CN108314723A - A kind of people source saltant type nerve growth factor and its preparation method and application - Google Patents
A kind of people source saltant type nerve growth factor and its preparation method and application Download PDFInfo
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- CN108314723A CN108314723A CN201810027039.0A CN201810027039A CN108314723A CN 108314723 A CN108314723 A CN 108314723A CN 201810027039 A CN201810027039 A CN 201810027039A CN 108314723 A CN108314723 A CN 108314723A
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- C—CHEMISTRY; METALLURGY
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- 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
- C07K14/475—Growth factors; Growth regulators
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Abstract
It is the tryptophan on the 221st amino acid position of wild type proNGF polypeptide chains instead of arginine the invention discloses a kind of people source saltant type nerve growth factor（proNGFR221W）, cause the tryptophan on ripe the 100th amino acid position of NGF polypeptide chains instead of arginine (ripe NGFR100W) formed.hNGFR100It can support the sensory neuron and sympathetic neuron of peripheral nervous system, while the nutritional status of central nervous system and peripheral nervous system partial nerve member can also be adjusted, but it will not be as its wild type NGFWTCause pain reaction, to solve long-standing problem and the critical issue for hindering NGF clinically to use, can be used for preventing or treat various periphery property and central sexual nerves and immunological diseases.
Description
Technical field
The invention belongs to field of genetic engineering, and in particular to a kind of people source saltant type nerve growth factor and preparation method thereof
And application.
Background technology
Nineteen fifty discovery, nerve growth factor（NGF）It is the battalion felt in peripheral nervous system with sympathetic nervous member
The factor is supported, while may also participate in the nutritional status of corpus straitum and matrix forebrain cholinergic neuron in regulation and control central nervous system.
With brain-derived neurotrophic factor（BDNF）, neurotrophin 3（NT-3）And neurotrophin（NT-4）Discovery,
NGF has been the important member of neurotrophin family now.NGF transmits signals to the god of response by following 2 kinds of receptors
In cell cytoplasm and core through member：The tyrosine receptor kinase A (TrkA) of 140 KD and the neurotrophin receptor of 75 KD
(p75NTR).NGF-TrkA signals can excite the neurotrophy of many classics to react.TrkB and TrkC can mediate NT-3 and NT-
4 signals.NGF- p75NTR signals are also the signal of interest access of NGF, not only contribute to the metabolism of sphingomyelins-ceramide, together
When also assist in adjust RhoA activity to regulate and control axon growth.In addition, there will be research has shown that, p75NTR signals can activate NF-
B, Akt and JNK signal path are to inducing cell apoptosis or promote cells survival and differentiation.
Based on powerful neurotrophic effect, currently, NGF has correlative study to the therapeutic effect of PNS and CNS diseases.
For example, in NGF to cholinergic neurons of basal forebrain（BFCNs）Find that NGF can be used for controlling in the research of neurotrophic function
Treat this kind of degenerative diseases of AD.Unfortunately, the biological characteristics of NGF limit its therapeutic domain.NGF cannot pass through blood brain
Barrier limits its systematicness and uses.Moreover, even low dose of NGF, can cause to ache after transmitting with vascular system
Pain side effect.In the recent period, a clinic II phase the study found that NGF is carried to after basal forebrain with virus be it is safe, not
There is pain side effect.The notable growth of BFCNs nerve fibres can favorably confirm the potential neurotrophic functions of NGF, but to recognizing
Know that aspect does not have an impact.In addition, some scientists are also being dedicated to studying NGF to diabetic peripheral neuropathy with integrated at present
Therapeutic effect.It being found in the clinical II phases are studied, the NGF of 0.1 and 0.3 μ g/kg dosage has certain therapeutic potential, but
It is super quick to be that there are dose-dependent pain at injection position.It is found in the large-scale clinic III phases are studied, 0.1 μ g/kg
The NGF of dosage no therapeutic effects compared with placebo.
In peripheral nervous system, NGF is not only neurotrophic factor, and is mediating inflammatory pain and nerve pain
The important molecule of pain.It finds in clinical studies, large dosage of NGF is terminated for AD sufferers, because NGF can cause greatly
Pain side effect.In another clinical research, with HIV by NGF for treating diabetic neuropathy and peripheral neuropathy
Also declaration stops, because NGF has serious side effect, including back pain, the injection site pain sensation is super quick, and myalgia and weight subtract
Gently.Therefore, the pain side effect of NGF has seriously limited its utilization in treatment neurodegenerative disease.It is led to solve pain
The side effect of cause, the effect that the NGF in Pain Process is elaborated will be particularly important with its receptor signal.
A series of heredity and clinical research confirmation, NGF can pass through TrkA and p75NTR mediated immunity pain.Than
If TrkA recessive mutations lead to hereditary sensory and self-disciplining neuropathy type IV (HSAN IV) (OMIM # 256800),
The also referred to as insensitive merging anhidrosis of idiopathic pain（CIPA）.Having strong evidence confirms, TrkA is in the pain for mediating NGF
Effect in sensibility effect：Alleviate the signal path of expression or the pharmacology inhibition TrkA mediations of TrkA, such as extracellular signal
Associated kinase (ERK), three kinases of phosphoinositide（PI3K）And phospholipase C γ (PLC γ) can alleviate the pain that NGF is mediated
Feel allergy.In addition, in p75NTR gene delection mouse, NGF still can induce hyperalgia, show that TrkA is the NGF induction pains sensation
The major downstream molecule of allergy.The evidence that p75NTR is acted in pain signal access is largely indirect evidence.Such as：Note
The Pain behaviour that p75NTR neutralizing antibodies can inhibit NGF inductions is penetrated, and NGF improves the reaction potence of sensory neuron.Cause
This, TrkA and p75NTR signals work in NGF induction pains.However, the two receptors are in mediated pain process
In be also being not very clear of how working.
