CN107325168A - A kind of growth and differentiation factor through modification and its preparation method and application - Google Patents
A kind of growth and differentiation factor through modification and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of growth and differentiation factor through modification, it is coupled and is formed with growth and differentiation factor by trim, the growth and differentiation factor is：(a) the spontaneous growth differentiation 11 of mammal is derived from；(b) substitution by the amino acid sequence of the spontaneous growth differentiation 11 by one or more amino acid residues and/or missing and/or addition and protein or the polypeptide as derived from spontaneous growth differentiation 11 with promotion neuron regeneration ability；Or (c) has at least 50% homology and with protein or the polypeptide as derived from the amino acid sequence shown in (a) or (b) for promoting neuron regeneration ability with the amino acid sequence shown in (a) or (b).The growth and differentiation factor through modification of the present invention can be preferably applied to prevent, improve and treat senile dementia, can also play a part of strengthening memory.In addition, the metabolite that growth and differentiation factor is regulated and controled has the purposes of diagnosis and examination senile dementia.
Description
Technical field
The present invention relates to a kind of modified protein, more particularly to a kind of growth and differentiation factor through modification and preparation method thereof and
Using.
Background technology
Alzheimer syndrome is called senile dementia, is characterized in that disease man memory and cognitive ability are gradually lost.
Patient is dead in 3 to 9 years after making a definite diagnosis, and accounts for 50% to the 56% of clinically dead case.For the cause of disease, current understanding is
The beta- amyloids and Tau albumen of abnormal folding are have accumulated in brain, so as to result in the generation of senile dementia
(Querfurth H.W.Alzheimer’s Disease.N Engl J Med 2010；362,329-344).
The medicine for the treatment of senile dementia is mainly Western medicine in the market, it is characterized in that must adhere to taking for a long time, so
And curative effect is small, partial symptoms can only be alleviated, it is impossible to the development of symptom management.Another defect of long-term taking is medicine
Side effect is big, while patient produces drug dependence (Becker, R.E.What can triumphs and tribulations
from drug research in Alzheimer's disease tell us about the development of
psychotropic drugs in generalLancet Psychiatry 2015；2(8):756-764).For this in the market
It is badly in need of better efficacy, the lower medicine of side effect.
GDF11 albumen is otherwise known as bone morphogenetic protein (BMP-11, Bone morphogenetic protein
11).Reference sequences number (NCBI Reference Sequence) of the people source GDF11 albumen on NCBI：NP_005802.1.
GDF11 albumen belongs to TGF-β superfamily protein member, and it has expression in many histoorgans, such as：Retina, smell
System, nervous system, pancreas, enteron aisle, kidney, skeletal muscle and heart (C.McPherron, Alexandra.Metabolic
functions of Myostatin and Gdf11.Immunol Endocrinol Metab Agents Med Chem,
Volume10,Number 4,December 2010,pp.217–231.).GDF11 is played in the early embryonic development stage to focus on
Act on, it can adjust the development of the histoorgans such as spinal cord, olfactory nerve, bone, kidney and pancreas.GDF11 can also be significantly improved
The human-subject test of brain, controls myocardial hypertrophy, and lifting muscle metabolism (Li, Q.and Z.-M.Hao, " GDF11:a
New Member of TGF-beta Superfamily."Progress in Biochemistry and Biophysics
2015,42 (7):616-623).GDF11 this effect, is presently considered to be what is realized by TGF-β signal transduction.GDF11 can
Combined with the acceptor (I types and II types) with TGF-β, activation TGF-β downstream signal transduction path (Rochette, L.,
M.Zeller,et al.."Growth and differentiation factor 11(GDF11):Functions in the
regulation of erythropoiesis and cardiac regeneration."Pharmacology&
Therapeutics,2015,156:26-33.)。
Current current research is found：Blood plasma to the young mouse of old mouse continuous injection can lift recognizing for old mouse
Know level (Villeda S.A.Young blood reverses age-related impairments in cognitive
function and synaptic plasticity in mice.Nature Medicine 2014；20:659-663).For
This, can may effectively lift the human-subject test of patients of senile dementia containing certain composition in blood plasma.Further study showed that：
The plasma fraction of old mouse and the plasma fraction of young mouse have significant difference, and maximum of which difference is old mouse
The content of growth and differentiation factor 11 (Growth differentiation factor 11, GDF11) is substantially less than year in blood plasma
Light mouse (Loffredo F.S.Growth Differentiation Factor 11Is a Circulating Factor
that Reverses Age-Related Cardiac Hypertrophy.Cell,2013,153,828-839)。
Although GDF11 is relevant with the human-subject test of brain, its raising to human-subject test is very limited, it is therefore desirable to find
The new drug of senile dementia can preferably be treated or effectively alleviated to a kind of effect.
The content of the invention
For the defect of prior art, an object of the present invention is to provide a kind of growth and differentiation factor through modification.
The second object of the present invention is the preparation method for providing the above-mentioned growth and differentiation factor through modification.
The third object of the present invention is the application for providing the above-mentioned growth and differentiation factor through modification.
The fourth object of the present invention is the purposes for providing the metabolite that the growth and differentiation factor is regulated and controled.
To achieve these goals, present invention employs following technical scheme：
A kind of growth and differentiation factor through modification, is coupled with growth and differentiation factor by trim and is formed, wherein, the growth
Differentiation factor is shown in following (a) or (b) or (c)：(a) the spontaneous growth differentiation 11 of mammal is derived from；(b) by institute
State the amino acid sequence of spontaneous growth differentiation 11 substitution and/or missing by one or more amino acid residues and/or add
Plus and with protein or the polypeptide as derived from spontaneous growth differentiation 11 for promoting neuron regeneration ability；(c) with (a) or
(b) amino acid sequence shown in has at least 50% homology and with promotion neuron regeneration ability by (a) or (b) Suo Shi
Amino acid sequence derived from protein or polypeptide.
In the above-mentioned growth and differentiation factor through modification, as a kind of preferred embodiment, the trim is that label is more
Peptide, plasma protein or the plasma protein fractions.
In the above-mentioned growth and differentiation factor through modification, as a kind of preferred embodiment, the tag polypeptide is His-
Tag, Flag-tag, HA-tag, c-Myc-tag, AVi-tag, MBP-tag, GST-tag, SNAP-tag, Halo-tag or
SUMO-tag。
In the above-mentioned growth and differentiation factor through modification, a kind of preferred embodiment, the growth through modification point are used as
The change factor is coupled by a growth and differentiation factor with one or more tag polypeptides to be formed；Preferably, it is described
Tag polypeptide is coupled by the N-terminal and/or C-terminal of covalent bond and the growth and differentiation factor.
In the above-mentioned growth and differentiation factor through modification, a kind of preferred embodiment, the growth through modification point are used as
The change factor is coupled by a growth and differentiation factor with one or more His-tag to be formed, wherein, the His-tag
Amino acid sequence as shown in SEQ ID NO.1 in sequence table or SEQ ID NO.2.
In the above-mentioned growth and differentiation factor through modification, a kind of preferred embodiment, the growth through modification point are used as
The change factor is coupled by a growth and differentiation factor with one or more GST-tag to be formed, wherein, the GST-tag
Amino acid sequence as shown in SEQ ID NO.3 in sequence table.
In the above-mentioned growth and differentiation factor through modification, a kind of preferred embodiment, the amino of the Flag-tag are used as
Acid sequence is as shown in SEQ ID NO.12 in sequence table, SEQ ID NO.13 in the amino acid sequence of the HA-tag such as sequence table
Shown, the amino acid sequence of the c-Myc-tag is as shown in SEQ ID NO.14 in sequence table, the amino acid of the AVi-tag
Sequence is as shown in SEQ ID NO.15 in sequence table, SEQ ID NO.16 in the amino acid sequence of the MBP-tag such as sequence table
Shown, the amino acid sequence of the SNAP-tag is as shown in SEQ ID NO.17 in sequence table, the amino acid of the Halo-tag
Sequence is as shown in SEQ ID NO.18 in sequence table, SEQ ID NO.19 in the amino acid sequence of the SUMO-tag such as sequence table
It is shown.
In the above-mentioned growth and differentiation factor through modification, as a kind of preferred embodiment, the plasma protein is white egg
In vain, fibrinogen, transferrins, immunoglobulin, the one or more turned in thyroprotein and Thyroid binding protein；
The fragment of the plasma protein is the Fc regions of immunoglobulin.
In the above-mentioned growth and differentiation factor through modification, a kind of preferred embodiment, the growth through modification point are used as
Changing the factor is coupled and is formed with one or more plasma proteins or the plasma protein fractions by a growth and differentiation factor
's；Preferably, the plasma protein or the plasma protein fractions by the N-terminal of covalent bond and the growth and differentiation factor and/
Or C-terminal coupling.
In the above-mentioned growth and differentiation factor through modification, as a kind of preferred embodiment, the growth and differentiation factor is
People source growth and differentiation factor 11, amino acid sequence is as shown in SEQ ID NO.4 in sequence table；Or the growth and differentiation factor is
Mouse source growth and differentiation factor 11, amino acid sequence is as shown in SEQ ID NO.5 in sequence table；Or the growth and differentiation factor is
The active fragment of people source growth and differentiation factor 11, amino acid sequence is as shown in SEQ ID NO.6 in sequence table；Or the growth
Differentiation factor is with the amino acid sequence in sequence table shown in SEQ ID NO.7.
A kind of nucleic acid molecules for encoding the above-mentioned growth and differentiation factor through modification.
A kind of recombinant expression carrier for including above-mentioned nucleic acid molecules.
In above-mentioned recombinant expression carrier, as a kind of preferred embodiment, the recombinant expression carrier is to be based on
The carrier of pET28a (+) plamid vector construction.
A kind of host cell for including above-mentioned nucleic acid molecules or including above-mentioned recombinant expression carrier.
In above-mentioned host cell, as a kind of preferred embodiment, the host cell is Escherichia coli.
The preparation method of the above-mentioned growth and differentiation factor through modification, the trim passes through chemical modification or amalgamation and expression
Mode is coupled with the growth and differentiation factor.
In above-mentioned preparation method, as a kind of preferred embodiment, the amalgamation and expression mode prepares described through modification
The method of growth and differentiation factor include：
The synthesis step of the gene order of the coding growth and differentiation factor through modification；The Growth and Differentiation through modification
The gene order of the factor is connected the step of forming recombinant vector with carrier is carrier；The step of converting of the recombinant vector；After conversion
The screening step of host cell；The expression step of induced fusion albumen；The collection step of fusion protein；And point of fusion protein
From purification step.
In above-mentioned preparation method, as a kind of preferred embodiment, the purification procedures using affinity column,
Cation exchange column and/or molecular sieve.
A kind of pharmaceutical composition, comprising：The above-mentioned growth and differentiation factor through modification or above-mentioned nucleic acid molecules or on
State recombinant expression carrier or above-mentioned host cell, and pharmaceutically acceptable carrier.
In pharmaceutical composition, as a kind of preferred embodiment, the pharmaceutically acceptable carrier is：Pharmaceutically may be used
Various conventional buffer solutions, protein, gelatin, monose, polysaccharide, amino acid, chelating agent, sugar alcohol, polyethylene glycol, the Yi Jibiao received
One or more in the activating agent of face.
A kind of sustained release preparation, comprising：The above-mentioned growth and differentiation factor through modification or above-mentioned nucleic acid molecules or above-mentioned
Recombinant expression carrier or above-mentioned host cell or aforementioned pharmaceutical compositions, and pharmaceutically acceptable biofacies are tolerant
Matter；Preferably, the formulation of the sustained release preparation is liposome, microballoon, hydrogel, Osmotic minipumps or microcapsules.
It is a kind of to be carried comprising the above-mentioned growth and differentiation factor through modification or above-mentioned nucleic acid molecules or above-mentioned recombination expression
The kit of body or above-mentioned host cell or aforementioned pharmaceutical compositions or above-mentioned sustained release preparation.
The above-mentioned growth and differentiation factor through modification or above-mentioned nucleic acid molecules or above-mentioned recombinant expression carrier or on
State host cell or aforementioned pharmaceutical compositions or above-mentioned sustained release preparation or mentioned reagent box prevention, improve or/and
Treat the application in senile dementia.
The above-mentioned growth and differentiation factor through modification or above-mentioned nucleic acid molecules or above-mentioned recombinant expression carrier or on
Stating host cell or aforementioned pharmaceutical compositions or above-mentioned sustained release preparation or mentioned reagent box is improving or is treating cognitive
Application in malfunction medicine.
In above-mentioned application, as a kind of preferred embodiment, the cognitive function is not normal to refer to the note related to aging
Recall power decline.
A kind of method for preventing, improving or treating senile dementia, this method is given always by certain method of administration
The above-mentioned growth and differentiation factor through modification of dementia disease patient or above-mentioned nucleic acid molecules or above-mentioned recombinant expression carrier or
The above-mentioned host cell of person or aforementioned pharmaceutical compositions or above-mentioned sustained release preparation.
A kind of to improve or the not normal method for the treatment of cognitive function, this method is to give cognitive work(by certain method of administration
Can the not normal above-mentioned growth and differentiation factor through modification of people or above-mentioned nucleic acid molecules or above-mentioned recombinant expression carrier or
Above-mentioned host cell or aforementioned pharmaceutical compositions or above-mentioned sustained release preparation.
In above-mentioned prevention, the method for improvement or treatment senile dementia and improvement or the not normal method for the treatment of cognitive function
In, it is characterised in that the method for administration is oral, schneiderian membrance administration, intravenous injection, intramuscular injection, drip-feed, abdominal cavity are noted
Penetrate, arteria hepatica injection or subcutaneous embedding.
A kind of method of extend above-mentioned growth and differentiation factor half-life period in vivo, the growth and differentiation factor is modified and prepared
Into the above-mentioned growth and differentiation factor through modification or above-mentioned nucleic acid molecules or above-mentioned recombinant expression carrier or above-mentioned host
Cell or aforementioned pharmaceutical compositions or above-mentioned sustained release preparation.
The metabolite that above-mentioned growth and differentiation factor (unmodified) is regulated and controled is in diagnosis and examination senile dementia disease
In purposes.Preferably, the metabolite that the growth and differentiation factor is regulated and controled is the one or more in following metabolite：
Propionyl Propionyl AC (C3), lysophosphatidyl choline LysoPC a C18:2nd, phosphatidyl PC aa C36:6th, phosphatidyl
C16:1-OH, phosphatidyl PC aa C38:0th, phosphatidyl PC aa C38:6th, phosphatidyl PC aa C40:1st, phosphatidyl PC aa
C40:2nd, phosphatidyl PC aa C40:6th, phosphatidyl PC ae C40:6.
