, culturing for 12 hours at 37 ℃ in a shaking way, transferring the bacterial liquid to a 250 ml triangular flask, culturing overnight at 37 ℃ in a shaking way, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.01mM, and performing shake culture at 23 ℃ to induce expression for 4-6 hours;

s5: bacterial membrane periplasmic protein extraction: centrifuging to collect thalli, resuspending in 20% sucrose solution containing 10mM Tris (pH7.5), standing at 0 ℃ for 5 minutes, adding 0.5M EDTA (ethylene diamine tetraacetic acid) with pH8.0, standing at 0 ℃ for 10 minutes, centrifuging to remove supernatant, washing precipitates with distilled water, suspending in 1M Tris solution with pH7.6, standing at 0 ℃ for 30 minutes, centrifuging to take supernatant, adding 1/10 volume of 100% trichloroacetic acid, standing at 0 ℃ for 15 minutes, centrifuging to remove supernatant, washing the precipitates with 70% cold ethanol twice, and freeze-drying for later use, wherein the precipitates contain soluble periplasmic protein of escherichia coli cells;

s6: HPLC purification, MASS-sequencing to identify the collected bs-5-YHEDA peak: salting out and removing protein precipitates by using 25% saturation ammonium sulfate, dialyzing the salted-out protein solution, performing Lysine Sepharose affinity chromatography, collecting each peak on an HPLC MonoQ I negative high-molecular exchange column, performing gradient elution at pH8.5, collecting each peak except a small amount of foreign protein, identifying components of positive reaction by using an anti-bs-5-HAYED antibody, and identifying by using a mass spectrum and a protein sequencer.

Technical Field

The invention relates to the technical field of synthesis of clinical chemical agents, in particular to a method for constructing and producing a bs-5-YHEDA peptide vector capable of reducing iron and removing free radicals for treating Alzheimer's disease.

Background

Senile dementia is also called Alzheimer disease, is a common neurodegenerative disease in the elderly, is clinically characterized by comprehensive dementia such as dysmnesia, aphasia, disuse, agnosia, visual space skill damage, executive dysfunction, personality and behavior change and the like, has unknown etiology so far, and brings great trouble to patients and families when the senile dementia occurs.

No effective treatment for senile dementia exists at present, and researches show that the content of iron accumulated in brain is gradually increased with the age, and the iron can promote free radicals to damage brain tissues and is an important reason for inducing AD.

At present, the Fmoc deprotection-amino acid condensation reaction method is repeatedly carried out on peptide resin, according to the designed amino acid sequence, from beginning to end, the first amino acid is connected to the resin, then the subsequent amino acids are condensed onto the existing sequence one by one in sequence, and once condensation reaction is carried out for each amino acid, until all the sequences are condensed onto the existing peptide sequence, the protein synthesis is completed, and the synthesized soluble polypeptide bs-5-YHEDA which can cross the blood brain barrier and enter the brain to chelate iron is proved to be capable of storing iron in the brain, reducing free radicals and protecting brain tissue neurons.

However, the preparation method has long period and high cost, the length of the synthesizable sequence is short, generally less than 100 amino acids, the requirement is difficult to meet, the synthesizable sequence is short, the amino acids with the required length and molecular weight cannot be obtained, the patient is inevitably difficult to pay expensive drug cost, the mass production is difficult to realize in the clinical process, and the basis cannot be laid for industrialization.

Disclosure of Invention

The invention aims to provide a method for constructing and producing a bs-5-YHEDA peptide vector capable of reducing iron and removing free radicals for treating Alzheimer's disease, and aims to solve the problems that the existing preparation method proposed in the background art is long in period, high in cost, short in synthesizable sequence, generally less than 100 amino acids in length, difficult to meet the requirements, short in synthesizable sequence, incapable of obtaining amino acids with required length and molecular weight, inevitably resulting in that patients are difficult to pay expensive medicine cost, difficult to realize mass production in the clinical process, and incapable of laying a foundation for industrialization.

In order to achieve the purpose, the invention provides the following technical scheme: a bs-5-YHEDA peptide vector capable of reducing iron and removing free radicals for treating Alzheimer disease is constructed, DNA containing a bs-5-YHEDA gene with an Ecor I restriction enzyme sequence at the 5 'end and a Hind III restriction enzyme sequence at the 3' end is cut by restriction enzymes Ecor I and Hind III and inserted between polyclonal fragment cuts of pIN III ompA3 vector cut by restriction enzymes Ecor I and Hind III and an ompA DNA of pIN III ompA3 vector and encoding leader peptide to form pIN III ompA3-bs-5-YHEDA vector;

pIN III ompA3-bs-5-YHEDA vector is transformed into JM101 competent Escherichia coli, after induction expression, a promoter in pIN III ompA3 vector can start transcription and translation of bs-5-YHEDA gene, and ompA DNA transcription and translation leader peptide can secrete bs-5-YHEDA into periplasm of thallus, and the leader peptide is automatically cut off.

