CN109111517A - A kind of modified growth and differentiation factor and its preparation method and application - Google Patents
A kind of modified growth and differentiation factor and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of modified growth and differentiation factors, are coupled and are formed with growth and differentiation factor by modifier, the growth and differentiation factor are as follows: (a) derives from the spontaneous growth differentiation 11 of mammal；(b) amino acid sequence of the spontaneous growth differentiation 11 by the substitution and/or deletion and/or addition of one or more amino acid residues and had into the protein as derived from spontaneous growth differentiation 11 or polypeptide that promote neuron regeneration ability；Or (c) with (a) or (b) shown in amino acid sequence there is at least 50% homology and have promotion neuron regeneration ability as (a) or (b) shown in protein or polypeptide derived from amino acid sequence.Modified growth and differentiation factor of the invention can be preferably applied to prevention, improve and treat senile dementia, can also play the role of enhancing memory.In addition, the metabolite that growth and differentiation factor is regulated and controled has the purposes of diagnosis and screening senile dementia.
Description
The application be on 06 27th, 2017 the applying date, application No. is 201710500854.X, entitled " a kind of
The divisional application of the patent application of modified growth and differentiation factor and its preparation method and application ".
Technical field
The present invention relates to a kind of modification albumen, in particular to a kind of modified growth and differentiation factor and preparation method thereof and
Using.
Background technique
Alzheimer syndrome is called senile dementia, its main feature is that sick 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 disease cause of disease, current understanding is
The beta- amyloid protein and Tau albumen folded extremely is had accumulated in brain, so as to cause the generation of senile dementia
(Querfurth H.W.Alzheimer's Disease.N Engl J Med 2010；362,329-344).
Treating the drug of senile dementia currently on the market is mainly Western medicine, it is characterized in that must adhere to taking for a long time, so
And curative effect is small, can only alleviate partial symptoms, not can control the development of the state of an illness.Another defect taken for a long time is drug
Side effect is big, while patient generates 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 general? Lancet Psychiatry 2015；2(8):756-764).Thus in the market
Curative effect is better for urgent need, the lower drug 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 source of people GDF11 albumen on NCBI: NP_005802.1.
GDF11 albumen belongs to TGF-β superfamily protein member, there is 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,
Volume 10,Number 4,December 2010,pp.217–231.).GDF11 is played in the early embryonic development stage to focus on
It acts on, the development of the histoorgans such as adjustable spinal cord, olfactory nerve, bone, kidney and pancreas.GDF11 can also be significantly improved
The human-subject test of brain controls myocardial hypertrophy, and promotes 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).This effect of GDF11 is presently considered to be and is realized by TGF-β signal transduction.GDF11 can
It is combined with the receptor (I type and II type) with TGF-β, activation TGF-β downstream signal transduction access (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 discovery: recognizing for old mouse can be promoted to the blood plasma of old mouse continuous injection youth 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 promote the human-subject test of patients of senile dementia containing certain ingredient in blood plasma.Further study showed that:
The plasma composition of old mouse and the plasma composition of young mouse have significant difference, and maximum 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)。
It is very limited to the raising of human-subject test although GDF11 is related with the human-subject test of brain, it is therefore desirable to find
A kind of effect preferably can treat or be effectively relieved the new drug of senile dementia.
Summary of the invention
In view of the drawbacks of the prior art, one of the objects of the present invention is to provide a kind of modified growth and differentiation factors.
The second object of the present invention is to provide the preparation method of above-mentioned modified growth and differentiation factor.
The third object of the present invention is to provide the application of above-mentioned modified growth and differentiation factor.
The fourth object of the present invention is to provide the purposes for the metabolite that the growth and differentiation factor is regulated and controled.
To achieve the goals above, the invention adopts the following technical scheme:
A kind of modified growth and differentiation factor, is coupled with growth and differentiation factor by modifier and is formed, wherein the growth
Differentiation factor is as follows (a) or (b) or (c) shown: (a) deriving from the spontaneous growth differentiation 11 of mammal；(b) by institute
The amino acid sequence of spontaneous growth differentiation 11 is stated by the substitution of one or more amino acid residues and/or missing and/or is added
Add and there is the protein as derived from spontaneous growth differentiation 11 or polypeptide that promote neuron regeneration ability；(c) with (a) or
(b) amino acid sequence shown in is at least 50% homology and has the by (a) or (b) shown of promotion neuron regeneration ability
Amino acid sequence derived from protein or polypeptide.