Whether a kind of determination tested patients are with senile dementia or with the method for suffering from senile dementia risk, bag
One or more concentration in above-mentioned metabolite in the blood sample for detecting the subject are included, if the metabolite
Concentration meets or exceeds threshold value set in advance, then judges the subject with senile dementia or with suffering from senile dementia
The risk of disease.Preferably, the concentration of the metabolite is that the method for adopting metabolism group is determined.
Compared to prior art, the present invention has the advantages that：
1st, the growth and differentiation factor through modification that the present invention is provided effectively extends on the premise of its basic function is ensured
Its half-life period in vivo.
2nd, while compared to literalness growth and differentiation factor 11 or its active fragment, Growth and Differentiation of the present invention through modification
The factor has preferably biological activity, can be preferably applied to prevent, improve and treat senile dementia, and it can be with addition
Play a part of strengthening memory.
3rd, the compound through modification and the medicine comprising the compound that the present invention is provided have the stability being metabolized in vivo,
It is used directly for or prevents, improves and treat senile dementia as pharmaceutical composition composition.
4th, growth and differentiation factor of the present invention is substantially reduced in old dementia patients in-vivo content, and signal leads to downstream
The content and activity of the relevant enzyme on road are reduced, and cause a variety of phosphatidyls and metabolism of carnitine product assay to reduce.This small point of metabolism
The change of sub- content can effectively predict the risk of senile dementia lurker, and senile dementia disease can be aided in
Diagnosis and examination.I.e. the downstream metabolites of growth and differentiation factor can be used for the auxiliary diagnosis of senile dementia.
Brief description of the drawings
Fig. 1 is the active fragment of coding His-tag and human growth and differentiation factor 7 11 synthesized by full genome synthetic method
The genome of the coupled product of (there is the amino acid sequence shown in SEQ ID No.6 in sequence table) N-terminal；
Fig. 2 is the active fragment of coding GST-tag and human growth and differentiation factor 7 11 synthesized by full genome synthetic method
The genome of the coupled product of (there is the amino acid sequence shown in SEQ ID No.6 in sequence table) N-terminal；
Fig. 3 is the His-tag with amino acid sequence shown in SEQ ID No.2 in sequence table in sequence table in embodiment 2
With human growth and differentiation factor 7 (i.e. human growth and differentiation factor 7 11 with the amino acid sequence in sequence table shown in SEQ ID No.6
Active fragment) N-terminal be coupled after with affinity chromatography purify obtained eluent SDS electrophoresis detections result.
Purifying refers to the supernatant containing coupled product that embodiment 1 is obtained crossing nickel post, finally with the 5mM Tris of the imidazoles containing 400mM
Hydrochloride buffer (pH8.0) elution pillar obtains eluent.Swimming lane 1：Protein molecular weight standard；Swimming lane 2-10 is coupling production
Thing passes through the eluent of ni-sepharose purification.
Fig. 4 is GST-tag and the life with the amino acid sequence shown in SEQ ID No.6 in sequence table in embodiment 4
The N-terminal of long differentiation factor (i.e. the active fragment of human growth and differentiation factor 7 11) is purified after being coupled with affinity chromatography
Eluent SDS electrophoresis detections result.Purifying refer to the supernatant containing coupled product for obtaining embodiment 3 cross containing
The affine pillar of fixed glutathione, finally with the 5mM Tris hydrochloride buffers of the glutathione dissociated containing 25mM
(pH8.0) pillar is eluted, eluent is obtained.Swimming lane 1：Protein molecular weight standard；Swimming lane 2-10 is coupled product by parent
The eluent purified with pillar.
Fig. 5 be in embodiment 6 20kD polyethylene glycol with the amino acid sequence shown in SEQ ID No.6 in sequence table
The N-terminal of human growth and differentiation factor 7 (i.e. the active fragment of human growth and differentiation factor 7 11) uses anion column SourceQ after being coupled
Purify the result of the SDS electrophoresis detections of obtained eluent.Purifying refers to the reaction solution mistake containing coupled product
SourceQ pillars, finally carry out gradient elution with the 5mM Tris hydrochloride buffers (pH8.0) containing 500mM sodium chloride, so that
Collect the eluent containing unit point modified outcome.Swimming lane 1：Protein molecular weight standard；Swimming lane 2-10 passes through for coupled product
The eluent of SourceQ purifying.
Fig. 6 be in embodiment 8 40kD polyethylene glycol with the amino acid sequence shown in SEQ ID No.6 in sequence table
The N-terminal of human growth and differentiation factor 7 (i.e. the active fragment of human growth and differentiation factor 7 11) uses anion column SourceQ after being coupled
Purify the result of the SDS electrophoresis detections of obtained eluent.Purifying refer to by embodiment 7 obtain containing coupled product
Reaction solution crosses SourceQ pillars, finally carries out gradient with the 5mM Tris hydrochloride buffers (pH8.0) containing 500mM sodium chloride
Elution, so as to collect the eluent containing unit point modified outcome.Swimming lane 1：Protein molecular weight standard；Swimming lane 2-10 is coupling
Product passes through the eluent that SourceQ is purified.
During Fig. 7 is embodiment 9, Examples 1 and 2 prepare after modification growth and differentiation factor (i.e. chemical compounds I or
His-tag-GDF11 the value-added activity figure of primary hippocampal cells) is promoted.
During Fig. 8 is embodiment 12, embodiment 3 and 4 prepare after modification growth and differentiation factor (i.e. compound ii or
GST-tag-GDF11 the value-added activity figure of primary hippocampal cells) is promoted.
During Fig. 9 is embodiment 15, embodiment 5 and 6 prepare after modification growth and differentiation factor (i.e. compound III or
20KD polyethylene glycol-GDF11) promote the value-added activity figure of primary hippocampal cells.
During Figure 10 is embodiment 18, embodiment 7 and 8 prepare after modification growth and differentiation factor (i.e. compounds Ⅳ or
40KD polyethylene glycol-GDF11) promote the value-added activity figure of primary hippocampal cells.
During Figure 11 is embodiment 10, chemical compounds I promotes the activity figure of primary hippocampal cells Synaptic formation.
During Figure 12 is embodiment 13, compound ii promotes the activity figure of primary hippocampal cells Synaptic formation.
During Figure 13 is embodiment 16, compound III promotes the activity figure of primary hippocampal cells Synaptic formation.
During Figure 14 is embodiment 19, compounds Ⅳ promotes the activity figure of primary hippocampal cells Synaptic formation.
During Figure 15 is embodiment 20, chemical compounds I, the lifting activity figure of II, III and IV pair of old mouse human-subject test.(a)
Time used in finding hiding platform changes over time curve；(b) time spent in target quadrant.
During Figure 16 is embodiment 21, chemical compounds I, the lifting activity figure of II, III and IV pair of senile dementia mouse human-subject test.
(a) time used in finding hiding platform changes over time curve；(b) time spent in target quadrant.
During Figure 17 is embodiment 10, the image of the primary hippocampal cells after different disposal.Toward the training of primary hippocampal cells
Support and be separately added into the chemical compounds I of equimolar amounts and the life with amino acid sequence shown in SEQ ID No.6 in sequence table in base
Long differentiation factor is GDF11 active fragments as experimental group.Control group hippocampal cell only adds DMEM culture mediums.Micro- after one day
Hippocampal cell is imaged under mirror.In figure, (a), (b), (c) is chemical compounds I, GDF11 active fragments and DMEM processing respectively
Primary hippocampal cells afterwards.
Figure 18 be embodiment 22 in, GDF11 content pair in normal population and senile dementia human albumin's extract
Than.
During Figure 19 is embodiment 23, the changes of contents contrast table of normal population and 10 kinds of senile dementia patients metabolism small molecule
Lattice.
Embodiment
In order to preferably be illustrated to the technical characteristic and effect of the present invention, below using embodiment to this hair
It is bright to be described in detail, but the present invention is not limited thereto.
The present invention provides a kind of growth and differentiation factor (the alternatively referred to as compound through modification) through modification, and the compound is
Compound after growth and differentiation factor is modified, it is coupled with growth and differentiation factor by trim and formed.The Growth and Differentiation because
Son is shown in following (a) or (b) or (c)：(a) the spontaneous growth differentiation 11 of mammal is derived from；(b) will be described natural
The amino acid sequence of growth and differentiation factor 11 by one or more amino acid residues (such as 2,8,10,15,20,
30,40,50,60) substitution and/or missing and/or addition and with promote neuron regeneration ability by natural
Protein or polypeptide derived from growth and differentiation factor 11；(c) there is at least 50% (ratio with the amino acid sequence shown in (a) or (b)
Such as 50%, 60%, 70%, 80%, 90%, 95%, 99%) homology and with promote neuron regeneration ability by (a) or
(b) protein or polypeptide derived from the amino acid sequence shown in.It is natural in the spontaneous growth differentiation 11 to refer to life all
It is coming or self-assembling formation.
Heretofore described growth and differentiation factor can be the natural growth point of people source, mouse source or other mammals
Change the factor 11, the growth and differentiation factor homology of its separate sources can reach 83.5%；It can also be spontaneous growth differentiation factor
11 amino acid sequence by one or more amino acid residues substitution and/or missing and/or addition and with promoting neuron
Protein or the polypeptide as derived from spontaneous growth differentiation 11 of power of regeneration, such as：The work of these growth and differentiation factors 11
Property fragment；Or there is more than 50% homology and with promotion neuron regeneration energy with these growth factors or active fragment
The protein or polypeptide that are derived by these growth and differentiation factor 11 or active fragments of power.When growth and differentiation factor 11 is people
Source growth and differentiation factor 11, amino acid sequence is as shown in SEQ ID NO.4 in sequence table；The growth and differentiation factor 11 is mouse source
Growth and differentiation factor 11, amino acid sequence is as shown in SEQ ID NO.5 in sequence table.The active fragment of growth and differentiation factor 11 is
Equally there is from growth and differentiation factor 11 and with growth and differentiation factor 11 peptide fragment for promoting neuron regeneration ability
(Katsimpardi L.Science 2014；344,630-634), such as SEQ ID NO.6 in sequence table, the SEQ ID
NO.6 amino acid sequence people source growth and differentiation factor 11 and the common ground of mouse source growth and differentiation factor 11, implementation of the invention
Example also further demonstrate amino acid sequence polypeptide shown in SEQ ID NO.6 have promote neuron regeneration ability (referring to Fig. 7-
14)；The growth and differentiation factor, which can also be with people source growth and differentiation factor 11, has more than 70% homology and with promotion
Protein in neuron regeneration ability protein or polypeptide, such as sequence table shown in SEQ ID NO.7, its amino acid sequence is
The common ground of people source Growth differentiation factor 8 and mouse source Growth differentiation factor 8.
The growth and differentiation factor through modification is even by an above-mentioned growth and differentiation factor and one or more trims
What connection was formed, coupling can be that covalent bond can also be non-covalent bond；It can be repaiied by chemical modification or amalgamation and expression to realize
The coupling of jewelry and growth and differentiation factor.
The trim can be tag polypeptide, plasma protein, the plasma protein fractions, macromolecule or small molecule.
In the present invention, the polypeptide refers to by the ol cpds of 2~100 amino acid molecular dehydrating condensations, its
Molecular weight is less than 10,000Da.The protein is formed by α amino and α carboxyls between the alpha amino acid encoded by nucleic acid
The peptide chain that peptide bond is formed by connecting, translated post-processing and generate have specific stereochemical structure, active macromolecular.High score
Son refer to beyond isolating protein by the main relative molecular weight with Covalent bonding together of numerous atoms or atomic group 10,000
Compound above.Small molecule refers to the molecule that molecular weight is less than 500.
When the trim is tag polypeptide, it is preferable that the tag polypeptide is His-tag, and (it has Flag-tag
Amino acid sequence in sequence table shown in SEQ ID NO.12), (it has in sequence table shown in SEQ ID NO.13 HA-tag
Amino acid sequence), c-Myc-tag (it has the amino acid sequence in sequence table shown in SEQ ID NO.14), AVi-tag (its
With the amino acid sequence in sequence table shown in SEQ ID NO.15), (it has SEQ ID NO.16 in sequence table to MBP-tag
Shown amino acid sequence), GST-tag, (it has the amino acid sequence in sequence table shown in SEQ ID NO.17 to SNAP-tag
Row), Halo-tag (it has the amino acid sequence in sequence table shown in SEQ ID NO.18), (it has sequence to SUMO-tag
Amino acid sequence in table shown in SEQ ID NO.19).It is highly preferred that the tag polypeptide is His-tag, its amino acid sequence
As shown in SEQ ID NO.1 in sequence table or SEQ ID NO.2, a label H is-tag by covalent bond be peptide bond with
N-terminal and/or the C-terminal coupling of the growth and differentiation factor.It is highly preferred that the tag polypeptide is GST-tag, its amino acid sequence
As shown in SEQ ID NO.3 in sequence table, a tag polypeptide GST-tag is peptide bond and the growth point by covalent bond
Change N-terminal and/or the C-terminal coupling of the factor.The molecular weight of albumen in itself can be increased plus above-mentioned label, metabolic rate can be reduced；
In addition, the stability of albumen can be increased in expression；Can also finally albumen be promoted to be folded in the correct way.
When the trim is the fragment of plasma protein or plasma protein, it is preferable that the plasma protein be albumin,
Fibrinogen, transferrins, immunoglobulin, the one or more turned in thyroprotein and Thyroid binding protein；Institute
The fragment for stating plasma protein is the Fc regions of immunoglobulin.The fragment of one plasma protein or plasma protein passes through covalent
Key is that the N-terminal and/or C-terminal of peptide bond and the growth and differentiation factor are coupled.
When the trim is the fragment of tag polypeptide or plasma protein or plasma protein, can by chemical modification or
The mode of amalgamation and expression realizes the coupling of trim and the growth and differentiation factor, that is, prepares the growth through modification point of the invention
Change the factor.
The method that the amalgamation and expression mode prepares the growth and differentiation factor through modification, includes successively：
The synthesis step of the gene order of the coding growth and differentiation factor through modification；The Growth and Differentiation through modification
The gene order of the factor is connected the step of forming recombinant vector with carrier is carrier；The step of converting of the recombinant vector；After conversion
The screening step of host cell；The expression step of induced fusion albumen；The collection step of fusion protein；The separation of fusion protein is pure
Change step.Preferably, the purification procedures use affinity column, cation exchange column and/or molecular sieve.