Recently, find that a kind of spontaneous NGF missense recessive mutations, these patients lose deep in a family of Sweden
The pain sensation, and frequently result in fracture and joint injury.This disease is defined as hereditary sensory autonomic neuropathy type V
(hereditary sensory autonomic neuropathy type V, HSAN V)、(Online Mendelian
Inheritance in Man (OMIM) # 608654) to these HSAN V sufferers carry out genetic analysis, find ngf gene
Site mutation (the 661C of sequence>T), lead to the tryptophan on ripe the 100th amino acid position of NGF polypeptide chains
Instead of arginine (ripe NGFR100W).IV types HSAN, HSAN patient V caused by being mutated unlike TrkA, which has, normally to be recognized
Know function, shows that NGF mutation do not influence its trophic function in nervous centralis.Our current researchs and other people grind
Study carefully the potential tool for showing that NGFR100W can be used as the difference for cracking NGF neurotrophic functions and pain function.
The initial features of NGFR100W disclose in the cell of culture, and R100 mutation may upset NGF precursors to maturation
NGF conversions, so as to cause the releases of more precursor NGF relatively.Difficulty is expressed in view of NGFR100W, Catanneo is with his
Colleague uses recombinant technique, and a series of residues, including the bis- mutation of NGFR100E and NGF are had detected on 100 this position
(NGFP61S/R100E).They find these NGFR100 mutant can normally combine with TrkA receptors, but cannot be incorporated into
p75NTR.These discoveries show the pain that cannot activate p75NTR signal paths that can effectively alleviate NGF inductions, also indicate that
TrkA signals there is no effect in pain.Inventors believe that NGFR100 mutation can develop into p75NTR antagonists,
For example NGFR100 treats preparation as a kind of painless NGF.
Invention content
Purpose one：For overcome the deficiencies in the prior art, the present invention provides a kind of people source saltant type nerve growth factor,
The people source saltant type nerve growth factor can support the sensory neuron and sympathetic neuron of peripheral nervous system, while also may be used
To adjust the nutritional status of central nervous system and peripheral nervous system partial nerve member, but people source saltant type nerve growth factor
Son will not be as its wild type nerve growth factorWTCause pain reaction, to solve long-standing problem and hinder nerve it is raw it is sub- because
Son（NGF）The critical issue clinically used, make its can be used for preventing or treat various periphery property and central sexual nerves with
And immunological diseases etc..
To achieve the goals above one, the technical solution adopted by the present invention is：A kind of people source saltant type nerve growth factor,
It is characterized in that：Including hNGFR100With hNGF (KKE/Δ 9/13), the amino acid of the people source saltant type nerve growth factor
Sequence is as shown in Figure 1, the hNGFR100Precursor（proNGFR221）Amino acid sequence as shown in Fig. 2, the KKE=K32/
K34/E35, Δ 9/13 are the amino acids basic for removing the positions 9-13.
Further, the hNGFR100For hNGFR100W、hNGFR100E、hNGFR100XOr hNGFR100P, the X be in addition to
Any amino acids basic except R, W, E, P and derivative, the KKE=K32A/K34A/E35A or KKE=K32B/K34B/
E35B。
Further, saltant type nerve growth factor in people source is in wild type growth factor of human nerve precursor（proNGF）It is more
Partial amino-acid residue side chain is replaced to be formed by 221st amino acid position of peptide chain, the wild type human nerve growth
Factor precursor（proNGF）Amino acid sequence be SEQ ID NO：NP_002497.2, the substitution original acid residue side chains
Substituted amino acid side chain include one to five substituted amino acid, substituted amino acid include hydrophobic amino acid (A, I, L, M, F,
P, W, Y, V), hydrophilic amino acid (R, D, N, C, E, Q, G, H, K, S, T), aliphatic lateral chain amino acid (G, A, V, L, I, P),
The amino acid (S, T, Y) of hydroxyl side chain, amino acid (C, M), the amino acid containing carboxylic acid and amide side chains of sulfur atom-containing side chain
In the amino acid (R, K, H) of (D, N, E, Q), the side chain containing basic group, the amino acid (H, F, Y, W) containing beta-branched side at least
It is a kind of.
Above-mentioned people source saltant type nerve growth factor is wild type human nerve growth factor precursor（proNGF）Polypeptide chain
Tryptophan on 221st amino acid position is instead of arginine（proNGFR221）, lead to ripe NGF polypeptide chains the 100th
Tryptophan on amino acid position is instead of arginine (ripe NGFR100)。hNGFR100It can support the feeling of peripheral nervous system
Neuron and sympathetic neuron, while the nutrition shape of central nervous system and peripheral nervous system partial nerve member can also be adjusted
State, but hNGFR100It will not be as its wild type NGFWTCause pain reaction.