In above-mentioned modified growth and differentiation factor, the modifier is that label is more as a preferred implementation manner,
Peptide, plasma protein or the plasma protein fractions.
In above-mentioned modified growth and differentiation factor, the tag polypeptide is His- as a preferred implementation manner,
Tag, Flag-tag, HA-tag, c-Myc-tag, AVi-tag, MBP-tag, GST-tag, SNAP-tag, Halo-tag or
SUMO-tag。
In above-mentioned modified growth and differentiation factor, the modified growth point as a preferred implementation manner,
Changing the factor is coupled and is formed by a growth and differentiation factor and one or more tag polypeptides；Preferably, described
Tag polypeptide is coupled by the N-terminal and/or C-terminal of covalent bond and the growth and differentiation factor.
In above-mentioned modified growth and differentiation factor, the modified growth point as a preferred implementation manner,
Changing the factor is to be coupled to be formed by a growth and differentiation factor and one or more His-tag, wherein the His-tag
Amino acid sequence as shown in SEQ ID NO.1 in sequence table or SEQ ID NO.2.
In above-mentioned modified growth and differentiation factor, the modified growth point as a preferred implementation manner,
Changing the factor is to be coupled to be formed by a growth and differentiation factor and one or more GST-tag, wherein the GST-tag
Amino acid sequence as shown in SEQ ID NO.3 in sequence table.
In above-mentioned modified growth and differentiation factor, the amino of the Flag-tag as a preferred implementation manner,
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 above-mentioned modified growth and differentiation factor, the plasma protein is white egg as a preferred implementation manner,
White, fibrinogen, immunoglobulin, turns one of thyroprotein and Thyroid binding protein or a variety of at transferrins；
The segment of the plasma protein is the region Fc of immunoglobulin.
In above-mentioned modified growth and differentiation factor, the modified growth point as a preferred implementation manner,
Changing the factor is coupled and is formed by a growth and differentiation factor and one or more plasma proteins or the plasma protein fractions
'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 above-mentioned modified growth and differentiation factor, the growth and differentiation factor is as a preferred implementation manner,
Source of people 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
Source of mouse 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 source of people 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 amino acid sequence shown in SEQ ID NO.7 in sequence table.
A kind of nucleic acid molecules encoding above-mentioned modified growth and differentiation factor.
A kind of recombinant expression carrier comprising above-mentioned nucleic acid molecules.
In above-mentioned recombinant expression carrier, the recombinant expression carrier is to be based on as a preferred implementation manner,
The carrier of pET28a (+) plamid vector construction.
A kind of host cell comprising above-mentioned nucleic acid molecules or comprising above-mentioned recombinant expression carrier.
In above-mentioned host cell, the host cell is Escherichia coli as a preferred implementation manner,.
The preparation method of above-mentioned modified growth and differentiation factor, the modifier pass through chemical modification or amalgamation and expression
Mode and the growth and differentiation factor are coupled.
In the above preparation method, as a preferred implementation manner, the amalgamation and expression mode prepare it is described through modifying
The method of growth and differentiation factor include:
Encode the synthesis step of the gene order of the modified growth and differentiation factor；The modified Growth and Differentiation
The gene order of the factor connect 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 the above preparation method, as a preferred implementation manner, the purification procedures using affinity column,
Cation exchange column and/or molecular sieve.
A kind of pharmaceutical composition includes: above-mentioned modified growth and differentiation factor or above-mentioned nucleic acid molecules or on
State recombinant expression carrier or above-mentioned host cell and pharmaceutically acceptable carrier.
In pharmaceutical composition, the pharmaceutically acceptable carrier as a preferred implementation manner, are as follows: pharmaceutically may be used
Various common buffers, the protein, gelatin, monosaccharide, polysaccharide, amino acid, chelating agent, sugar alcohol, polyethylene glycol, Yi Jibiao received
One of face activating agent is a variety of.
A kind of sustained release preparation includes: above-mentioned modified growth and differentiation factor 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 dosage form of the sustained release preparation is liposome, microballoon, hydrogel, Osmotic minipumps or microcapsules.
One kind is carried comprising above-mentioned modified growth and differentiation factor or above-mentioned nucleic acid molecules or above-mentioned recombinant expression
The kit of body or above-mentioned host cell or aforementioned pharmaceutical compositions or above-mentioned sustained release preparation.
Above-mentioned modified growth and differentiation factor 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.