When the trim is macromolecule, macromolecule is pharmaceutically conventional macromolecule, it is preferable that the macromolecule is
Polyethylene glycol (PEG)；The polyethylene glycol is linear or bifurcated molecule, and molecular weight is 1-100KD, it is highly preferred that described poly-
The molecular weight 5-40KD of ethylene glycol, further, the polyethylene glycol are monomethyl polyethylene glycol.PEG can by covalent bond with
The free amino of growth and differentiation factor, sulfydryl or carboxyl coupling.Trim and life can be realized by way of chemical modification
The coupling of long differentiation factor, that is, prepare the growth and differentiation factor of the invention through modification.The chemical modification mode prepares the warp
The method of the growth and differentiation factor of modification includes：Blend step：By the growth and differentiation factor and trim with appropriate mole
Than mixing；Reactions steps：Reaction reagent is added into mixed solution, is chemically reacted, so as to obtain reaction solution；Purifying
Step：The reaction solution is purified, so as to obtain the growth and differentiation factor through modification.Preferably, it is described to chemically react
The pH value that reaction solution is controlled in journey is 3-10；The purification step uses affinity column, cation exchange column and/or molecule
Sieve.If it is intended to trim carries out unit point modification to growth and differentiation factor, then need to modify polyethylene glycol from special unit point
(polyethylene glycol of single-point modification is entitled：MPEG-butyrALD, buys from the triumphant positive biology in Beijing, article No. is KZ-SC) come real
Existing.
When the trim is small molecule, it is preferable that the small molecule is carbohydrate, phosphoric acid, free group such as methyl, second
Base or phenyl ring.Small molecule is coupled by the free amino in covalent bond and growth and differentiation factor, sulfydryl or carboxyl, and different is small
Molecule coupling labeled group may be different.I.e. growth and differentiation factor is glycosylated, phosphorylation, acylation, ethylizes or methylate.Specifically
Preparation method be enzymic catalytic reaction method：Growth and differentiation factor is mixed with small numerator modified thing, corresponding enzymatic is added anti-
Should.
When trim is the fragment of tag polypeptide, plasma protein or plasma protein, the present invention also provides coding above-mentioned warp
The nucleic acid molecules of the growth and differentiation factor of modification, including DNA molecular and RNA molecule.Its nucleic acid molecules can be by full genome
The method of synthesis is obtained, and the nucleic acid molecules of trim can also will be encoded by designing identical restriction enzyme site, with coding blood plasma
The nucleic acid molecules of the fragment of albumen or plasma protein link together.
When trim is the fragment of tag polypeptide, plasma protein or plasma protein, the present invention also provides and includes above-mentioned core
The recombinant expression carrier of acid molecule.Preferably, the recombinant expression carrier is to be based on pET28a (+), pET15b, pGEX-6p-1,
The carrier of the plamid vector constructions such as pGEX-4T-1.
When trim is the fragment of tag polypeptide, plasma protein or plasma protein, the present invention also provides and includes above-mentioned core
Acid molecule or the host cell for including above-mentioned recombinant expression carrier.Preferably, the host cell is Escherichia coli
(Escherichia coli)。
Present invention also offers a kind of pharmaceutical composition, comprising：The above-mentioned growth and differentiation factor through modification, above-mentioned nucleic acid point
Sub, above-mentioned recombinant expression carrier or above-mentioned host cell；And pharmaceutically acceptable carrier.The pharmaceutically acceptable carrier
For：Pharmaceutically acceptable various conventional buffer solutions are (such as：Organic acid buffer liquid such as citrate buffer solution, inorganic acid buffer
Such as phosphate buffer, physiological saline), protein (such as albumin and immunoglobulin), gelatin, monose, polysaccharide, amino acid, chela
One in mixture (such as EDTA), sugar alcohol (such as mannitol), polyethylene glycol and surfactant (such as TWEEN and PLURONICS)
Plant or a variety of.
Preferably, described pharmaceutical composition, is counted using cumulative volume as 100 microlitres, including following component：0.01-0.2M phosphoric acid
Buffer solution (pH 7.0), 0.01-0.1M glucose, 0.001-0.05M mannitol, the Growth and Differentiation through modification described in 1-5 micrograms
The factor.It is highly preferred that described pharmaceutical composition, is counted using cumulative volume as 100 microlitres, including following component：0.1M phosphate buffers
(pH 7.0), 0.05M glucose, 0.01M mannitol, 2 microgram growth and differentiation factors.
The formulation of described pharmaceutical composition can be：Tablet, capsule, oral liquid, syrup, particle, dripping pill；It is liquid drugs injection, lyophilized
Powder pin, aseptic powder injection, injection liquor, spray agent.Preferably parenteral solution formulation or spray agent.
Described pharmaceutical composition can be administered as follows：Orally, schneiderian membrance administration, intravenous injection, intramuscular injection,
Drip-feed, intraperitoneal injection, arteria hepatica injection and subcutaneous embedding.Unit dosage form is：Growth through modification described in 1 microgram point
Change the factor/kg body weight.
Present invention also offers a kind of sustained release preparation, comprising：The above-mentioned growth and differentiation factor through modification, above-mentioned nucleic acid point
Sub, above-mentioned recombinant expression carrier, above-mentioned host cell or aforementioned pharmaceutical compositions；And pharmaceutically acceptable biofacies
Tolerant matter；Preferably, the formulation of the sustained release preparation is liposome, microballoon, hydrogel, Osmotic minipumps or microcapsules.
The above-mentioned growth and differentiation factor through modification, above-mentioned nucleic acid molecules, above-mentioned restructuring are included present invention also offers a kind of
Expression vector, above-mentioned host cell, the kit of aforementioned pharmaceutical compositions or above-mentioned sustained release preparation.It can also be wrapped in kit
Include other materials, such as negative control or positive control etc..
The above-mentioned growth and differentiation factor through modification, above-mentioned nucleic acid molecules, above-mentioned recombinant expression carrier, above-mentioned host cell,
Aforementioned pharmaceutical compositions, above-mentioned sustained release preparation or mentioned reagent box are in prevention, improvement or/and treatment senile dementia
Application.
The above-mentioned growth and differentiation factor through modification, above-mentioned nucleic acid molecules, above-mentioned recombinant expression carrier, above-mentioned host cell,
Aforementioned pharmaceutical compositions, above-mentioned sustained release preparation or mentioned reagent box answering in improving or treating cognitive function arrhythmic agents
With.The cognitive function is not normal to refer to the failure of memory related to aging.
A kind of method for preventing, improving or treating senile dementia, this method is given always by certain method of administration
The above-mentioned growth and differentiation factor through modification of dementia disease patient, above-mentioned nucleic acid molecules, above-mentioned recombinant expression carrier, above-mentioned host are thin
Born of the same parents, aforementioned pharmaceutical compositions, above-mentioned sustained release preparation.
A kind of to improve or the not normal method for the treatment of cognitive function, this method is to give cognitive work(by certain method of administration
The not normal above-mentioned growth and differentiation factor through modification of people of energy, above-mentioned nucleic acid molecules, above-mentioned recombinant expression carrier, above-mentioned host are thin
Born of the same parents, aforementioned pharmaceutical compositions, above-mentioned sustained release preparation.
Preferably, the method for administration is oral, schneiderian membrance administration, intravenous injection, intramuscular injection, drip-feed, abdominal cavity
Injection, arteria hepatica injection or subcutaneous embedding.
A kind of method for extending above-mentioned growth and differentiation factor Half-life in vivo, growth and differentiation factor modification is prepared into
The above-mentioned growth and differentiation factor through modification, above-mentioned nucleic acid molecules, above-mentioned recombinant expression carrier, above-mentioned host cell, said medicine
Composition or above-mentioned sustained release preparation.
Below by embodiment prepared by the compound in the present invention and application is described in detail.
The various culture mediums and common agents used in the present invention are prepared using conventional method, point being related in embodiment
For example unreceipted specific experimental condition of sub- biologic operation and method, refer to SambrookJ etc. and edit, Science Press, and 2002,
The specification of Molecular Cloning:A Laboratory guide (third edition) or corresponding product.
Embodiment 1：What the N-terminal progress coupling of His-tag and sub 11 active fragments of human growth and differentiation factor 7 was prepared contains
His-tag-GDF11 (chemical compounds I) liquid
His-tag-GDF11 manufactured in the present embodiment, its amino acid sequence referring to sequence table SEQ ID NO.8, wherein
His-tag has the amino acid sequence shown in SEQ ID NO.2, and the active fragment of human growth and differentiation factor 7 11 has SEQ ID
C-terminal is that free carboxy on histidine is coupled to human growth and differentiation factor 7 in amino acid sequence shown in NO.6, His-tag sequences
On the alpha- amino of N-terminal, peptide bond is formed.
The specific preparation method of the present embodiment chemical compounds I is as follows：
(1) gene of the method composite coding chemical compounds I synthesized by full genome：The gene has SEQ ID in sequence table
Base sequence shown in NO.10, the gene contains (referring to Fig. 1 and SEQ ID NO.10) successively from 5 ' ends to 3 ' ends：Nco I enzymes
Enzyme site (the 1st the-the 6 base of base), the nucleotide sequence (the 7th the-the 32 base of base) for encoding His-tag, encoding human growth
The nucleotide sequence (the 33rd the-the 362 base of base) of the active fragment of differentiation 11, Xho I restriction enzyme site (the 363rd bases-the
368 bases)；This step is completed by Sangon Biotech (Shanghai) Co., Ltd., and the gene is connected to pUC57 plasmid vectors
On.
(2) by the pUC57 plasmids of the gene (there is the base sequence shown in SEQ ID NO.10) containing coded compounds I
Carrier carries out restriction enzyme cleavage：Restriction enzyme is Nco I and Xho I, and cleavage reaction temperature is 37 DEG C, and the time is
2 hours, the gene content of cleavage reaction was 50ng；Obtain small fragment：Gene order A (i.e. the genes of coded compounds I)；(3)
PET28a (+) plasmid vector is subjected to double digestion with Nco I and Xho I：Cleavage reaction temperature is 37 DEG C, and the time is 4 hours, is cut
The plasmid content for cutting reaction is 50ng；Obtain large fragment：The Frame sequence B of plasmid；(4) digestion products purifying and connection：Respectively
Agarose electrophoresis purifying is carried out to the Frame sequence B of said gene sequence A and plasmid, by gene order A and plasmid after purification
Frame sequence B be attached with T4 ligases, condition of contact be 16 DEG C overnight, gene order A and plasmid in coupled reaction
Frame sequence B mol ratio is 4：1, obtain connection product；
(5) connection product is converted into e. coli bl21 (DE3), the condition of conversion is 42 DEG C of thermal shocks 90 seconds；(6) will conversion
Escherichia coli afterwards are uniformly applied to the solid LB flat boards containing ammonia benzyl chloramphenicol resistance, and (wherein the content of ammonia benzyl mycin is 50 micro-
Grams per milliliter) on, 37 DEG C of incubated overnights now can grow many bacterium colonies on LB flat boards；(7) 4 to 5 bacterium colonies are chosen with toothpick to 1 liter
LB liquid medium (wherein the content of ammonia benzyl mycin is 50 mcg/mls) containing ammonia benzyl chloramphenicol resistance is inner, and 37 DEG C are rocked training
Support, the speed setting rocked is 220rpm.Culture to bacterium solution 600nm place absorbance be 0.4-0.6 between when, addition IPTG lure
Protein expression is led, the IPTG ultimate densities of addition are 0.6mM；The condition of inducible protein expression is 37 DEG C, 220rpm；
(8) induce after 5 hours, Escherichia coli are collected by centrifugation, centrifugal condition is 1500g, 15 minutes；
(9) Escherichia coli of collection are suspended with 5mM Tris hydrochloride buffers, then carries out ultrasonication, finally by
12000g is centrifuged in 15min precipitating residues, the supernatant now obtained containing a large amount of His-tag and the work of human growth and differentiation factor 7 11
Property the compound i.e. His-tag-GDF11 (compound I) that is coupled in N-terminal of fragment, its supernatant can directly upper nickel post enter
The purifying of row embodiment 2.
Embodiment 2：Purified after the N-terminal of the active fragment of His-tag modified humans growth and differentiation factor 11 with nickel post
His-tag inside the present embodiment has the amino acid sequence shown in SEQ ID NO.2, human growth and differentiation factor 7 tool
There is the amino acid sequence shown in SEQ ID NO.6, His-tag is coupled to the N-terminal of the active fragment of growth and differentiation factor 11.Specifically
Ground, the growth and differentiation factor of the His-tag modifications of the present embodiment is the His-tag-GDF11 that embodiment 1 is obtained.
The present embodiment specific implementation method is followed successively by：
(1) nickel post is balanced：Containing 2 milliliters of glucan pearls in nickel post, with 20 milliliters of 5mM Tris hydrochloric acid
(pH8.0) pillar balance was carried out；
(2) supernatant containing coupled product that embodiment 1 is obtained is crossed into nickel post, flow velocity is 2ml/min, coupling here
Product is the growth and differentiation factor that N-terminal is coupled with His-tag；
(3) pillar is rinsed with the 5mM Tris hydrochloride buffers (pH8.0) containing 20mM imidazoles, flow velocity is 2ml/min, is gone
Except foreign protein；
(4) pillar is eluted with the 5mM Tris hydrochloride buffers (pH8.0) containing 400mM imidazoles again, flow velocity is 2ml/
Min, collects the growth and differentiation factor being coupled in eluent, eluent containing a large amount of N-terminals with His-tag, as a result referring to Fig. 3, even
The size of co-product is 13kDa.
Embodiment 3：Sub 11 active fragments of GST-tag and human growth and differentiation factor 7 N-terminal carry out coupling prepare containing
GST-tag-GDF11 (compound ii) liquid
GST-tag inside the present embodiment has the amino acid sequence shown in SEQ ID NO.3, human growth and differentiation factor 7 11
Active fragment has an amino acid sequence shown in SEQ ID NO.6, and the carboxyls of GST-tag C-terminals is that peptide bond is coupled by covalent bond
To the N-terminal of sub 11 active fragments of human growth and differentiation factor 7, coupled product is GST-tag-GDF11, the amino acid sequence of the modified protein
Row are as shown in SEQ ID NO.9.