The common part that can lack or add one or several amino acid includes purification tag（Such as Avi-tag,
His-tag）, the Met of N-terminal, N-terminal signal peptide etc., these are all well-known to those skilled in the art.When in prokaryotes
Expression, albumen n end generally require the nucleotide sequence of coding Met；For the secreting, expressing in eucaryote, albumen n end is logical
Often need signal peptide.Those skilled in the art know, by changing the coding gene sequence of known peptide and being conducted into table
Up to carrier, the polypeptide that can be prepared substitution, be inserted into or be added to amino acid residue, these methods are recorded in extensively《Molecule gram
Grand experiment guide（The third edition）》(Beijing：Science Press, 2002) etc. in document well known in the art.Preferably replace,
Missing adds one to five amino acid, more preferably one to three amino acid.In substituted amino acid residue, preferably
It is substituted by other amino acid similar with original acid residue side chains property.For example, the similar amino acid of side chain properties has respectively
Hydrophobic amino acid (A, I, L, M, F, P, W, Y, V), hydrophilic amino acid (R, D, N, C, E, Q, G, H, K, S, T), aliphatic side
The amino acid (G, A, V, L, I, P) of chain, the amino acid (S, T, Y) of hydroxyl side chain, sulfur atom-containing side chain amino acid (C, M),
Amino acid (D, N, E, Q) containing carboxylic acid and amide side chains, the side chain containing basic group amino acid (R, K, H), contain beta-branched side
Amino acid (H, F, Y, W).
Goal of the invention two：The present invention provides a kind of nucleic acid of encoding human source saltant type nerve growth factor, feature exists
In：The nucleic acid of the people source saltant type nerve growth factor is DNA molecular or RNA molecule form, and the DNA molecular includes recombination
DNA and artificial synthesized cDNA, the DNA of recombination be coding strand or template strand, polynucleotide sequence such as Fig. 3 institutes of the nucleic acid
Show, the precursor of the nucleic acid of the people source saltant type nerve growth factor（proNGFR221）As shown in figure 4, wild type human nerve growth
Factor precursor（proNGF）Polynucleotide sequence such as SEQ ID NO：Shown in NP_002497.2, people source saltant type nerve
The amino acid sequence of growth factor as shown in Figure 1, people source saltant type nerve growth factor precursor（proNGFR221）Amino acid
Sequence is as shown in Figure 2.
Above-mentioned nucleic acid can be DNA form, can also be rna form, preferably DNA form.DNA form includes recombination
DNA and artificial synthesized cDNA, DNA can be coding strand or template strand.Skill is recombinated by routine techniques, such as PCR method, DNA
Art or artificial synthesized method, those skilled in the art can be readily available the nucleotides sequence of the encoding fusion protein of the present invention
Row or its segment.In fact, according to the polynucleotide sequence of design, currently have been able to prepare by commercial channel specification
Corresponding DNA.Once obtaining nucleic acid, so that it may to be cloned into carrier, then convert or be transfected into corresponding cell, then pass through
Conventional host cell is proliferated, therefrom isolated a large amount of nucleotide sequence.In the specific implementation mode of the present invention,
The polynucleotide sequence of the nucleic acid of the present invention is as shown in figure 3, its precursor proNGFR221As shown in figure 4, wild type proNGF multinuclears
Nucleotide sequence such as SEQ ID NO：Shown in NP_002497.2.
Goal of the invention three：The present invention provides it is a kind of include codified people source saltant type nerve growth factor nucleic acid
Carrier, it is characterised in that：It is included in the prokaryotic expression carrier of expression in bacterium；The Pichia pastoris of the expression in yeast carries
Body, Hansenula vectors；The baculovirus vector expressed in insect cell；The bovine vaccine of expression in the cell of mammal
Viral vectors, retroviral vector, adenovirus vector, adeno-associated virus carrier.
The present invention provides carriers, contain above-mentioned nucleic acid.Term " recombinant expression carrier " herein, " expression carries
Body " only claims " carrier " sometimes, can interact be replaced herein, refers to bacterial plasmid commonly used in the art, clay, phagocytosis
Grain, yeast plasmid, plant cell virus, animal virus and various other viral vectors.In the present invention be applicable in carrier include but
It is not limited to：The carrier (prokaryotic expression carrier) of the expression in bacterium, in yeast expression carrier (such as Pichia pastoris carry
Body, Hansenula vectors etc.), the baculovirus vector expressed in insect cell, in mammalian cell expression load
Body (vaccinia virus vector, retroviral vector, adenovirus vector, adeno-associated virus carrier etc.), in plant expression plant
Object viral vectors and in mammal galactophore expression various carriers.Preferred expression carrier includes selectable marker gene,
Such as the ampicillin resistance gene of bacterium, tetracycline resistance gene, kalamycin resistance gene, streptomycin resistance gene, chlorine
Mycin resistant gene；Neomycin resistance gene, the Zeocin resistant genes of saccharomycete, the defect selection marker of saccharomycete, such as
His, Leu, Trp etc.；The neomycin resistance gene of eukaryocyte, Zeocin resistant genes, dihydrofolate reductase gene and glimmering
Photoprotein marker gene etc..In the specific implementation mode of the present invention, carrier of the invention is pcDNA, is suitble to dynamic in lactation
It is expressed in object cell.
Goal of the invention four：The present invention provides it is a kind of include carrier described in goal of the invention three as above cell, it is special
Sign is：The cell is the human embryonic kidney epithelial cells that conversion or transfection have the people source saltant type nerve growth factor nucleic acid（
HEK293）.
Above-mentioned cell can be prokaryotic cell, can also be eukaryocyte, as bacterial cell, yeast cells, plant cell,
Insect cell, mammalian cell etc..How those skilled in the art known by carrier high-efficiency converts or to be transfected into host thin if being
In born of the same parents, method therefor includes but not limited to：Calcium Chloride Method, electroporation are used for bacterial cell, electroporation, protoplast fusion
Method, liposome, coprecipitation of calcium phosphate, electro fusion method and microinjection., it is surprising that the present inventor is from current
Be found that in host cell as astronomical figure rat bone marrow tumour cell can secreting, expressing be transfected into it is therein the present invention
hNGFR100, directly purified using the less culture supernatant of relative impurity ingredient therefore without smudge cells.Cause
This, the preferably described cell of the present invention is the human embryonic kidney epithelial cells that conversion or transfection have the above-mentioned nucleic acid of the present invention（ HEK293）With
Other human archeocyte strains for manufacturing biologics.