A production method for constructing a bs-5-YHEDA peptide vector capable of producing iron-reducing and free radical-removing medicines for treating Alzheimer's disease comprises the following steps:

s1: construction of bs-5-HAYED Artificial Gene: the DNA sequence encoding the bs-5-YHEDA peptide was 5'-cat gcc tac cag gat cat gcc tac cag gat cat gcc tac cag gat cat gcc taccag gat cat gcc tac cag gat cag tca gac atc gtc gca caa tcc tcc taa-3' according to the gene code;

adding EcoR I restriction enzyme recognition sequence ' G ^ AA TTC ' and HIND III restriction enzyme recognition sequence ' A ^ AG CTT ' at two ends respectively for facilitating splicing and insertion, and adding a stop codon taa at the 3' end;

s2: construction of secretion expression vector pIN III ompA 3-bs-5-YHEDA: entrusted company to synthesize the above DNA sequence, cut the bs-5-HAYED artificial gene with EcoR I and Hind III restriction enzymes, extract the secretory expression vector pIN III ompA3 plasmid stored in DH5 alpha E.coli, cut the multiple cloning site of pIN III ompA3 plasmid with the above restriction enzymes, and attach the cut bs-5-HAYED artificial gene into pIN IIIompA3 cut with T4 DNA ligase to finally become pIN III ompA3-bs-5-YHEDA vector;

s3: transformation and positive colony screening: with CaCl₂Method for preparing JM101 competent cells, pIN III ompA3-bs-5-YHEDA vector plasmid was transformed into JM101 competent cells, plated on a plate containing ampicillin medium, and Amp was selected^RThe white colonies of (4) and identified by PCR and electrophoresis;

s4: selecting colony amplification and inducing expression: selecting a single colony, placing the single colony in a 10ml medium test tube, culturing for 12 hours at 37 ℃ in a shaking way, transferring the bacterial liquid to a 250 ml triangular flask, culturing overnight at 37 ℃ in a shaking way, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.01mM, and performing shake culture at 23 ℃ to induce expression for 4-6 hours;

s5: bacterial membrane periplasmic protein extraction: centrifuging to collect thalli, resuspending in 20% sucrose solution containing 10mM Tris (pH7.5), standing at 0 ℃ for 5 minutes, adding 0.5M EDTA (ethylene diamine tetraacetic acid) with pH8.0, standing at 0 ℃ for 10 minutes, centrifuging to remove supernatant, washing precipitates with distilled water, suspending in 1M Tris solution with pH7.6, standing at 0 ℃ for 30 minutes, centrifuging to take supernatant, adding 1/10 volume of 100% trichloroacetic acid, standing at 0 ℃ for 15 minutes, centrifuging to remove supernatant, washing the precipitates with 70% cold ethanol twice, and freeze-drying for later use, wherein the precipitates contain soluble periplasmic protein of escherichia coli cells;

s6: HPLC purification, MASS-sequencing to identify the collected bs-5-YHEDA peak: salting out and removing protein precipitates by using 25% saturation ammonium sulfate, dialyzing the salted-out protein solution, performing Lysine Sepharose affinity chromatography, collecting each peak on an HPLCMono Q I negative high-molecular exchange column, performing gradient elution at pH8.5, collecting each peak except a small amount of hybrid protein, identifying components of positive reaction by using an anti-bs-5-HAYED antibody, and identifying by using a mass spectrum and a protein sequencer.

Compared with the prior art, the invention has the beneficial effects that: the bs-5-YHEDA peptide carrier capable of producing the iron-reducing free radicals for treating the Alzheimer disease is constructed and produced by developing and preparing the bs-5-YHEDA peptide capable of crossing a blood brain barrier through a condensation method, entering AD brain tissues for reducing iron and removing free radicals, translating the bs-5-YHEDA into a corresponding DNA sequence to construct an artificial gene, recombining the artificial gene to a secretion expression carrier pIN III ompA3, transforming the artificial gene to escherichia coli expression bacteria to produce the bs-5-YHEDA peptide, secreting the cell periphery by virtue of ompA peptide expressed by the carrier, precipitating by salting out, dialyzing and purifying by HPLC, synthesizing polypeptides with sufficient length, saving the production cost, realizing mass production, being used for clinic and laying a foundation for industrialization.