The present embodiment specific implementation method is as follows：
(1) the method composite coding GST-tag-GDF11 synthesized by full genome gene：The gene has in sequence table
Base sequence shown in SEQ ID NO.11, the gene contains (referring to Fig. 2 and SEQ ID NO.11) successively from 5 ' ends to 3 ' ends：
Nco I restriction enzyme sites (the 1st the-the 6 base of base), the nucleotide sequence (the 7th the-the 660 base of base) for encoding GST-tag, volume
The nucleotide sequences (the 661st the-the 990 base of base) of sub 11 active fragments of code human growth and differentiation factor 7, Xho I restriction enzyme sites (the
991 the-the 996 bases of base)；This step is completed by Sangon Biotech (Shanghai) Co., Ltd., and the gene is connected to
On pUC57 plasmid vectors.
(2) gene comprising coding GST-tag-GDF11 (had into the base sequence shown in SEQ ID NO.11 in sequence table
Row) pUC57 plasmid vectors carry out restriction enzyme cleavage：Restriction enzyme is Nco I and Xho I, cleavage reaction temperature
Spend for 37 DEG C, the time is 2 hours, the gene content of cleavage reaction is 50ng；Obtain small fragment：Gene order A (coding GST-
Tag-GDF11 gene)；
(3) pET28a (+) plasmid vector is subjected to double digestion with Nco I and Xho I：Cleavage reaction temperature is 37 DEG C, when
Between be 4 hours, the plasmid content of cleavage reaction obtains large fragment for 50ng：The Frame sequence B of plasmid；
(4) purifying and connection of the gene and plasmid after limitation cleavage：Respectively to said gene sequence A and the frame of plasmid
Frame sequence B carries out agarose electrophoresis purifying, and gene order A and the Frame sequence B of plasmid after purification is carried out with T4 ligases
Connection, condition of contact is 16 DEG C and stayed overnight that the Frame sequence B of gene order A and plasmid mol ratio is 4 in coupled reaction：1, obtain
To connection product；
(5) connection product is converted into e. coli bl21 (DE3), the condition of conversion is 42 DEG C of thermal shocks 90 seconds；
(6) Escherichia coli after conversion are uniformly applied to solid LB flat boards (the wherein ammonia benzyl containing ammonia benzyl chloramphenicol resistance
The content of mycin is 50 mcg/mls) on, 37 DEG C of incubated overnights now can grow many bacterium colonies on LB flat boards；
(7) 4 to 5 bacterium colonies are chosen with toothpick to 1 liter of LB liquid medium (wherein ammonia benzyl mycin containing ammonia benzyl chloramphenicol resistance
Content be 50 mcg/mls) it is inner, 37 DEG C are rocked culture, the speed setting rocked be 220rpm.Culture is to bacterium solution in 600nm
When locating absorbance between 0.4-0.6, the expression of IPTG inducible proteins is added, the IPTG ultimate densities of addition are 0.6mM；Induce egg
The condition expressed in vain is 37 DEG C, 220rpm；
(8) induce after 5 hours, Escherichia coli are collected by centrifugation, centrifugal condition is 1500g, 15 minutes；
(9) Escherichia coli of collection are suspended with 5mM Tris hydrochloride buffers, then carries out ultrasonication, finally by
12000g is centrifuged in 15min precipitating residues, supernatant now containing a large amount of GST-tag and sub 11 active tablets of human growth and differentiation factor 7
The compound i.e. GST-tag-GDF11 (compound ii) that section is coupled in N-terminal, supernatant is used directly for purifying.
Embodiment 4：With containing the affine of glutathione after the N-terminal of the active fragment of GST-tag modified humans growth and differentiation factor 11
Post is purified.
GST-tag inside the present embodiment has SEQ ID NO.3 sequences, and human growth and differentiation factor 7 11 active fragments of son have
SEQ ID NO.6 sequences, GST-tag is coupled to the N-terminal of growth and differentiation factor.Specifically, the His-tag of the present embodiment is modified
Growth and differentiation factor is the GST-tag-GDF11 that embodiment 3 is obtained.
The present embodiment specific implementation method is followed successively by：
(1) affinity column containing mating type glutathione is balanced：The affinity column of QIAGEN mating type glutathione
In containing 2 milliliters of glucan pearls, pillar balance was carried out with 20 milliliters of 5mM Tris hydrochloric acid (pH8.0)；
(2) supernatant containing coupled product prepared by embodiment 3 is crossed into pillar, flow velocity is 2ml/min, coupling here
Product is the growth and differentiation factor that N-terminal is coupled with GST-tag；
(3) pillar is rinsed with the 5mMTris hydrochloride buffers (pH8.0) containing 200mM sodium chloride, flow velocity is 2ml/min,
Remove foreign protein；
(4) pillar is eluted with the 5mM Tris hydrochloride buffers (pH8.0) containing the 25mM glutathione dissociated, flow velocity is
2ml/min, collects the growth and differentiation factor being coupled in eluent, eluent containing a large amount of N-terminals with GST-tag, purification result ginseng
See Fig. 4, the size of coupled product is 38kDa.
Embodiment 5：20kDa polyethylene glycol carries out coupling in N-terminal with sub 11 active fragments of human growth and differentiation factor 7 and prepared
Liquid containing 20kDa polyethylene glycol-GDF11 (compound III)
Sub 11 active fragments of human growth and differentiation factor 7 that the present embodiment is used have the amino acid sequence shown in SEQ ID NO.6
Row.
The present embodiment specific implementation method is as follows：
(1) take sub 11 active fragments of 2 microlitres of human growth and differentiation factor 7s (i.e. with the amino acid sequence shown in SEQ ID NO.6,
Purchased from PeproTech, 120-11), protein concentration is measured at a wavelength of 280 nm, and 5mg/ml then is arrived into concentration regulation；
(2) sub 11 active fragments of human growth and differentiation factor 7 that 10ml concentration is 5mg/ml are taken, 20kDa is added thereto is used for egg
(mPEG-ButyrALD, molecular weight 20kDa, Nektar, the dressing agent can only be with albumen N for the polyethylene glycol that white N-terminal is specifically modified
The alpha amino coupleds of end dissociative) solid 100mg, is stirred to being completely dissolved, polyethylene glycol here is used for spy in room temperature condition
The N-terminal of the different active fragment of modified human growth and differentiation factor 11, the polyethylene glycol and sub 11 active fragments of above-mentioned human growth and differentiation factor 7
The mol ratio of mixing is 5:1, obtain the mixture of 20kDa polyethylene glycol and sub 11 active fragments of human growth and differentiation factor 7；
(3) reducing agent sodium cyanoborohydride (CH3BNNa) is added into mixture, and (reducing agent is in the mixture after addition
Concentration is 50mM), it is stored at room temperature 24 hours, the obtained reaction solution containing 20kDa polyethylene glycol-GDF11.Now more than 90%
Sub 11 active fragments of human growth and differentiation factor 7 by single polyethyleneglycol modified, i.e., one polyethylene glycol and a growth and differentiation factor
11 molecules are coupled, and conjugation sites are the alpha-amidos at growth and differentiation factor 11N ends.At this moment, reaction solution is used directly for
Anion-exchange column is purified.
Embodiment 6：Anion column is used after the N-terminal of sub 11 active fragments of the polyethyleneglycol modified human growth and differentiation factor 7s of 20kDa
SourceQ is purified
The reaction solution containing 20kDa polyethylene glycol-GDF11 (compound III) that embodiment 5 is obtained is chromatographed with SourceQ
Post (GE Healthcare) is purified.Specific method is as follows：
(1) chromatographic column is balanced with containing 5mM Tris hydrochloride buffers (pH8.0)；
(2) reaction solution containing 20kDa polyethylene glycol-GDF11 that embodiment 5 is obtained is subjected to loading after balancing；
(3) after loading, gradient elution is carried out with A liquid and B liquid mixed liquor, wherein A liquid is 5mM Tris hydrochloride buffers
(pH8.0), B liquid is the 5mM Tris hydrochloride buffers (pH8.0) containing 500mM sodium chloride, and specific gradient elution method is：
Pillar 5min only is crossed with A liquid at the beginning, the ratio of A liquid is gradually reduced after this, the ratio of B liquid is simultaneously gradually lifted,
So that by the unit volume eluent of pillar, the ratio of B liquid is interior in 50min, is progressively linearly lifted from 0% to 100%,
So as to effectively establish a NaCl concentration gradient for crossing pillar, carrying out gradient elution, (this step can pass through Akta eggs
White purification system software set, GE Healthcare).Timing when gradient elution, unreacted polyethylene glycol substantially on
Neutral, so being eluted out at first after 10min, there is people's growth that the order of protein peak is modified for many sites in elution
The active fragment of differentiation 11 (elution starts rear 18min appearances), sub 11 active fragments of human growth and differentiation factor 7 of unit point modification
Sub 11 active fragments of (elution starts rear 30min appearances) and unmodified human growth and differentiation factor 7 (elution starts rear 45min appearances).
Different elution protein peaks can be collected by 280nm ultraviolet detection, the eluent for collecting second protein peak is to obtain only
The alpha-amido of the active fragment N-terminal of growth and differentiation factor 11 is by polyethyleneglycol modified compound, so as to isolate and purify out unit point
Sub 11 active fragments of the human growth and differentiation factor 7 of modification.Purification result is referring to 5, and the size of coupled product is 33kDa.
Embodiment 7：40kDa polyethylene glycol carries out coupling in N-terminal with sub 11 active fragments of human growth and differentiation factor 7 and prepared
Liquid containing 40kDa polyethylene glycol-GDF11 (compounds Ⅳ)
Sub 11 active fragments of human growth and differentiation factor 7 that the present embodiment is used have the amino acid sequence shown in SEQ ID NO.6
Row.The present embodiment specific implementation method is as follows：
(1) 2 microlitres of active fragments of human growth factor 11 are taken (i.e. with the amino acid sequence shown in SEQ ID NO.6, to be purchased from
PeproTech, 120-11), protein concentration is measured at a wavelength of 280 nm, and 5mg/ml then is arrived into concentration regulation；
(2) sub 11 active fragments of human growth and differentiation factor 7 that 10ml concentration is 5mg/ml are taken, the poly- second two of 40kDa is added thereto
Alcohol solid 100mg, is stirred to being completely dissolved, so as to obtain sub 11 active fragments of human growth and differentiation factor 7 and 40kDa in room temperature condition
The mixture of polyethylene glycol；Here polyethylene glycol is polyethylene glycol (mPEG-ButyrALD, molecule of special modified protein N-terminal
Measure 40kDa, Nektar, the alpha amino coupleds that the dressing agent can only dissociate with albumen n end), polyethylene glycol and people's Growth and Differentiation
The mol ratio of the active fragment of the factor 11 mixing is 5:1；
(3) it is reducing agent sodium cyanoborohydride (the CH3BNNa) (concentration of reducing agent in the mixture after addition to add concentration
For 50mM), with salt acid for adjusting pH to 5, it is stored at room temperature 24 hours.The obtained reaction solution containing 40kDa polyethylene glycol-GDF11.
Now in the reaction solution 11 active fragments of more than 90% human growth and differentiation factor 7 by single polyethyleneglycol modified, i.e., one poly- second
Glycol is coupled with an active fragment molecule of growth and differentiation factor 11, conjugation sites be growth and differentiation factor 11N ends α-
Amino.At this moment, reaction solution is used directly for anion-exchange column purifying.
Embodiment 8：Anion column is used after the N-terminal of sub 11 active fragments of the polyethyleneglycol modified human growth and differentiation factor 7s of 40kDa
SourceQ is purified
The reaction solution containing 40kDa polyethylene glycol-GDF11 (compounds Ⅳ) that embodiment 7 is obtained is chromatographed with SourceQ
Post (GE Healthcare) is purified.Specific method is as follows：
(1) chromatographic column is balanced with containing 5mM Tris hydrochloride buffers (pH8.0)；
(2) reaction solution containing 40kDa polyethylene glycol-GDF11 that embodiment 7 is obtained is subjected to loading after balancing；
(3) after loading, gradient elution is carried out with A liquid and B liquid mixed liquor, wherein A liquid is 5mMTris hydrochloride buffers
(pH8.0), B liquid is the 5mM Tris hydrochloride buffers (pH8.0) containing 500mM sodium chloride, the method for specific gradient elution
For：Pillar 5min only is crossed with A liquid at the beginning, the ratio of A liquid is gradually reduced after this, the ratio of B liquid is simultaneously gradually lifted
Example so that by the unit volume eluent of pillar, the ratio of B liquid in 50min in, from 0% progressively linear lifting to
100%, so as to effectively establish a NaCl concentration gradient for crossing pillar, carrying out gradient elution, (this step can pass through
Akta protein purification system software sets, GE Healthcare).Timing when gradient elution, unreacted polyethylene glycol
Substantially without electric charge, so being eluted out at first after 6min.Elution occurs what the order of protein peak was modified for many sites
Human growth and differentiation factor 7 11 active fragments of son (elution starts rear 15min appearances), human growth and differentiation factor 7 11 of unit point modification are lived
Property fragment (elution starts rear 22min appearances) and sub 11 active fragments of unmodified human growth and differentiation factor 7 (elution starts rear 45min
Appearance).Different elution protein peaks can be collected by 280nm ultraviolet detection, the eluent for collecting second protein peak is
The alpha-amido of the only active fragment N-terminal of growth and differentiation factor 11 is obtained by polyethyleneglycol modified compound, so as to isolate and purify out
Sub 11 active fragments of human growth and differentiation factor 7 of unit point modification.Purification result is referring to Fig. 6, and the size of coupled product is 53kDa.
Embodiment 9：N-terminal is prepared into the human growth and differentiation factor 7 that His-tag is coupled sub 11 active fragments, i.e. Examples 1 and 2
The His-tag-GDF11 arrived promotes the value-added activity of primary hippocampal cells
The present embodiment specific implementation method is as follows：
The His-tag-GDF11 coupled products after purification of 2 microlitres of taking-up, determine its absorbance under ultraviolet 280nm, from
And calculate the protein concentration of coupled product.Primary hippocampal cells (the original used in the present invention originated with 24 orifice plate culture rats
For the method described in the cultural method reference documents below of hippocampal cell：Guo,W.,Y.Ji,et al.(2014)."
Neuronal activity alters BDNF-TrkB signaling kinetics and downstream
functions."J Cell Sci 127(Pt 10):2249-2260.), culture used medium is DMEM, and condition of culture is
37 DEG C, 5% carbon dioxide.When cell density reaches every hole 4 × 105During individual cell, toward a some holes of 24 orifice plates in (6 holes)
The coupled product microgram containing albumen 500 added in coupled product, each hole is added, experiment A groups-His-tag-GDF11 is used as.Together
When add 500 micrograms toward (6 holes) in the other hole of 24 orifice plates sub 11 active fragments of human growth and differentiation factor 7 (it has
SEQ ID NO.6 amino acid sequence), it is used as experiment B groups-GDF11.Finally, a remaining some holes (6 holes) is no in 24 orifice plates
Process, be used as blank control group.