Goal of the invention five：The present invention provides a kind of preparation methods of people source saltant type nerve growth factor, and method is such as
Under：
First, 1. 2. the amino acid sequence of designer source saltant type nerve growth factor is as shown in Figure 1, utilize human embryo kidney (HEK) epithelium thin
Born of the same parents（ HEK293）The base of cell codon expression frequency optimization coding the people source saltant type nerve growth factor protein matter
Cause, the polynucleotide sequence of the gene is as shown in figure 3,3. to go out polynucleotide sequence by PCR method sectionally smooth joins as shown in Figure 3
DNA, and be cloned into pcDNA plasmids to form hNGFR100- avitag plasmids convert Escherichia coli（E. Coli）XL-1,4.
Extract hNGFR100Plasmid is identified through digestion and sequencing, wherein containing polynucleotide sequence such as with in-frame sequence
SEQ ID NO：DNA shown in 1；
Second, designer source saltant type nerve growth factor precursor（proNGFR100）Amino acid sequence as shown in Fig. 2, 2. sharp
Use human embryonic kidney epithelial cells（ HEK293）Cell codon expression frequency optimization coding the people source saltant type nerve growth factor
Sub- precursor（proNGFR100）The polynucleotide sequence of the gene of protein, the gene is spelled as shown in figure 4, being 3. segmented by PCR methods
Polynucleotide sequence DNA as shown in Figure 4 is picked out, and is cloned into pcDNA plasmids to form pcDNA/1c plasmids, large intestine is converted
Bacillus（E. Coli）4. XL-1 extracts proNGFR100Plasmid is identified through digestion and sequencing, wherein with in-frame suitable
Sequence contains polynucleotide sequence DNA as shown in Figure 4；
Third, by hNGFR100- avitag plasmids and pcDNA/1c plasmids are transfected into HEK293 cells according to electrotransformation method respectively
Middle carry out secreting, expressing, has been transfected hNGF respectivelyR100Or proNGFR100The Positive transfections cell and its culture of plasmid；
4th, hNGF will have been transfected respectivelyR100- hNGF (KKE/ Δs 9/13) or proNGFR100The Positive transfections cell of plasmid and
The culture supernatant of its culture uses Protein A affinity chromatographys, SP-Sepharose ion-exchange chromatographies and Q- successively
People source saltant type nerve growth factor hNGF is obtained after sepharose chromatographic purifyingsR100。
Above-mentioned preparation method comprising following steps：The object of the invention one is expressed with the cell described in the object of the invention four
The hNGFR100, and detach the hNGFR100.Express the hNGF described in objects of the present invention oneR100Cell can be with
It cultivates and/or is induced to express required hNGF by conventional methodR100.The hNGF of expressionR100Can in the cell, cell membrane
It is upper or be secreted into periplasmic, extracellular.Separation（Purifying）Method include but not limited to：Split bacterium（Ultrasonic wave splits bacterium, osmotic pressure
Split bacterium）, it centrifuges, saltouts, molecular sieve chromatography, ion-exchange chromatography, adsorption chromatography (affinity chromatography, metal chelate affinity chromatography), reversely
Chromatography, high performance liquid chromatography, Capillary Electrophoresis, preparative isoelectric focusing and denaturation, renaturation process etc..Since rat bone marrow tumour is thin
Born of the same parents can secreting, expressing be transfected into the hNGF of the present invention thereinR100, therefore the culture supernatant for preferably directly expressing cell
To detach.It is preferred that in the fifth aspect of the invention, separation process includes using the culture supernatant that cell is expressed successively
Protein A affinity chromatographys, SP-Sepharose ion-exchange chromatographies and Q-sepharose chromatographic purifyings.
Goal of the invention six：The present invention provides one kind for preventing or treating periphery property, central, nerve and be immunized
The pharmaceutical composition of disease, it is characterised in that：Including people source saltant type nerve growth factor and further include people source saltant type god
Carrier through growth factor.
Further, the carrier includes nontoxic filler, nontoxic diluent, nontoxic adjuvant or nontoxic preparation
Auxiliary material.
Further, pharmaceutical composition is made for the agent of tablet, capsule, pulvis, emulsion agent, injection or spray-type
Type.
The present invention provides hNGFR100The pharmaceutical composition of clinical application comprising described in objects of the present invention one
hNGFR100And pharmaceutically acceptable carrier.Above-mentioned pharmaceutical composition can be used to prevent or treat various periphery property in
Pivot nerve and immunological diseases etc., such as due to diabetic peripheral neuropathy with integrated etc..It is used herein pharmaceutically
The carrier of receiving refers to nontoxic filler, diluent, adjuvant or other pharmaceutical adjuncts.It, can be with according to techniques known
According to therapeutic purposes, administration route need pharmaceutical composition is made various dosage forms, preferably the composition is unit dosage shape
Formula, such as tablet, capsule, pulvis, emulsion agent, injection or spray-type.
Using the above scheme, the application of people source saltant type nerve growth factor of the invention is to prepare and can prevent or treat various
Periphery property and central sexual nerves and immunological diseases etc.（Such as diabetic peripheral neuropathy with integrated）Drug.