Drawings

FIG. 1 is a schematic diagram showing the structure of the bs-5-YHEDA gene of the present invention;

FIG. 2 is a schematic diagram showing the construction of secretion expression vector pIN III ompA3-bs-5-YHEDA according to the present invention;

FIG. 3 is a schematic diagram of the enzyme-linked assay of the present invention;

FIG. 4 is a schematic diagram illustrating the restriction enzyme digestion identification of plasmid extracted from JM101 according to the present invention;

FIG. 5 is a schematic diagram showing the result of PCR amplification of the bs-5-YHEDA gene according to the present invention;

FIG. 6 is a schematic diagram showing the PCR sequencing result of the plasmid bs-5-YHEDA gene of the present invention;

FIG. 7 is a schematic representation of HPLC purification according to the present invention;

FIG. 8 is a schematic diagram of the identification of bs-5-YHEDA expressed in HPLC-purified periplasmic protein by Western Blot according to the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a technical scheme that: a bs-5-YHEDA peptide vector capable of producing iron-reducing free radicals to treat Alzheimer disease is constructed, DNA containing a bs-5-YHEDA gene with an EcoR I restriction enzyme sequence at the 5 'end and a HindIII restriction enzyme sequence at the 3' end is cut by restriction enzymes Ecor I and Hind III and inserted between polyclonal fragment cuts of pIN III ompA3 vector cut by restriction enzymes Ecor I and Hind III and after ompA DNA of pIN III ompA3 vector encoding leader peptide, pIN III ompA3-bs-5-YHEDA vector is formed;

pIN III ompA3-bs-5-YHEDA vector is transformed into JM101 competent Escherichia coli, after induction expression, a promoter in pIN III ompA3 vector can start transcription and translation of bs-5-YHEDA gene, and ompA DNA transcription and translation leader peptide can secrete bs-5-YHEDA into periplasm of thallus, and the leader peptide is automatically cut off.

A production method for constructing a bs-5-YHEDA peptide vector capable of producing iron-reducing and free radical-removing medicines for treating Alzheimer's disease comprises the following steps:

s1: construction of bs-5-HAYED Artificial Gene: the DNA sequence encoding the bs-5-YHEDA peptide was 5'-cat gcc tac cag gat cat gcc tac cag gat cat gcc tac cag gat cat gcc taccag gat cat gcc tac cag gat cag tca gac atc gtc gca caa tcc tcc taa-3' according to the gene code;

adding EcoR I restriction enzyme recognition sequence ' G ^ AA TTC ' and HIND III restriction enzyme recognition sequence ' A ^ AG CTT ' at two ends respectively for facilitating splicing and insertion, and adding a stop codon taa at the 3' end;

s2: construction of secretion expression vector pIN III ompA 3-bs-5-YHEDA: entrusted company to synthesize the above DNA sequence, cut the bs-5-HAYED artificial gene with EcoR I and Hind III restriction enzymes, extract the secretory expression vector pIN III ompA3 plasmid stored in DH5 alpha E.coli, cut the multiple cloning site of pIN III ompA3 plasmid with the above restriction enzymes, and attach the cut bs-5-HAYED artificial gene into pIN IIIompA3 cut with T4 DNA ligase to finally become pIN III ompA3-bs-5-YHEDA vector;

s3: transformation and positive colony screening: with CaCl₂Method for preparing JM101 competent cells, pIN IIIompA3-bs-5-YHEDA vector plasmid was transformed into JM101 competent cells, plated on a plate containing ampicillin medium, and Amp was selected^RThe white colonies of (4) and identified by PCR and electrophoresis;

s4: selecting colony amplification and inducing expression: selecting a single colony, placing the single colony in a 10ml medium test tube, culturing for 12 hours at 37 ℃ in a shaking way, transferring the bacterial liquid to a 250 ml triangular flask, culturing overnight at 37 ℃ in a shaking way, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.01mM, and performing shake culture at 23 ℃ to induce expression for 4-6 hours;

s5: bacterial membrane periplasmic protein extraction: centrifuging to collect thalli, resuspending in 20% sucrose solution containing 10mM Tris (pH7.5), standing at 0 ℃ for 5 minutes, adding 0.5M EDTA (ethylene diamine tetraacetic acid) with pH8.0, standing at 0 ℃ for 10 minutes, centrifuging to remove supernatant, washing precipitates with distilled water, suspending in 1M Tris solution with pH7.6, standing at 0 ℃ for 30 minutes, centrifuging to take supernatant, adding 1/10 volume of 100% trichloroacetic acid, standing at 0 ℃ for 15 minutes, centrifuging to remove supernatant, washing the precipitates with 70% cold ethanol twice, and freeze-drying for later use, wherein the precipitates contain soluble periplasmic protein of escherichia coli cells;

s6: HPLC purification, MASS-sequencing to identify the collected bs-5-YHEDA peak: salting out and removing protein precipitates by using 25% saturation ammonium sulfate, dialyzing the salted-out protein solution, performing Lysine Sepharose affinity chromatography, collecting each peak on an HPLCMono Q I negative high-molecular exchange column, performing gradient elution at pH8.5, collecting each peak except a small amount of hybrid protein, identifying components of positive reaction by using an anti-bs-5-HAYED antibody, and identifying by using a mass spectrum and a protein sequencer.