Continue to cultivate cell 24 hours under conditions of original, then under an optical microscope to experiment A groups, experiment B groups
Cell count is carried out respectively with blank control group, respectively experiment with computing A groups, the experiment white cellular control unit of B group cell number duties
The ratio of number.It can be found that coupled product (His-tag-GDF11) 11 active fragments than human growth and differentiation factor 7 can more promote
Primary hippocampal cells rise in value, and the activity of coupled product is specific ginseng more than active 4 times of sub 11 active fragments of human growth and differentiation factor 7
See Fig. 7.
Embodiment 10：His-tag is Examples 1 and 2 in the product that N-terminal is coupled with sub 11 active fragments of human growth and differentiation factor 7
The His-tag-GDF11 prepared promotes the activity of primary hippocampal cells Synaptic formation
The present embodiment specific implementation method is as follows：
The His-tag-GDF11 coupled products after purification of 2 microlitres of taking-up, determine its absorbance under ultraviolet 280nm, from
And calculate the protein concentration of coupled product.With 24 orifice plate culture primary hippocampal cells, culture used medium is DMEM, cultivates bar
Part is 37 DEG C, 5% carbon dioxide.When cell density reaches every hole 4 × 105During individual cell, toward the partial hole of 24 orifice plates in plus
Enter the coupled product microgram containing albumen 500 added in coupled product, each hole, be used as experiment A groups-His-tag-GDF11 (A groups
6 holes of coprocessing).Simultaneously toward human growth and differentiation factor 7 11 active fragments of son that 500 micrograms are added in the other part hole of 24 orifice plates
(it has SEQ ID NO.6 amino acid sequence), is used as experiment B groups (6 holes of B groups coprocessing).Finally, it is remaining in 24 orifice plates
A some holes do not process, be used as blank control group (blank control group totally 6 holes).
Continue to cultivate cell 24 hours under conditions of original, then under an optical microscope to experiment A groups, experiment B groups
Cynapse counting is carried out respectively with blank control group, respectively experiment with computing A groups, the experiment white control group cynapse of B group cynapse number duties
The ratio of number.It can be found that coupled product (N-terminal and sub 11 active fragments of the human growth and differentiation factor 7 that His-tag is coupled) compares life
The long active fragment of differentiation 11 can more promote the formation of primary hippocampal cynapse, and the activity of coupled product is human growth and differentiation factor 7
More than active 5 times of 11 active fragments, referring specifically to Figure 11.Different groups of hippocampal cell is imaged under the microscope, joined
Figure 17 is seen, from Figure 17, the experiment B groups (activity of human growth and differentiation factor 7 11 of the amino acid sequence i.e. with SEQ ID NO.6
Fragment processing) more cynapses substantially are formed than blank control group, experiment A groups (His-tag-GDF11 processing) are substantially again than real
Test B and form more cynapses.
Embodiment 11：GST-tag is embodiment 3 and 4 in the product that N-terminal is coupled with sub 11 active fragments of human growth and differentiation factor 7
The long-acting benefits of the GST-tag-GDF11 prepared in blood
GST-tag inside the present embodiment has SEQ ID NO.3 sequences, and human growth and differentiation factor 7 11 active fragments of son have
Amino acid sequence shown in SEQ ID NO.6, GST-tag is coupled to the N-terminal of growth and differentiation factor.
The present embodiment is by measuring GST-tag-GDF11 coupled products and sub 11 active tablets of unmodified human growth and differentiation factor 7
Half-life period of the section (it has SEQ ID NO.6 amino acid sequence) in Mice Body, so as to examine life after GST-tag modifications
The long-acting benefit of the long active fragment of differentiation 11.It is specific as follows：From the adult mice of 12 C75BL/6 strains, average body
Weight is 30g or so.Mouse is divided into two groups, every group 6, passes through unmodified human growth and differentiation factor 7 11 of tail vein injection respectively
Active fragment and GST-tag-GDF11 coupled products, the dosage of injection is 20mg/kg body weight.After injection, used at following time point
Blood pin is taken to take blood in mice eye：5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 9 hours, 12 is small
When, 15 hours, 18 hours, 21 hours, 24 hours, 36 hours, 72 hours.Take after blood, blood plasma, centrifugal rotational speed is collected by centrifugation in room temperature
For 1000g, centrifugation time is 30 minutes.
Survey 11 active fragments of son of human growth and differentiation factor 7 in blood plasma and GST-tag-GDF11 coupled products respectively with ELISA
Concentration.Measurement result is found：Half-life period of human growth and differentiation factor 7 11 active fragments of son in Mice Body is average 5 hours, GST-
Half-life period of the tag-GDF11 coupled products in Mice Body is average 24 hours.This shows the coupled product energy of GST-tag modifications
Sub 11 active fragments of human growth and differentiation factor 7 are significantly improved in the half-life period of Mice Body intracellular metabolite, so that the treatment with the longer time
Effect.
Embodiment 12：GST-tag is embodiment 3 and 4 in the product that N-terminal is coupled with sub 11 active fragments of human growth and differentiation factor 7
The GST-tag-GDF11 prepared promotes the value-added activity of primary hippocampal cells
GST-tag inside the present embodiment has the amino acid sequence shown in SEQ ID NO.3, human growth and differentiation factor 7 11
Active fragment has the amino acid sequence shown in SEQ ID NO.6, and GST-tag is coupled to the sub- active fragment of human growth and differentiation factor 7
N-terminal.
The present embodiment specific implementation method is as follows：The GST-tag-GDF11 coupled products of 2 microlitres of taking-up, in ultraviolet 280nm
Lower its absorbance of measure, so as to calculate the protein concentration of coupled product.With 24 orifice plate culture primary hippocampal cells, culture is used to train
It is DMEM to support base, and condition of culture is 37 DEG C, 5% carbon dioxide.When cell density reaches every hole 4 × 105During individual cell, toward 24
The coupled product microgram containing albumen 500 added in GST-tag-GDF11 coupled products, each hole is added in one some holes of orifice plate,
It is used as experiment A groups.Simultaneously toward human growth and differentiation factor 7 11 active fragments of son that 500 micrograms are added in the other hole of 24 orifice plates
(it has SEQ ID NO.6 amino acid sequence), is used as experiment B groups.Finally, a remaining some holes is not done and located in 24 orifice plates
Reason, is used as blank control group.
Continue to cultivate cell 24 hours under conditions of original, then under an optical microscope to experiment A groups, experiment B groups
Cell count is carried out respectively with blank control group, respectively experiment with computing A groups, the experiment white cellular control unit of B group cell number duties
The ratio of number.It can be found that coupled product (GST-tag-GDF11) 11 active fragments than human growth and differentiation factor 7 can more promote
Primary hippocampal cells rise in value, and the activity of coupled product is active 2 times of the sub 11 active fragment active fragments of human growth and differentiation factor 7
It is many, referring specifically to Fig. 8.
Embodiment 13：GST-tag is embodiment 3 and 4 in the product that N-terminal is coupled with sub 11 active fragments of human growth and differentiation factor 7
The GST-tag-GDF11 prepared promotes the activity of primary hippocampal cells Synaptic formation
GST-tag inside the present embodiment has the amino acid sequence shown in SEQ ID NO.3, human growth and differentiation factor 7 11
Active fragment has the amino acid sequence shown in SEQ ID NO.6, and GST-tag is coupled to the N-terminal of human growth and differentiation factor 7.
The present embodiment specific implementation method is as follows：The GST-tag-GDF11 coupled products of 2 microlitres of taking-up, in ultraviolet 280nm
Lower its absorbance of measure, so as to calculate the protein concentration of coupled product.With 24 orifice plate culture primary hippocampal cells, culture is used to train
It is DMEM to support base, and condition of culture is 37 DEG C, 5% carbon dioxide.When cell density reaches every hole 4 × 105During individual cell, toward 24
The coupled product microgram containing albumen 500 added in GST-tag-GDF11 coupled products, each hole is added in one some holes of orifice plate,
It is used as experiment A groups.Simultaneously toward human growth and differentiation factor 7 11 active fragments of son that 500 micrograms are added in the other hole of 24 orifice plates
(it has SEQ ID NO.6 amino acid sequence), is used as experiment B groups.
Finally, a remaining some holes is not processed in 24 orifice plates, is used as blank control group.Continue to train under conditions of original
Support cell 24 hours, cynapse counting then carried out respectively to experiment A groups, experiment B groups and blank control group under an optical microscope,
Difference experiment with computing A groups, the experiment white control group number of synapses purpose ratio of B group cynapse number duties.It can be found that coupled product
(GST-tag-GDF11) 11 active fragments than human growth and differentiation factor 7 can more promote the formation of primary hippocampal cynapse, coupled product
Activity be the sub 11 active fragments activity of human growth and differentiation factor 7 more than 3 times, referring specifically to Figure 12.
Embodiment 14：20kDa polyethylene glycol is to implement in the product that N-terminal is coupled with sub 11 active fragments of human growth and differentiation factor 7
The long-acting effectiveness of 20kDa polyethylene glycol-GDF11 prepared by example 5 and 6 in blood
Coupled product inside the present embodiment is sub 11 active tablets of human growth and differentiation factor 7 of N-terminal and 20kD polyethylene glycol conjugations
Section, sub 11 active fragments of the human growth and differentiation factor 7 being related to have the amino acid sequence shown in SEQ ID NO.6.
The present embodiment is by measuring 20kDa polyethylene glycol-GDF11 coupled products and unmodified human growth and differentiation factor 7 11
Half-life period of the active fragment (it has SEQ ID NO.6 amino acid sequence) in Mice Body, so as to examine the poly- second two of 20kD
The long-acting benefits of GDF11 after alcohol modification.Adult mice specific as follows from 12 C75BL/6 strains, average weight is 30g left
It is right.Mouse is divided into two groups, every group 6, passes through sub 11 active fragments of the unmodified human growth and differentiation factor 7 of tail vein injection respectively
With 20kDa polyethylene glycol-GDF11 coupled products, the dosage of injection is 20mg/kg body weight.After injection, at following time point with taking
Blood pin takes blood in mice eye：5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 9 hours, 12 hours,
15 hours, 18 hours, 21 hours, 24 hours, 36 hours, 72 hours.Take after blood, blood plasma is collected by centrifugation in room temperature, centrifugal rotational speed is
1000g, centrifugation time is 30 minutes.
Survey human growth and differentiation factor 7 11 active fragments of son and 20kDa polyethylene glycol-GDF11 couplings in blood plasma respectively with ELISA
The concentration of product.Measurement result is found：Half-life period of human growth and differentiation factor 7 11 active fragments of son in Mice Body is small for average 5
When, half-life period of the coupled product in Mice Body is average 18 hours.This shows the polyethyleneglycol modified coupled product energy of 20kD
Sub 11 active fragments of human growth and differentiation factor 7 are significantly improved in the half-life period of Mice Body intracellular metabolite, so that the treatment with the longer time
Effect.
Embodiment 15：20kDa polyethylene glycol is to implement in the product that N-terminal is coupled with sub 11 active fragments of human growth and differentiation factor 7
20kDa polyethylene glycol-GDF11 prepared by example 5 and 6 promotes the value-added activity of primary hippocampal cells
Coupled product inside the present embodiment is sub 11 active tablets of human growth and differentiation factor 7 of N-terminal and 20kD polyethylene glycol conjugations
Section, sub 11 active fragments of the human growth and differentiation factor 7 being related to have the amino acid sequence shown in SEQ ID NO.6.
The present embodiment specific implementation method is as follows：The coupled product of 2 microlitres of taking-up, determines its extinction under ultraviolet 280nm
Degree, so as to calculate the protein concentration of coupled product.With 24 orifice plate culture primary hippocampal cells, culture used medium is DMEM,
Condition of culture is 37 DEG C, 5% carbon dioxide.When cell density reaches every hole 4 × 105During individual cell, toward some of 24 orifice plates
The coupled product microgram containing albumen 500 added in coupled product, each hole is added in hole, experiment A groups are used as.Simultaneously toward 24 orifice plates
Other hole in add sub 11 active fragments of human growth and differentiation factor 7 of 500 micrograms (it have SEQ ID NO.6 amino acid
Sequence), it is used as experiment B groups.Finally, a remaining some holes is not processed in 24 orifice plates, is used as blank control group.
Continue to cultivate cell 24 hours under conditions of original, then under an optical microscope to experiment A groups, experiment B groups
Cell count is carried out respectively with blank control group, respectively experiment with computing A groups, the experiment white cellular control unit of B group cell number duties
The ratio of number.It can be found that coupled product (20kDa polyethylene glycol-GDF11) 11 active fragments than human growth and differentiation factor 7 are more
Primary hippocampal cells can be promoted to rise in value, the activity of coupled product is 1.5 times of left sides of the sub 11 active fragments activity of human growth and differentiation factor 7
The right side, referring specifically to Fig. 9.
Embodiment 16：20kDa polyethylene glycol is to implement in the product that N-terminal is coupled with sub 11 active fragments of human growth and differentiation factor 7
20kDa polyethylene glycol-GDF11 prepared by example 5 and 6 promotes the activity of primary hippocampal cells Synaptic formation
Coupled product inside the present embodiment is sub 11 active tablets of human growth and differentiation factor 7 of N-terminal and 20kD polyethylene glycol conjugations
Section, sub 11 active fragments of the human growth and differentiation factor 7 being related to have the amino acid sequence shown in SEQ ID NO.6.
The present embodiment specific implementation method is as follows：20kDa polyethylene glycol-GDF11 the coupled products of 2 microlitres of taking-up, in purple
Its absorbance is determined under outer 280nm, so as to calculate the protein concentration of coupled product.With 24 orifice plate culture primary hippocampal cells, training
It is DMEM to support used medium, and condition of culture is 37 DEG C, 5% carbon dioxide.When cell density reaches every hole 4 × 105It is individual thin
During born of the same parents, toward a some holes of 24 orifice plates in add the coupled product microgram containing albumen 500 added in coupled product, each hole, as
Test A groups.Simultaneously toward human growth and differentiation factor 7 11 active fragments (its tool that 500 micrograms are added in the other hole of 24 orifice plates
Have SEQ ID NO.6 amino acid sequence), it is used as experiment B groups.Finally, a remaining some holes is not processed in 24 orifice plates, as
Blank control group.