The beneficial effects of the present invention are：People source saltant type nerve growth factor can support the feeling of peripheral nervous system
Neuron and sympathetic neuron, while the nutrition shape of central nervous system and peripheral nervous system partial nerve member can also be adjusted
State, but hNGFR100It will not cause pain reaction as its wild type NGFWT, to solve long-standing problem and hinder NGF in clinic
The critical issue of upper utilization；The hNGF of the present inventionR100Specific mammalian cell secreting, expressing can be utilized, is composed in glycosylation
While close to the mankind, it is more convenient the purifying in later stage.
The invention will be further described below in conjunction with the accompanying drawings.
Description of the drawings
Attached drawing 1 is hNGF of the present inventionR100WAmino acid sequence figure；
Attached drawing 2 is proNGF of the present inventionR221WAmino acid sequence figure；
Attached drawing 3 is hNGF of the present inventionR100WPolynucleotide sequence figure；
Attached drawing 4 is proNGF of the present inventionR221WPolynucleotide sequence figure；
Attached drawing 5 is hNGF of the present inventionR100WPolynucleotide sequence and amino acid sequence compares figure；
Attached drawing 6 is proNGF of the present inventionR221WPolynucleotide sequence and amino acid sequence compares figure.
Specific implementation mode
In order to make it easy to understand, the present invention will be described in detail by specific embodiment and attached drawing below.It needs
It is emphasized that these descriptions are only exemplary description, and it is not meant to limit the scope of the invention.According to this explanation
The discussion of book, many variations of the invention, change are all apparent for those skilled in the art.In addition, of the invention
Open source literature is referred to, these documents are that their entire contents are included in carry out herein in order to more clearly describe the present invention
With reference to as its full text is already contained among this paper.
The present invention is illustratively illustrated by specific embodiment, wherein if any not most place, reference can be made to《Point
Sub- cloning experimentation guide（The third edition）》（Science Press, Beijing）Equal laboratory manuals and commercially available reagent and kit make
With explanation.
The present invention devises a kind of people source saltant type nerve growth factor comprising hNGFR100With hNGF (KKE/Δs 9/
13), the amino acid sequence of people source saltant type nerve growth factor is as shown in Figure 1, the hNGFR100Precursor（proNGFR221）'s
Amino acid sequence is as shown in Fig. 2, the KKE=K32/K34/E35, Δ 9/13 are the amino acids basic for removing the positions 9-13.It is preferred that
, hNGFR100For hNGFR100W、hNGFR100E、hNGFR100XOr hNGFR100P, wherein X is the appointing except P in addition to R, W, E
What amino acids basic and derivative, KKE=K32A/K34A/E35A or KKE=K32B/K34B/E35B.The embodiment of the present invention with
hNGFR100It is illustrated for example with hNGF (K32A/K34A/E35A).
The hNGF of 1 present invention of embodimentR100WDesign
We devise amino acid sequence hNGF as shown in Figure 1R100WProtein, and obtained HEK293 is studied according to us
Cell codon expression frequency optimization encodes the gene of the protein, and polynucleotide sequence is as shown in figure 3, root is conventional
PCR method sectionally smooth joins go out polynucleotide sequence DNA as shown in Figure 3, and are cloned into pcDNA plasmids（It is purchased from Invitrogen
Company））In to form hNGFR100W- avitag plasmids convert Escherichia coli（E. Coli） XL-1.Extract hNGFR100WMatter
Grain is identified through digestion and sequencing, wherein containing polynucleotide sequence such as SEQ ID NO with in-frame sequence：Shown in 1
DNA, in theory being capable of express amino acid sequence protein as shown in Figure 1.
In addition, we devise amino acid sequence proNGF as shown in Figure 2R100WProtein, and studied according to us
The HEK293 cell codon expression frequency optimizations that arrive encode the gene of the protein, polynucleotide sequence such as Fig. 4 institutes
Show, polynucleotide sequence DNA as shown in Figure 4 is gone out according to conventional PCR method sectionally smooth joins, and be cloned into pcDNA plasmids（It is commercially available
From Invitrogen companies）In to form pcDNA/1c plasmids, convert Escherichia coli（E. Coli） XL-1.Extract hNGF
(K32A/K34A/E35A)-proNGFR100WPlasmid is identified through digestion and sequencing, wherein being contained with in-frame sequence more
Nucleotide sequence DNA as shown in Figure 4, in theory being capable of express amino acid sequence protein as shown in Figure 2.
The above plasmid returns to us and carries out next step experiment.
The hNGF of 2 present invention of embodimentR100W- hNGF (K32A/K34A/E35A), expression and purifying two above matter
Grain is transfected into HEK293 cells according to conventional electrotransformation method respectively（It is purchased from Shanghai Edinburg biotechnology and develops limited public affairs
Department）Middle carry out secreting, expressing.In brief, hNGF (K32A/K34A/E35A)-hNGFR100WOr proNGFR100WPlasmid respectively takes 40
μ g distinguish digestion to linearize with restriction enzyme Sal I., and ice bath is spare.It, will under conditions of 37 °C, 5% CO2
HEK293 cell culture is in 10% inactivated fetal bovine serum（Gibco, Australia）, 1% penicillin and streptomysin（Invitrogen,
Carlsbad, CA, USA）The sugared culture solutions of DMEM high（Gibco, Australia）.When reaching exponential phase of growth and survival rate
When more than 90%, with ice-cold PBS（pH7.2）PBS is resuspended in after washing（pH7.2）In（A concentration of 1 x 107A cell/ml）,
Then the hNGF of linearisation is converted respectively with Gene Pulser electrotransformations instrumentR100WOr proNGFR100WPlasmid is each（Electrotransformation parameter
For：1500 volts, 3 μ Fd）.Ice bath after five minutes, is added DMEM cultures based on 37 °C of cultures, is then transferred to and is added to 5 μ g/ml
Blasticidin）DMEM culture mediums in cultivate about 3-4 weeks, until there is the thin of significant cell death situation and survival
Born of the same parents are in discrete shape, have been transfected hNGF respectivelyR100WOr proNGFR100WThe Positive transfections cell and its culture of plasmid.