Continue to cultivate cell 24 hours under conditions of original, then under an optical microscope to experiment A groups, experiment B groups
Cynapse counting is carried out respectively with blank control group, respectively experiment with computing A groups, the experiment white control group cynapse of B group cynapse number duties
The ratio of number.It can be found that coupled product (20kDa polyethylene glycol-GDF11) 11 active fragments than human growth and differentiation factor 7 are more
The formation of primary hippocampal cynapse can be promoted, the activity of coupled product is 1.2 times of the sub 11 active fragments activity of human growth and differentiation factor 7
Left and right, referring specifically to Figure 13.
Embodiment 17：40kDa polyethylene glycol is to implement in the product that N-terminal is coupled with sub 11 active fragments of human growth and differentiation factor 7
The long-acting effectiveness of 40kDa polyethylene glycol-GDF11 prepared by example 7 and 8 in blood
Coupled product inside the present embodiment is sub 11 active tablets of human growth and differentiation factor 7 of N-terminal and 20kD polyethylene glycol conjugations
Section, sub 11 active fragments of the human growth and differentiation factor 7 being related to have the amino acid sequence shown in SEQ ID NO.6.
By measuring 40kDa polyethylene glycol-GDF11 coupled products and sub 11 active fragments of unmodified human growth and differentiation factor 7
The half-life period of (it has SEQ ID NO.6 amino acid sequence) in Mice Body, so that after examining 40kD polyethyleneglycol modified
The long-acting benefits of GDF11.It is specific as follows：From the adult mice of 12 C75BL/6 strains, average weight is 30g or so.Will be small
Mouse is divided into two groups, every group 6, passes through sub 11 active fragments of the unmodified human growth and differentiation factor 7 of tail vein injection and 40kDa respectively
Polyethylene glycol-GDF11 coupled products, the dosage of injection is 20mg/kg body weight.After injection, at following time point with taking blood pin to exist
Mice eye takes blood：5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 9 hours, 12 hours, 15 is small
When, 18 hours, 21 hours, 24 hours, 36 hours, 72 hours.Take after blood, blood plasma is collected by centrifugation in room temperature, centrifugal rotational speed is
1000g, centrifugation time is 30 minutes.
Survey human growth and differentiation factor 7 11 active fragments of son and 40kDa polyethylene glycol-GDF11 couplings in blood plasma respectively with ELISA
The concentration of product.Measurement result is found：Half-life period of human growth and differentiation factor 7 11 active fragments of son in Mice Body is small for average 5
When, half-life period of the coupled product in Mice Body is average 16 hours.This shows the polyethyleneglycol modified coupled product energy of 40kD
Sub 11 active fragments of human growth and differentiation factor 7 are significantly improved in the half-life period of Mice Body intracellular metabolite, so that the treatment with the longer time
Effect.
Embodiment 18：40kDa polyethylene glycol is to implement in the product that N-terminal is coupled with sub 11 active fragments of human growth and differentiation factor 7
40kDa polyethylene glycol-GDF11 prepared by example 7 and 8 promotes the value-added activity of primary hippocampal cells
Coupled product inside the present embodiment is sub 11 active tablets of human growth and differentiation factor 7 of N-terminal and 40kD polyethylene glycol conjugations
Section, sub 11 active fragments of the human growth and differentiation factor 7 being related to have the amino acid sequence shown in SEQ ID NO.6.
The present embodiment specific implementation method is as follows：40kDa polyethylene glycol-GDF11 the coupled products of 2 microlitres of taking-up, in purple
Its absorbance is determined under outer 280nm, so as to calculate the protein concentration of coupled product.With 24 orifice plate culture primary hippocampal cells, training
It is DMEM to support used medium, and condition of culture is 37 DEG C, 5% carbon dioxide.When cell density reaches every hole 4 × 105It is individual thin
During born of the same parents, toward a some holes of 24 orifice plates in add the 40kDa polyethylene glycol-GDF11 coupled products added in coupled product, each hole
Containing the microgram of albumen 500, experiment A groups are used as.Simultaneously toward the human growth and differentiation factor 7 that 500 micrograms are added in the other hole of 24 orifice plates
Sub 11 active fragments (it has SEQ ID NO.6 amino acid sequence), are used as experiment B groups.Finally, remaining one in 24 orifice plates
Some holes is not processed, and is used as blank control group.
Continue to cultivate cell 24 hours under conditions of original, then under an optical microscope to experiment A groups, experiment B groups
Cell count is carried out respectively with blank control group, respectively experiment with computing A groups, the experiment white cellular control unit of B group cell number duties
The ratio of number.It can be found that coupled product (40kDa polyethylene glycol-GDF11) 11 active fragments than human growth and differentiation factor 7 are more
Primary hippocampal cells can be promoted to rise in value, the activity of coupled product is 1.8 times of left sides of the sub 11 active fragments activity of human growth and differentiation factor 7
The right side, referring specifically to Figure 10.
Embodiment 19：40kDa polyethylene glycol is to implement in the product that N-terminal is coupled with sub 11 active fragments of human growth and differentiation factor 7
40kDa polyethylene glycol-GDF11 prepared by example 7 and 8 promotes the activity of primary hippocampal cells Synaptic formation
Coupled product inside the present embodiment is sub 11 active tablets of human growth and differentiation factor 7 of N-terminal and 40kD polyethylene glycol conjugations
Section, sub 11 active fragments of the human growth and differentiation factor 7 being related to have the amino acid sequence shown in SEQ ID NO.6.
The present embodiment specific implementation method is as follows：40kDa polyethylene glycol-GDF11 the coupled products of 2 microlitres of taking-up, in purple
Its absorbance is determined under outer 280nm, so as to calculate the protein concentration of coupled product.With 24 orifice plate culture primary hippocampal cells, training
It is DMEM to support used medium, and condition of culture is 37 DEG C, 5% carbon dioxide.When cell density reaches every hole 4 × 105It is individual thin
During born of the same parents, toward a some holes of 24 orifice plates in add the coupled product microgram containing albumen 500 added in coupled product, each hole, as
Test A groups.Simultaneously toward human growth and differentiation factor 7 11 active fragments (its tool that 500 micrograms are added in the other hole of 24 orifice plates
Have SEQ ID NO.6 amino acid sequence), it is used as experiment B groups.Finally, a remaining some holes is not processed in 24 orifice plates, as
Blank control group.
Continue to cultivate cell 24 hours under conditions of original, then under an optical microscope to experiment A groups, experiment B groups
Cynapse counting is carried out respectively with blank control group, respectively experiment with computing A groups, the experiment white control group cynapse of B group cynapse number duties
The ratio of number.It can be found that coupled product (40kDa polyethylene glycol-GDF11) 11 active fragments than human growth and differentiation factor 7 are more
The formation of primary hippocampal cynapse can be promoted, the activity of coupled product is 1.4 times of the sub 11 active fragments activity of human growth and differentiation factor 7
Left and right, referring specifically to Figure 14.
Embodiment 20：Chemical compounds I (His-tag-GDF11 prepared by Examples 1 and 2), the compound ii (system of embodiment 3 and 4
Standby GST-tag-GDF11), compound III (embodiment 5 and 6 prepare 20KD polyethylene glycol-GDF11), compounds Ⅳ (implemented
40KD polyethylene glycol-GDF11 prepared by example 7 and 8) to the activity that is lifted into aged mice (18 monthly ages) cognitive ability.
Specific method is as follows：Chemical compounds I-IV is injected into different mouse (C57BL/6, CD1 product by caudal vein respectively
System) in vivo.Administration group ensures 25 micro- grams/times of dosage, the administration 8 times (average to be administered once for every three days) in 24 days,
Administration group 1-4 is the male mouse of 20 18 months C57BL/6, and chemical compounds I-IV, which is sequentially allocated, gives 1-4 administration groups.It is 20 without administration group
Only the male mouse of the C57BL/6 of 18 months, receive 8 Saline injections for 24 days before Behaviors survey.Negative control group is 20 3
The male mouse of the C57BL/6 of individual month, receive 8 Saline injections for 24 days equally before Behaviors survey.Behaviors survey is water fan
Test the learning ability and memory for detecting mouse in palace.
Water maze laboratory is carried out between 8 a.m. at 1 point in afternoon.Water maze spatial memory training period is 4 days, 4 times a day,
Training interval time is 10 minutes every time.In an experiment, every four mouse are randomly divided into a training group.For each training
Group, the position of platform of water maze is probabilistically assigned, and keeps constant in whole training.In training, mouse is released from optional position
It is put into water maze, and allows it to search for hiding platform in 120 seconds.If mouse did not find platform in 120 seconds, it
Platform will be directed into.The distance for finding the time used in platform in training every time and being passed through is recorded automatically by intelligent video camera head
Get off.Water maze test is carried out after last time is trained 48 hours, and every mouse is released into the water fan without placement platform
Gong Zhong, and allow its freely activity 60 second.Its travelling route is automatically recorded by intelligent video camera head.When the test phase is than training period
Between it is short one times, with avoid mouse produce Depressive behavior.Mouse spends in three quadrants of time and other that target quadrant is spent
Time is recorded, for the assessment to mouse memory power.Test result is referring to Figure 15, wherein figure (a) is training first day
By the 4th day, every group of mouse found the time used in hiding platform, it is as can be seen from the figure polyethyleneglycol modified after Gdf11 can be bright
The aobvious Spatial learning ability for improving mouse；Figure (b) is that mouse, can be with from figure in the time used in target quadrant in water maze test
Find out 20kD it is polyethyleneglycol modified after Gdf11 can significantly improve the spatial memory of mouse.
Embodiment 21：Chemical compounds I (His-tag-GDF11 prepared by Examples 1 and 2), the compound ii (system of embodiment 3 and 4
Standby GST-tag-GDF11), compound III (embodiment 5 and 6 prepare 20KD polyethylene glycol-GDF11), compounds Ⅳ (implemented
Example 7 and 8 prepare 40KD polyethylene glycol-GDF11) mouse alzheimer's disease model treatment in activity.
5XFAD senile dementia mouse models are ordered in U.S. jackson laboratory, and according to zoopery standard
Bred and raised.Each experimental mouse model all carries out identified for genes by rat-tail, it is ensured that app gene and PS1 genes
Stable mutation.Chemical compounds I-IV is injected into mouse body respectively by caudal vein.Administration group ensures 25 micro- grams/times of administration
Dosage, the administration before Behaviors survey in 24 days 8 times, administration group 1-4 is 20 14 week old 5XFAD mouse, chemical compounds I-
IV is sequentially allocated and gives 1-4 administration groups.Without the 5XFAD mouse that administration group is 20 14 week old, receive within 24 days before Behaviors survey
8 Saline injections.Negative control group is the male mouse of C57BL/6 of 20 14 week old, equally receives Saline injections.Behaviouristics
Test is that water maze laboratory is used to detect the learning ability and memory of mouse.Water maze laboratory be the same as Example 20.Test result
Referring to Figure 16, wherein figure (a) is spatial memory learning ability curve map, after as can be seen from the figure 20kD is polyethyleneglycol modified
Gdf11 can significantly improve the Spatial learning ability of Elderly dementia patients；It is Senlie dementia model mouse spatial memory to scheme (b)
Test and appraisal figure, as can be seen from the figure His-tag modifications can significantly improve the spatial memory of Elderly dementia patients.
Embodiment 22
Normal person 20, the senile dementia patients through making a definite diagnosis 20.
First, 3 milliliters of vein everyone blood plasma is gathered with the heparin tube of sodium citrate, 1 milliliter of centrifuging and taking supernatant, up and down
4 DEG C are transported to use for laboratory in protein extracting after reverse mixing.The blood plasma isolated averagely is dispensed into the lab
In 1.5mlEP pipes, -80 DEG C of refrigerators are preserved.
Secondly, blood plasma is taken out from -80 DEG C of refrigerators, is inserted in and thaws on ice, mixing is shaken after defrosting and is inserted in ice.In transfer
Buffer solution (48 mMs of every liter of trishydroxymethylaminomethanes, 39 mMs of every liter of glycine, 0.03% lauryl sodium sulfate),
Under conditions of confining liquid (5% skimmed milk power, 0.1%TBST), carried out by using polyvinyladine floride film and half-dried transferring system
Western blotting, is visualized using ECL solution (GE Healthcare, rpn2108).Checked under same volume normal
People organizes the change with the protein expression level of old dementia patients group, and one-dimensional native gel electrophoresis separates target protein laggard
Row Western blot.The skimmed milk power common goat-anti rabbit-anti that is incubated with horseradish peroxidase-labeled of the GDF11 antibody 5%
Body is combined.GDF11 concentrations versus's result is referring to Figure 18 in the blood plasma of normal person and patients of senile dementia, normal person's
GDF11 concentration is apparently higher than old dementia patients.
Embodiment 23
Everyone blood plasma small molecule extract in embodiment 22 is used for small molecule Mass Spectrometer Method, what it was detected has bright
The small molecule and its variation tendency such as Figure 19 of aobvious changes of contents.
First, extract
1) blood plasma is taken out from -80 DEG C of refrigerators, is inserted in and thaws on ice, mixing is shaken after defrosting and is inserted in ice；
2) filter paper that filter paper breaks into 6mm is laid in 96 orifice plates with card punch；
3) 5 μ L blood plasma are drawn with liquid-transfering gun to add on filter paper, adds 100ul100% methanol；
4) sample is stored at room temperature 40min；
5) static rear whole supernatants of drawing are interior to EP pipes, and 4 DEG C of centrifugation, 12000rpm, 20min；
6) drawn after centrifuging with rifle in the special specimen bottle of the supreme machine of 80ul supernatants, machine in wait.
2nd, upper machine testing
Liquid chromatogram and mass spectrometry parameters are set so that sample applied sample amount is 5 microlitres.Liquid chromatogram uses following parameter：Stream
Speed is 0.5mL/min, and using BEH C18 pillars (2.1 millimeters high, 50 millimeters of internal diameters) (GE Healthcare), mobility is used
(volume ratio is 2 to the mixture of acetonitrile and isopropanol：1)；Mass spectrum uses following parameter：Air pressure inside is 25psi, and ionizer is
ESI, ionizer voltage is set to 5000V, and temperature setting is 300 degree, Q2 rooms collision energy 40eV.
Testing result shows, in GDF11 metabolite, propionyl Propionyl AC (C3), lysophosphatidyl choline
LysoPC a C18:2nd, phosphatidyl PC aa C36:6th, phosphatidyl C16:1-OH, phosphatidyl PC aa C38:0th, phosphatidyl PC aa
C38:6th, phosphatidyl PC aa C40:1st, phosphatidyl PC aa C40:2nd, phosphatidyl PC aa C40:6th, phosphatidyl PC ae C40:6
Concentration there is significant difference in normal person and old dementia patients, referring specifically to Figure 19, it is possible thereby to determine, above-mentioned metabolism
Product can be used for diagnosis and examination senile dementia disease, i.e., using above-mentioned metabolite as senile dementia mark.When
The concentration of above-mentioned one or more metabolites meets or exceeds threshold value set in advance in the blood sample of the subject, then sentences
The fixed subject is with senile dementia or with the risk for suffering from senile dementia.The concentration of the metabolite is to use
What metabolism group method was determined.
SEQUENCE LISTING
<110>Field, it is wild
<120>A kind of growth and differentiation factor through modification and its preparation method and application
<130> C1CNCN160063
<160> 19
<170> PatentIn version 3.5
<210> 1
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> His-tag-1
<400> 1
His His His His His His
1 5
<210> 2
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> His-tag-2
<400> 2
Met Gly Ser Ser His His His His His His
1 5 10
<210> 3
<211> 218
<212> PRT
<213> Artificial Sequence
<220>
<223> GST-tag
<400> 3
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys
210 215
<210> 4
<211> 407
<212> PRT
<213> Homo sapiens
<400> 4
Met Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu
1 5 10 15
Glu Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala
20 25 30
Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly Glu Arg Ser
35 40 45
Ser Arg Pro Ala Pro Ser Val Ala Pro Glu Pro Asp Gly Cys Pro Val
50 55 60
Cys Val Trp Arg Gln His Ser Arg Glu Leu Arg Leu Glu Ser Ile Lys
65 70 75 80
Ser Gln Ile Leu Ser Lys Leu Arg Leu Lys Glu Ala Pro Asn Ile Ser
85 90 95
Arg Glu Val Val Lys Gln Leu Leu Pro Lys Ala Pro Pro Leu Gln Gln
100 105 110
Ile Leu Asp Leu His Asp Phe Gln Gly Asp Ala Leu Gln Pro Glu Asp
115 120 125
Phe Leu Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val Ile Ser
130 135 140
Met Ala Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro Leu
145 150 155 160
Cys Cys His Phe His Phe Ser Pro Lys Val Met Phe Thr Lys Val Leu
165 170 175
Lys Ala Gln Leu Trp Val Tyr Leu Arg Pro Val Pro Arg Pro Ala Thr
180 185 190
Val Tyr Leu Gln Ile Leu Arg Leu Lys Pro Leu Thr Gly Glu Gly Thr
195 200 205
Ala Gly Gly Gly Gly Gly Gly Arg Arg His Ile Arg Ile Arg Ser Leu
210 215 220
Lys Ile Glu Leu His Ser Arg Ser Gly His Trp Gln Ser Ile Asp Phe
225 230 235 240
Lys Gln Val Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly
245 250 255
Ile Glu Ile Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr
260 265 270
Ser Leu Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu Arg
275 280 285
Val Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys
290 295 300
Asp Glu His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val
305 310 315 320
Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr
325 330 335
Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys
340 345 350
Tyr Pro His Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala
355 360 365
Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr
370 375 380
Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val
385 390 395 400
Val Asp Arg Cys Gly Cys Ser
405
<210> 5
<211> 405
<212> PRT
<213> Rattus rattus
<400> 5
Met Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu
1 5 10 15
Glu Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala
20 25 30
Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly Glu Arg Ser Ser Arg
35 40 45
Pro Ala Pro Ser Ala Pro Pro Glu Pro Asp Gly Cys Pro Val Cys Val
50 55 60
Trp Arg Gln His Ser Arg Glu Leu Arg Leu Glu Ser Ile Lys Ser Gln
65 70 75 80
Ile Leu Ser Lys Leu Arg Leu Lys Glu Ala Pro Asn Ile Ser Arg Glu
85 90 95
Val Val Lys Gln Leu Leu Pro Lys Ala Pro Pro Leu Gln Gln Ile Leu
100 105 110
Asp Leu His Asp Phe Gln Gly Asp Ala Leu Gln Pro Glu Asp Phe Leu
115 120 125
Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val Ile Ser Met Ala
130 135 140
Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro Leu Cys Cys
145 150 155 160
His Phe His Phe Ser Pro Lys Val Met Phe Thr Lys Val Leu Lys Ala
165 170 175
Gln Leu Trp Val Tyr Leu Arg Pro Val Pro Arg Pro Ala Thr Val Tyr
180 185 190
Leu Gln Ile Leu Arg Leu Lys Pro Leu Thr Gly Glu Gly Thr Ala Gly
195 200 205
Gly Gly Gly Gly Gly Arg Arg His Ile Arg Ile Arg Ser Leu Lys Ile
210 215 220
Glu Leu His Ser Arg Ser Gly His Trp Gln Ser Ile Asp Phe Lys Gln
225 230 235 240
Val Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly Ile Glu
245 250 255
Ile Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr Ser Leu
260 265 270
Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu Arg Val Leu
275 280 285
Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys Asp Glu
290 295 300
His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp Phe
305 310 315 320
Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr Lys Ala
325 330 335
Asn Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys Tyr Pro
340 345 350
His Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala Gly Pro
355 360 365
Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr Phe Asn
370 375 380
Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val Val Asp
385 390 395 400
Arg Cys Gly Cys Ser
405
<210> 6
<211> 109
<212> PRT
<213> Homo sapiens
<400> 6
Asn Leu Gly Leu Asp Cys Asp Glu His Ser Ser Glu Ser Arg Cys Cys
1 5 10 15
Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile
20 25 30
Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu
35 40 45
Tyr Met Phe Met Gln Lys Tyr Pro His Thr His Leu Val Gln Gln Ala
50 55 60
Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser
65 70 75 80
Pro Ile Asn Met Leu Tyr Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly
85 90 95
Lys Ile Pro Gly Met Val Val Asp Arg Cys Gly Cys Ser
100 105
<210> 7
<211> 514
<212> PRT
<213> Homo sapiens
<400> 7
Asp Phe Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys
1 5 10 15
Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile
20 25 30
Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu
35 40 45
Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala
50 55 60
Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser
65 70 75 80
Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly
85 90 95
Lys Ile Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser Met Val Leu
100 105 110
Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu Glu Leu Arg
115 120 125
Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala Ala Ala Ala Ala
130 135 140
Ala Ala Ala Ala Gly Val Gly Gly Glu Arg Ser Ser Arg Pro Ala Pro
145 150 155 160
Ser Ala Pro Pro Glu Pro Asp Gly Cys Pro Val Cys Val Trp Arg Gln
165 170 175
His Ser Arg Glu Leu Arg Leu Glu Ser Ile Lys Ser Gln Ile Leu Ser
180 185 190
Lys Leu Arg Leu Lys Glu Ala Pro Asn Ile Ser Arg Glu Val Val Lys
195 200 205
Gln Leu Leu Pro Lys Ala Pro Pro Leu Gln Gln Ile Leu Asp Leu His
210 215 220
Asp Phe Gln Gly Asp Ala Leu Gln Pro Glu Asp Phe Leu Glu Glu Asp
225 230 235 240
Glu Tyr His Ala Thr Thr Glu Thr Val Ile Ser Met Ala Gln Glu Thr
245 250 255
Asp Pro Ala Val Gln Thr Asp Gly Ser Pro Leu Cys Cys His Phe His
260 265 270
Phe Ser Pro Lys Val Met Phe Thr Lys Val Leu Lys Ala Gln Leu Trp
275 280 285
Val Tyr Leu Arg Pro Val Pro Arg Pro Ala Thr Val Tyr Leu Gln Ile
290 295 300
Leu Arg Leu Lys Pro Leu Thr Gly Glu Gly Thr Ala Gly Gly Gly Gly
305 310 315 320
Gly Gly Arg Arg His Ile Arg Ile Arg Ser Leu Lys Ile Glu Leu His
325 330 335
Ser Arg Ser Gly His Trp Gln Ser Ile Asp Phe Lys Gln Val Leu His
340 345 350
Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly Ile Glu Ile Asn Ala
355 360 365
Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr Ser Leu Gly Pro Gly
370 375 380
Ala Glu Gly Leu His Pro Phe Met Glu Leu Arg Val Leu Glu Asn Thr
385 390 395 400
Lys Arg Ser Arg Arg Asn Leu Gly Leu Asp Cys Asp Glu His Ser Ser
405 410 415
Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe
420 425 430
Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys
435 440 445
Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys Tyr Pro His Thr His
450 455 460
Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr
465 470 475 480
Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr Phe Asn Asp Lys Gln
485 490 495
Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val Val Asp Arg Cys Gly
500 505 510
Cys Ser
<210> 8
<211> 119
<212> PRT
<213> Artificial Sequence
<220>
<223> his-tag-2+GDF11-3
<400> 8
Met Gly Ser Ser His His His His His His Asn Leu Gly Leu Asp Cys
1 5 10 15
Asp Glu His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val
20 25 30
Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr
35 40 45
Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys
50 55 60
Tyr Pro His Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala
65 70 75 80
Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr
85 90 95
Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val
100 105 110
Val Asp Arg Cys Gly Cys Ser
115
<210> 9
<211> 327
<212> PRT
<213> Artificial Sequence
<220>
<223> GST-tag+GDF11-3
<400> 9
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Asn Leu Gly Leu Asp Cys
210 215 220
Asp Glu His Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val
225 230 235 240
Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr
245 250 255
Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys
260 265 270
Tyr Pro His Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala
275 280 285
Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr
290 295 300
Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val
305 310 315 320
Val Asp Arg Cys Gly Cys Ser
325
<210> 10
<211> 368
<212> DNA
<213> Artificial Sequence
<220>
<223>The gene of coded compounds I
<400> 10
ccatgggcag cagccatcat catcatcatc acaacctggg tctggactgc gacgagcact 60
caagcgagtc ccgctgctgc cgatatcccc tcacagtgga ctttgaggct ttcggctggg 120
actggatcat cgcacctaag cgctacaagg ccaactactg ctccggccag tgcgagtaca 180
tgttcatgca aaaatatccg catacccatt tggtgcagca ggccaatcca agaggctctg 240
ctgggccctg ttgtaccccc accaagatgt ccccaatcaa catgctctac ttcaatgaca 300
agcagcagat tatctacggc aagatccctg gcatggtggt ggatcgctgt ggctgctctt 360
aactcgag 368
<210> 11
<211> 996
<212> DNA
<213> Artificial Sequence
<220>
<223>The gene of coded compounds II
<400> 11
ccatggatgt cccctatact aggttattgg aaaattaagg gccttgtgca acccactcga 60
cttcttttgg aatatcttga agaaaaatat gaagagcatt tgtatgagcg cgatgaaggt 120
gataaatggc gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct tccttattat 180
attgatggtg atgttaaatt aacacagtct atggccatca tacgttatat agctgacaag 240
cacaacatgt tgggtggttg tccaaaagag cgtgcagaga tttcaatgct tgaaggagcg 300
gttttggata ttagatacgg tgtttcgaga attgcatata gtaaagactt tgaaactctc 360
aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga tcgtttatgt 420
cataaaacat atttaaatgg tgatcatgta acccatcctg acttcatgtt gtatgacgct 480
cttgatgttg ttttatacat ggacccaatg tgcctggatg cgttcccaaa attagtttgt 540
tttaaaaaac gtattgaagc tatcccacaa attgataagt acttgaaatc cagcaagtat 600
atagcatggc ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca tcctccaaaa 660
aacctgggtc tggactgcga cgagcactca agcgagtccc gctgctgccg atatcccctc 720
acagtggact ttgaggcttt cggctgggac tggatcatcg cacctaagcg ctacaaggcc 780
aactactgct ccggccagtg cgagtacatg ttcatgcaaa aatatccgca tacccatttg 840
gtgcagcagg ccaatccaag aggctctgct gggccctgtt gtacccccac caagatgtcc 900
ccaatcaaca tgctctactt caatgacaag cagcagatta tctacggcaa gatccctggc 960
atggtggtgg atcgctgtgg ctgctcttaa ctcgag 996
<210> 12
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Flag-tag
<400> 12
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
<210> 13
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> HA-tag
<400> 13
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
1 5
<210> 14
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> c-Myc-tag
<400> 14
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
<210> 15
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> AVi-tag
<400> 15
Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu
1 5 10 15
<210> 16
<211> 396
<212> PRT
<213> Artificial Sequence
<220>
<223> MBP-tag
<400> 16
Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15
Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys
20 25 30
Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu
35 40 45
Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu
50 55 60
His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
65 70 75 80
Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr
85 90 95
Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln
100 105 110
Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys
115 120 125
Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn
130 135 140
Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala
145 150 155 160
Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn
165 170 175
Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly
180 185 190
Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205
Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu
210 215 220
Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu
225 230 235 240
Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp
245 250 255
Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val
260 265 270
Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu
275 280 285
Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu
290 295 300
Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn
305 310 315 320
Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu
325 330 335
Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys
340 345 350
Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala
355 360 365
Val Arg Thr Ala Val Ile Asn Ala Ala Ser Gly Arg Gln Thr Val Asp
370 375 380
Glu Ala Leu Lys Asp Ala Gln Thr Arg Ile Thr Lys
385 390 395
<210> 17
<211> 100
<212> PRT
<213> Artificial Sequence
<220>
<223> SNAP-tag
<400> 17
Gly Pro Gly Ser Asp Lys Asp Cys Glu Met Lys Arg Thr Thr Leu Asp
1 5 10 15
Ser Pro Leu Gly Lys Leu Glu Leu Ser Gly Cys Glu Gln Gly Leu His
20 25 30
Glu Ile Ile Phe Leu Gly Lys Gly Thr Ser Ala Ala Asp Ala Val Glu
35 40 45
Val Pro Ala Pro Ala Ala Val Leu Gly Gly Pro Glu Pro Leu Met Gln
50 55 60
Ala Thr Ala Trp Leu Asn Ala Tyr Phe His Gln Pro Glu Ala Ile Glu
65 70 75 80
Glu Phe Pro Val Pro Ala Leu His His Pro Val Phe Gln Gln Glu Ser
85 90 95
Phe Thr Arg Gln
100
<210> 18
<211> 293
<212> PRT
<213> Artificial Sequence
<220>
<223> Halo-tag
<400> 18
Met Ser Glu Ile Gly Thr Gly Phe Pro Phe Asp Pro His Tyr Val Glu
1 5 10 15
Val Leu Gly Glu Arg Met His Tyr Val Asp Val Gly Pro Arg Asp Gly
20 25 30
Thr Pro Val Leu Phe Leu His Gly Asn Pro Thr Ser Ser Tyr Leu Trp
35 40 45
Arg Asn Ile Ile Pro His Val Ala Pro Ser His Arg Cys Ile Ala Pro
50 55 60
Asp Leu Ile Gly Met Gly Lys Ser Asp Lys Pro Asp Leu Asp Tyr Phe
65 70 75 80
Phe Asp Asp His Val Arg Tyr Leu Asp Ala Phe Ile Glu Ala Leu Gly
85 90 95
Leu Glu Glu Val Val Leu Val Ile His Asp Trp Gly Ser Ala Leu Gly
100 105 110
Phe His Trp Ala Lys Arg Asn Pro Glu Arg Val Lys Gly Ile Ala Cys
115 120 125
Met Glu Phe Ile Arg Pro Ile Pro Thr Trp Asp Glu Trp Pro Glu Phe
130 135 140
Ala Arg Glu Thr Phe Gln Ala Phe Arg Thr Ala Asp Val Gly Arg Glu
145 150 155 160
Leu Ile Ile Asp Gln Asn Ala Phe Ile Glu Gly Ala Leu Pro Lys Cys
165 170 175
Val Val Arg Pro Leu Thr Glu Val Glu Met Asp His Tyr Arg Glu Pro
180 185 190
Phe Leu Lys Pro Val Asp Arg Glu Pro Leu Trp Arg Phe Pro Asn Glu
195 200 205
Leu Pro Ile Ala Gly Glu Pro Ala Asn Ile Val Ala Leu Val Glu Ala
210 215 220
Tyr Met Asn Trp Leu His Gln Ser Pro Val Pro Lys Leu Leu Phe Trp
225 230 235 240
Gly Thr Pro Gly Val Leu Ile Pro Pro Ala Glu Ala Ala Arg Leu Ala
245 250 255
Glu Ser Leu Pro Asn Cys Lys Thr Val Asp Ile Gly Pro Gly Leu His
260 265 270
Tyr Leu Gln Glu Asp Asn Pro Asp Leu Ile Gly Ser Glu Ile Ala Arg
275 280 285
Trp Leu Pro Ala Leu
290
<210> 19
<211> 99
<212> PRT
<213> Artificial Sequence
<220>
<223> SUMO-tag
<400> 19
Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro
1 5 10 15
Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser
20 25 30
Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu
35 40 45
Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg
50 55 60
Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp
65 70 75 80
Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile
85 90 95
Gly Gly Ser
Claims (31)
1. a kind of growth and differentiation factor through modification, is coupled with growth and differentiation factor by trim and is formed, wherein, the growth point
It is shown in following (a) or (b) or (c) to change the factor：(a) the spontaneous growth differentiation 11 of mammal is derived from；(b) will be described
The amino acid sequence of spontaneous growth differentiation 11 passes through the substitution and/or missing and/or addition of one or more amino acid residues
And with protein or the polypeptide as derived from spontaneous growth differentiation 11 for promoting neuron regeneration ability；(c) with (a) or
(b) amino acid sequence shown in has at least 50% homology and with promotion neuron regeneration ability by (a) or (b) Suo Shi
Amino acid sequence derived from protein or polypeptide.