The medium supernatant of two kinds of cell difference cultivation stages is drawn, respectively according to ELISA detections wherein amino acid sequence
Arrange the expression quantity of protein as illustrated in fig. 1 and 2.Illustrated according to manufacturer, utilizes CalTag Laboratories (South
San Francisco, the U.S.) ELISA detection reagents are detected, read OD on microplate readout instrument (Bio-Tek)405,
Calculate the expression quantity of gained protein.As a result as illustrated in fig. 1 and 2, both protein are all enough by HEK293 secreting, expressings, and
Expression quantity can exceed that the relatively high expression level of 100mg/L.
The supernatant for taking two kinds of cell culture plateaus of 3L respectively, is splined on and uses PBS（pH7.2）The Protein A of balance
Affinity column（It is purchased from GE Healthcare companies）, then with the PBS of 5 times of column volumes（pH7.2）It washes, discards outflow
Liquid.Then with the 0.1M acetate buffers of 3 times of column volumes（pH3.0）Albumen is eluted, eluent is collected and 2M Tris are added in
With pH to 5.5.Then, the eluent of neutralization is splined on SP-Sepharose ion exchange columns with the flow velocity of 4 mL/min
（It is purchased from GE Healthcare companies）, with 0.05M acetate buffers（pH5.5）It washes, discards efflux.Then with containing 0.15M
The phosphate buffer of NaCl（pH7.4）Elute albumen, with spectrophotometer detect efflux, when OD280 be more than 0.2 start persistently
It collects, until OD280 falls back to 0.4 again.The eluent is splined on Q-sepharose chromatographic columns with the flow velocity of 4 mL/min
（It is purchased from GE Healthcare companies）, with spectrophotometer detect efflux, when OD280 be more than 0.2 start persistent collection,
Until OD280 falls back to 0.4 again.The efflux of collection respectively under the reducing conditions with SDS-PAGE is carried out under non reducing conditions
Detection, amino acid sequence protein as illustrated in fig. 1 and 2 can access effective purifying.
3 precursor NGF of embodimentR100WCut into NGFR100WAnd NGFR100WPurify proNGFR100WIt is NGFR100WPrecursor.Cause
This, proNGFR100WSequence is sheared to obtain active NGFR100W.With the albumen of trypsin-like substrate interaction
Enzyme spcificity shear protein, there is no the active sites of digestive protein molecules.Trypsin like proteases shear peptide bond, form band
Positive charge amino acid, such as arginine and lysine.As trypsin like proteases, some serine proteases（Serine
Endopeptidase）Take part in proNGFR100WIt is converted into NGFR100WProcess.It is preferred that serine trypsase be used to shear
Presequence, and other protease can be used for substituting the function of serine protease.It is worth noting that, shearing is not only needle
To trypsase itself, but also include other substrates for having trypsin-like.In general, if proNGFR100W/ NGFR100W
Ratio it is relatively rational in the case of, ripe beta-NGF would not be by this proteolytic cleavage.On the contrary, albuminate and folding
The sequence of folded intermediate product exposure is just easier to be fallen by proteolytic cleavage.
In order to preferably by proNGFR100WCut into NGFR100W, trypsase is with respect to proNGFR100WThe ratio of mutation is
From 1:200 to 1:100,000；Desirably 1:5000 to 1:20,000；Most preferably 1:10,000（W/W）.Optimal
Under conditions of change, room temperature down cut 8 to 23 hours, preferably 18 hours.Under conditions of set by the patent, proNGFR100W
It is sheared, and is generated almost without by-product completely, also without finding any polymer.
proNGFR100WThe NGF that mutation shearing generatesR100WIt will be further purified with chromatography etc..Step is further purified to want
It asks trypsase and NGFR100 WThe related impurities separation that trypsin digestion generates.Purification step will reduce HCPs, endotoxin
With DNA etc..The method of any protein purification can use.The best way is purification by chromatography, for example is detached with agarose
Column.Final product beta-NGF will use SDS-PAGE, rp-HPLC, the methods of SE-HPLC and IEX-HPLC to identify its purity.
Phosphate buffer will be used for proNGFR100WIt is digested to NGFR100WProcess, it can't protease always
Activity.Sodium phosphate buffer is added in MEP- eluents to final concentration of 25Mm.PH value is 6.5.For protein breakdown, pancreas egg
White enzyme (Roche, GMP grade) is according to 1:10,000(W/W) (trypsin:ProNGFR100W t) ratio addition.Egg
White cracking condition is that 18 hours are cracked under normal temperature condition.After trypsin digestion, beta-NGF is reprinted in SP agaroses
Trypsase is filtered on HP columns, shears by-product and related impurities.Pillar by 25mM sodium phosphate buffer（pH6.5）Balance.
Trypsin digestion reaction is reprinted on SP agarose HP columns (2 g beta-NGF/L media), and the albumen not combined will be by
Equilibrium liquid elutes.Elution process is classified into (3 cv, 25 %, 25 mM sodium phosphates pH, 6.5/1 M chlorinations of three steps
Sodium elutes (buffer solution B), and 10 cv, 25% to 50% buffer solution Bs carry out linear gradient elution, 3 cv of and, 100 % buffer solutions
B (flow velocity is 60 cm/h)).