2. the growth and differentiation factor according to claim 1 through modification, it is characterised in that
The trim is tag polypeptide, plasma protein or the plasma protein fractions.
3. the growth and differentiation factor according to claim 2 through modification, it is characterised in that
The tag polypeptide be His-tag, Flag-tag, HA-tag, c-Myc-tag, AVi-tag, MBP-tag, GST-tag,
SNAP-tag, Halo-tag or SUMO-tag.
4. the growth and differentiation factor according to claim 2 through modification, it is characterised in that
The growth and differentiation factor through modification is by a growth and differentiation factor and one or more tag polypeptides
What coupling was formed；Preferably, the tag polypeptide is coupled by the N-terminal and/or C-terminal of covalent bond and the growth and differentiation factor.
5. the growth and differentiation factor according to claim 4 through modification, it is characterised in that
The growth and differentiation factor through modification is to be coupled shape by a growth and differentiation factor and one or more His-tag
Into, wherein, the amino acid sequence of the His-tag is as shown in SEQ ID NO.1 in sequence table or SEQ ID NO.2.
6. the growth and differentiation factor according to claim 4 through modification, it is characterised in that the Growth and Differentiation through modification
The factor is coupled with one or more GST-tag by a growth and differentiation factor and formed, wherein, the GST-tag's
Amino acid sequence is as shown in SEQ ID NO.3 in sequence table.
7. the growth and differentiation factor according to claim 3 through modification, it is characterised in that the amino acid of the Flag-tag
Sequence is as shown in SEQ ID NO.12 in sequence table, SEQ ID NO.13 institutes in the amino acid sequence such as sequence table of the HA-tag
Show, the amino acid sequence of the c-Myc-tag is as shown in SEQ ID NO.14 in sequence table, the amino acid sequence of the AVi-tag
Row are as shown in SEQ ID NO.15 in sequence table, SEQ ID NO.16 institutes in the amino acid sequence such as sequence table of the MBP-tag
Show, the amino acid sequence of the SNAP-tag is as shown in SEQ ID NO.17 in sequence table, the amino acid sequence of the Halo-tag
Row are as shown in SEQ ID NO.18 in sequence table, SEQ ID NO.19 institutes in the amino acid sequence such as sequence table of the SUMO-tag
Show.
8. the growth and differentiation factor according to claim 2 through modification, it is characterised in that the plasma protein is white egg
In vain, fibrinogen, transferrins, immunoglobulin, the one or more turned in thyroprotein and Thyroid binding protein；
The fragment of the plasma protein is the Fc regions of immunoglobulin.
9. the growth and differentiation factor according to claim 2 through modification, it is characterised in that the Growth and Differentiation through modification
The factor is coupled and formed with one or more plasma proteins or the plasma protein fractions by a growth and differentiation factor；
Preferably, the plasma protein or the plasma protein fractions pass through covalent bond and the N-terminal and/or C of the growth and differentiation factor
End coupling.
10. the growth and differentiation factor according to claim 1 through modification, it is characterised in that the growth and differentiation factor is
People source growth and differentiation factor 11, amino acid sequence is as shown in SEQ ID NO.4 in sequence table；Or the growth and differentiation factor is
Mouse source growth and differentiation factor 11, amino acid sequence is as shown in SEQ ID NO.5 in sequence table；Or the growth and differentiation factor is
The active fragment of people source growth and differentiation factor 11, amino acid sequence is as shown in SEQ ID NO.6 in sequence table；Or the growth
Differentiation factor is with the amino acid sequence in sequence table shown in SEQ ID NO.7.
11. encode the nucleic acid molecules of any growth and differentiation factors through modification of claim 1-10.
12. a kind of recombinant expression carrier for including nucleic acid molecules described in claim 11.
13. recombinant expression carrier according to claim 12, it is characterised in that the recombinant expression carrier is to be based on
The carrier of pET28a (+) plamid vector construction.
14. one kind is comprising nucleic acid molecules described in claim 11 or includes any recombinant expression carriers of claim 12-13
Host cell.
15. host cell according to claim 14, it is characterised in that the host cell is Escherichia coli.
16. the preparation method of any described growth and differentiation factors through modification of claim 1-10, it is characterised in that described to repair
Jewelry is coupled by way of chemical modification or amalgamation and expression with the growth and differentiation factor.
17. preparation method according to claim 16, it is characterised in that the amalgamation and expression mode prepares described through modification
The method of growth and differentiation factor include：
The synthesis step of the gene order of the coding growth and differentiation factor through modification；
The gene order of the growth and differentiation factor through modification is connected the step of forming recombinant vector with carrier is carrier；
The step of converting of the recombinant vector；
The screening step of host cell after conversion；
The expression step of induced fusion albumen；
The collection step of fusion protein；
The purification procedures of fusion protein.
18. preparation method according to claim 17, it is characterised in that the purification procedures use affinity chromatography
Post, cation exchange column and/or molecular sieve.
19. a kind of pharmaceutical composition, comprising：Claim 1-10 any described growth and differentiation factor or power through modification
Profit requires recombinant expression carrier described in nucleic acid molecules described in 11 or claim 12 or 13 or claims 14 or 15 institute
State host cell, and pharmaceutically acceptable carrier.
20. pharmaceutical composition according to claim 19, it is characterised in that the pharmaceutically acceptable carrier is：Medicine
Acceptable various conventional buffer solutions on, protein, gelatin, monose, polysaccharide, amino acid, chelating agent, sugar alcohol, polyethylene glycol,
And the one or more in surfactant.
21. a kind of sustained release preparation, comprising：Claim 1-10 any described growth and differentiation factor or right through modification
It is required that described in recombinant expression carrier described in nucleic acid molecules described in 11 or claim 12 or 13 or claims 14 or 15
Host cell or any described pharmaceutical compositions of claim 19-20, and pharmaceutically acceptable biofacies are tolerant
Matter；Preferably, the formulation of the sustained release preparation is liposome, microballoon, hydrogel, Osmotic minipumps or microcapsules.
22. it is a kind of comprising described in any described growth and differentiation factors or claim 11 through modification of claim 1-10
Host cell described in recombinant expression carrier described in nucleic acid molecules or claim 12 or 13 or claims 14 or 15 or
The kit of sustained release preparation described in any described pharmaceutical compositions of person's claim 19-20 or claim 21.
23. nucleic acid molecules described in any described growth and differentiation factors or claim 11 through modification of claim 1-10,
Or host cell described in recombinant expression carrier described in claim 12 or 13 or claims 14 or 15 or right will
Ask sustained release preparation described in any described pharmaceutical compositions of 19-20 or claim 21 or the examination described in claim 22
Application of the agent box in prevention, improvement or/and treatment senile dementia.
24. nucleic acid molecules described in any described growth and differentiation factors or claim 11 through modification of claim 1-10,
Or host cell described in recombinant expression carrier described in claim 12 or 13 or claims 14 or 15 or right will
Ask sustained release preparation described in any described pharmaceutical compositions of 19-20 or claim 21 or the examination described in claim 22
Application of the agent box in improving or treating cognitive function arrhythmic agents.
25. application according to claim 24, it is characterised in that the cognitive function is not normal to refer to the note related to aging
Recall power decline.
26. a kind of method for preventing, improving or treating senile dementia, this method is to give old age by certain method of administration
Dementia patients rights requires nucleic acid point described in any described growth and differentiation factors or claim 11 through modification of 1-10
Host cell or right described in recombinant expression carrier described in son or claim 12 or 13 or claims 14 or 15
It is required that sustained release preparation described in any described pharmaceutical compositions of 19-20 or claim 21.
27. a kind of improve or the not normal method for the treatment of cognitive function, this method is to give cognitive work(by certain method of administration
Nucleic acid point described in any described growth and differentiation factors or claim 11 through modification of people's claim 1-10 that can be not normal
Host cell or right described in recombinant expression carrier described in son or claim 12 or 13 or claims 14 or 15
It is required that sustained release preparation described in any described pharmaceutical compositions of 19-20 or claim 21.
28. according to the method for claim 26 or 27, it is characterised in that the method for administration is oral, schneiderian membrance administration, vein
Injection, intramuscular injection, drip-feed, intraperitoneal injection, arteria hepatica injection or subcutaneous embedding.
29. a kind of method of the growth and differentiation factor half-life period in vivo extended described in claim 1, by the Growth and Differentiation because
Son modification is prepared into nucleic acid described in any described growth and differentiation factors or claim 11 through modification of claim 1-10
Host cell or power described in recombinant expression carrier described in molecule or claim 12 or 13 or claims 14 or 15
Profit requires sustained release preparation described in any described pharmaceutical compositions of 19-20 or claim 21.
30. the metabolite that the growth and differentiation factor described in claim 1 is regulated and controled is in diagnosis and examination senile dementia disease
Purposes.Preferably, the metabolite that the growth and differentiation factor is regulated and controled is the one or more in following metabolite：Third
Acyl Propionyl AC (C3), lysophosphatidyl choline LysoPC a C18:2nd, phosphatidyl PC aa C36:6th, phosphatidyl C16:
1-OH, phosphatidyl PC aa C38:0th, phosphatidyl PC aa C38:6th, phosphatidyl PC aa C40:1st, phosphatidyl PC aa C40:2、
Phosphatidyl PC aa C40:6th, phosphatidyl PC ae C40:6.
31. whether a kind of determination tested patients are with senile dementia or with the method for suffering from senile dementia risk, bag
The concentration of the metabolite one or more in the blood sample for detecting the subject described in claim 30 is included, if the generation
The concentration for thanking to product meets or exceeds threshold value set in advance, then judges the subject with senile dementia or with suffering from
The risk of senile dementia.Preferably, the concentration of the metabolite is that the method for adopting metabolism group is determined.
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| CN108359013A (en) * | 2018-02-07 | 2018-08-03 | 上海交通大学 | A kind of artificial antigen submission nanometer formulation and its application for inside and outside specific amplification T cell |
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| CN113845583B (en) * | 2020-06-28 | 2023-08-11 | 江苏中新医药有限公司 | Modified recombinant human nerve growth factor and preparation method thereof |
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| WO2015038938A1 (en) * | 2013-09-13 | 2015-03-19 | The California Institute For Biomedical Research | Modified therapeutic agents and compositions thereof |
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| CN108129572A (en) * | 2018-01-04 | 2018-06-08 | 河南大学 | Application of the people GDF11-Fc fusion proteins in ulcerative colitis is treated |
| CN108359013A (en) * | 2018-02-07 | 2018-08-03 | 上海交通大学 | A kind of artificial antigen submission nanometer formulation and its application for inside and outside specific amplification T cell |
| CN109485716A (en) * | 2018-11-13 | 2019-03-19 | 广东美赛尔细胞生物科技有限公司 | A kind of modified growth and differentiation factor and the preparation method and application thereof |
| CN109485716B (en) * | 2018-11-13 | 2021-02-02 | 广东美赛尔细胞生物科技有限公司 | Modified growth differentiation factor and preparation method and application thereof |
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| CN109111517A (en) | 2019-01-01 |
| CN109134664B (en) | 2022-08-26 |
| CN109134664A (en) | 2019-01-04 |
| CN109111517B (en) | 2022-12-20 |
| CN107325168B (en) | 2018-10-30 |
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