SEQUENCE LISTING
<110>Wenzhou Medical University
Wenzhou City biological medicine collaborative innovation center
<120>A kind of people source saltant type nerve growth factor and its preparation method and application
<130> 2018
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 120
<212> PRT
<213>Artificial sequence
<400> 1
Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp
1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val
50 55 60
Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys
65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln
85 90 95
Ala Ala Trp Trp Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu
100 105 110
Ser Arg Lys Ala Val Arg Arg Ala
115 120
<210> 2
<211> 241
<212> PRT
<213>Artificial sequence
<400> 2
Met Ser Met Leu Phe Tyr Thr Leu Ile Thr Ala Phe Leu Ile Gly Ile
1 5 10 15
Gln Ala Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile
20 25 30
Pro Gln Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu
35 40 45
Arg Arg Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala
50 55 60
Gly Gln Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg
65 70 75 80
Arg Leu Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu
85 90 95
Ala Ala Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro
100 105 110
Phe Asn Arg Thr His Arg Ser Lys Arg Ser Ser Ser His Pro Ile Phe
115 120 125
His Arg Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly
130 135 140
Asp Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu
145 150 155 160
Gly Glu Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu
165 170 175
Thr Lys Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile
180 185 190
Asp Ser Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val
195 200 205
Lys Ala Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Trp Phe Ile Arg
210 215 220
Ile Asp Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg
225 230 235 240
Ala
<210> 3
<211> 427
<212> DNA
<213>Artificial sequence
<400> 3
tcatcatccc atcccatctt ccacaggggc gaattctcgg tgtgtgacag tgtcagcgtg 60
tgggttgggg ataagaccac cgccacagac atcaagggca aggaggtgat ggtgttggga 120
gaggtgaaca ttaacaacag tgtattcaaa cagtactttt ttgagaccaa gtgccgggac 180
ccaaatcccg ttgacagcgg gtgccggggc attgactcaa agcactggaa ctcatattgt 240
accacgactc acacctttgt caaggcgctg accatggatg gcaagcaggc tgcctggtgg 300
tttatccgga tagatacggc ctgtgtgtgt gtgctcagca ggaaggctgt gagaagagcc 360
tgacctgccg acacgctccc tccccctgcc ccttctacac tctcctgggc ccctccctac 420
ctcaacc 427
<210> 4
<211> 790
<212> DNA
<213>Artificial sequence
<400> 4
atgtccatgt tgttctacac tctgatcaca gcttttctga tcggcataca ggcggaacca 60
cactcagaga gcaatgtccc tgcaggacac accatccccc aagtccactg gactaaactt 120
cagcattccc ttgacactgc ccttcgcaga gcccgcagcg ccccggcagc ggcgatagct 180
gcacgcgtgg cggggcagac ccgcaacatt actgtggacc ccaggctgtt taaaaagcgg 240
cgactccgtt caccccgtgt gctgtttagc acccagcctc cccgtgaagc tgcagacact 300
caggatctgg acttcgaggt cggtggtgct gcccccttca acaggactca caggagcaag 360
cggtcatcat cccatcccat cttccacagg ggcgaattct cggtgtgtga cagtgtcagc 420
gtgtgggttg gggataagac caccgccaca gacatcaagg gcaaggaggt gatggtgttg 480
ggagaggtga acattaacaa cagtgtattc aaacagtact tttttgagac caagtgccgg 540
gacccaaatc ccgttgacag cgggtgccgg ggcattgact caaagcactg gaactcatat 600
tgtaccacga ctcacacctt tgtcaaggcg ctgaccatgg atggcaagca ggctgcctgg 660
tggtttatcc ggatagatac ggcctgtgtg tgtgtgctca gcaggaaggc tgtgagaaga 720
gcctgacctg ccgacacgct ccctccccct gccccttcta cactctcctg ggcccctccc 780
tacctcaacc 790
Claims (10)
1. a kind of people source saltant type nerve growth factor, it is characterised in that：Including hNGFR100With hNGF (KKE/Δ 9/13), institute
The amino acid sequence of people source saltant type nerve growth factor is stated as shown in Figure 1, the hNGFR100Precursor proNGFR221Amino
Acid sequence is as shown in Fig. 2, the KKE=K32/K34/E35, Δ 9/13 are the amino acids basic for removing the positions 9-13.
2. people source saltant type nerve growth factor according to claim 1, it is characterised in that：The hNGFR100For
hNGFR100W、hNGFR100E、hNGFR100XOr hNGFR100P, the X is any amino acids basic except P in addition to R, W, E
And derivative, the KKE=K32A/K34A/E35A or KKE=K32B/K34B/E35B.
3. people source saltant type nerve growth factor according to claim 1 or 2, it is characterised in that：People source saltant type
Nerve growth factor is in wild type growth factor of human nerve precursor（proNGF）It is taken on 221st amino acid position of polypeptide chain
It is formed by for partial amino-acid residue side chain, the wild type human nerve growth factor precursor（proNGF）Amino acid sequence
For SEQ ID NO：The substituted amino acid side chain of NP_002497.2, the substitution original acid residue side chains include one to five
Substituted amino acid, substituted amino acid include hydrophobic amino acid (A, I, L, M, F, P, W, Y, V), hydrophilic amino acid (R, D, N,
C, E, Q, G, H, K, S, T), the amino acid (G, A, V, L, I, P) of aliphatic lateral chain, hydroxyl side chain amino acid (S, T, Y), contain
The ammonia of the amino acid (C, M) of sulphur atom side chain, the amino acid (D, N, E, Q) containing carboxylic acid and amide side chains, the side chain containing basic group
Base acid (R, K, H), at least one of the amino acid (H, F, Y, W) containing beta-branched side.
4. a kind of nucleic acid of encoding human source saltant type nerve growth factor, it is characterised in that：The people source saltant type nerve growth
The nucleic acid of the factor is DNA molecular or RNA molecule form, and the DNA molecular includes the DNA and artificial synthesized cDNA of recombination, weight
The DNA of group is coding strand or template strand, and the polynucleotide sequence of the nucleic acid is as shown in figure 3, people source saltant type nerve growth factor
The precursor proNGF of the nucleic acid of sonR221As shown in figure 4, the polynucleotide sequence of wild type human nerve growth factor precursor proNGF
Such as SEQ ID NO：Shown in NP_002497.2, the amino acid sequence of the people source saltant type nerve growth factor as shown in Figure 1,
The precursor proNGF of people source saltant type nerve growth factorR221Amino acid sequence it is as shown in Figure 2.
5. a kind of carrier including the people source saltant type nerve growth factor nucleic acid described in claim 4, it is characterised in that：Including
The prokaryotic expression carrier of expression in bacterium；The pichia vector, Hansenula vectors of expression in yeast；In insect
The baculovirus vector expressed in cell；In mammalian cell the vaccinia virus vector, retroviral vector of expression,
Adenovirus vector, adeno-associated virus carrier.
6. a kind of includes the cell of the people source saltant type nerve growth factor subcarrier described in claim 5, it is characterised in that：It should
Cell is the human embryonic kidney epithelial cells HEK293 that conversion or transfection have the people source saltant type nerve growth factor nucleic acid.
7. a kind of preparation method of people source saltant type nerve growth factor, method are as follows：
First, 1. 2. the amino acid sequence of designer source saltant type nerve growth factor is as shown in Figure 1, utilize human embryo kidney (HEK) epithelium thin
The gene of born of the same parents HEK293 cell codon expression frequency optimizations coding the people source saltant type nerve growth factor protein matter, should
The polynucleotide sequence of gene as shown in figure 3,3. go out polynucleotide sequence DNA as shown in Figure 3 by PCR method sectionally smooth joins,
And it is cloned into pcDNA plasmids to form hNGFR100- avitag plasmids convert Escherichia coli（E. Coli）4. XL-1 is extracted
Go out hNGFR100Plasmid is identified through digestion and sequencing, wherein containing polynucleotide sequence such as SEQ ID with in-frame sequence
NO：DNA shown in 1；
Second, designer source saltant type nerve growth factor precursor（proNGFR100）Amino acid sequence as shown in Fig. 2, 2. utilizing
Human embryonic kidney epithelial cells（ HEK293）Cell codon expression frequency optimization coding the people source saltant type nerve growth factor
Precursor（proNGFR100）The gene of protein, 3. the polynucleotide sequence of the gene is as shown in figure 4, pass through PCR method sectionally smooth joins
Go out polynucleotide sequence DNA as shown in Figure 4, and be cloned into pcDNA plasmids to form pcDNA/1c plasmids, converts large intestine bar
Bacterium（E. Coli）4. XL-1 extracts proNGFR100Plasmid is identified through digestion and sequencing, wherein with in-frame sequence
Contain polynucleotide sequence DNA as shown in Figure 4；
Third, by hNGFR100- avitag plasmids and pcDNA/1c plasmids are transfected into HEK293 cells according to electrotransformation method respectively
Middle carry out secreting, expressing, has been transfected hNGF respectivelyR100Or proNGFR100The Positive transfections cell and its culture of plasmid；
4th, hNGF will have been transfected respectivelyR100- hNGF (KKE/ Δs 9/13) or proNGFR100The Positive transfections cell of plasmid and its
The culture supernatant of culture uses Protein A affinity chromatographys, SP-Sepharose ion-exchange chromatographies and Q- successively
People source saltant type nerve growth factor is obtained after sepharose chromatographic purifyings.
8. a kind of for preventing or treating by diabetes or HIV or chemotherapy or the periphery property of old amnesia initiation, central, god
Pharmaceutical composition through property and immunological diseases, it is characterised in that：Including such as claim 1 or claim 2 or according to right
It is required that 7 prepare gained people source saltant type nerve growth factor and further include people source saltant type nerve growth factor carrier.
9. according to claim 8 caused for preventing or treating by diabetes or HIV or chemotherapy or old amnesia
The pharmaceutical composition of periphery property, central, nerve and immunological diseases, it is characterised in that：The carrier includes nontoxic fills out
Fill agent, nontoxic diluent, nontoxic adjuvant or nontoxic pharmaceutical adjunct.
10. according to claim 8 caused for preventing or treating by diabetes or HIV or chemotherapy or old amnesia
The pharmaceutical composition of periphery property, central, nerve and immunological diseases, it is characterised in that：Pharmaceutical composition be made for tablet,
The dosage form of capsule, pulvis, emulsion agent, injection or spray-type.
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| US20170260565A1 (en) * | 2011-11-07 | 2017-09-14 | The Broad Institute, Inc. | Propeptide-luciferase fusion proteins and methods of use thereof |
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| CN115813951A (en) * | 2022-11-10 | 2023-03-21 | 广州连达科技有限公司 | Transgenic stem cells and their use for treating insomnia or sleep disorders |
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