Above-mentioned modified growth and differentiation factor 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 cognition
Application in malfunction drug.
In above-mentioned application, the cognitive function is not normal as a preferred implementation manner, refers to note relevant to aging
Recall power decline.
A method of prevention improves or treats senile dementia, and this method is given always by certain administration route
The above-mentioned modified growth and differentiation factor 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 method of improve or treatment cognitive function is not normal, this method is to give cognition function by certain administration route
Can the not normal above-mentioned modified growth and differentiation factor 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 the not normal method of above-mentioned prevention, the method for improvement or treatment senile dementia and improvement or treatment cognitive function
In, which is characterized in that the administration route is oral, schneiderian membrance administration, intravenous injection, intramuscular injection, intravenous drip, abdominal cavity note
It penetrates, arteria hepatica injection or subcutaneous embedding.
A method of extending above-mentioned growth and differentiation factor half-life period in vivo, the growth and differentiation factor is modified and is prepared
At above-mentioned modified growth and differentiation factor 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 screening senile dementia disease
In purposes.Preferably, the metabolite that the growth and differentiation factor is regulated and controled is one of following metabolite or a variety of:
Propionyl Propionyl AC (C3), lysophosphatidyl choline LysoPC a C18:2, phosphatidyl PC aa C36:6, phosphatidyl
C16:1-OH, phosphatidyl PC aa C38:0, phosphatidyl PC aa C38:6, phosphatidyl PC aa C40:1, phosphatidyl PC aa
C40:2, phosphatidyl PC aa C40:6, phosphatidyl PC ae C40:6.
Whether a kind of determining tested patients suffer from senile dementia or with the method for suffering from senile dementia risk, packets
One of above-mentioned metabolite or a variety of concentration in the blood sample for detecting the subject are included, if the metabolite
Concentration meets or exceeds preset threshold value, then determines that the subject suffers from senile dementia or has and suffer from senile dementia
The risk of disease.Preferably, the concentration of the metabolite is that the method for adopting metabolism group measures.
Compared with the prior art, the invention has the following beneficial effects:
1, modified growth and differentiation factor provided by the invention effectively extends under the premise of guaranteeing its basic function
Its half-life period in vivo.
2, simultaneously compared to literalness growth and differentiation factor 11 or its active fragment, the modified Growth and Differentiation of the present invention
The factor has preferably biological activity, can be preferably applied to prevention, improve and treatment senile dementia, and furthermore it can be with
Play the role of enhancing memory.
3, modified compound provided by the invention and the drug comprising the compound have the stability being metabolized in vivo,
It is used directly for or prevents as pharmaceutical composition ingredient, improves and treat senile dementia.
4, growth and differentiation factor of the present invention is substantially reduced in old dementia patients in-vivo content, and signal is logical downstream
The content and activity of the relevant enzyme on road reduce, and a variety of phosphatidyls and metabolism of carnitine product assay is caused to reduce.This small point of metabolism
The variation of sub- content can effectively predict the risk of senile dementia lurker, and can assist senile dementia disease
Diagnosis and screening.That is the downstream metabolites of growth and differentiation factor can be used for the auxiliary diagnosis of senile dementia.
Detailed description of the invention
Fig. 1 is the active fragment of the coding His-tag and human growth and differentiation factor 7 11 that are synthesized by full genome synthetic method
The genome of the coupled product of (with amino acid sequence shown in SEQ ID No.6 in sequence table) N-terminal；
Fig. 2 is the active fragment of the coding GST-tag and human growth and differentiation factor 7 11 that are synthesized by full genome synthetic method
The genome of the coupled product of (with 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 sub (i.e. human growth and differentiation factor 7 11 of the human growth and differentiation factor 7 with amino acid sequence shown in SEQ ID No.6 in sequence table
Active fragment) N-terminal be coupled after the result of the SDS electrophoresis detection of eluent purified with affinity chromatography.
Purifying refers to that the supernatant containing coupled product for obtaining embodiment 1 crosses nickel column, 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 that coupling produces
Object passes through the eluent of ni-sepharose purification.
Fig. 4 is GST-tag and the life with 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) purifies to obtain after being coupled with affinity chromatography
Eluent SDS electrophoresis detection 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 buffer of the glutathione to dissociate 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
With the eluent of pillar purifying.
Fig. 5 be in embodiment 6 20kD polyethylene glycol with there is 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
The result of the SDS electrophoresis detection of the eluent purified.Purifying refers to the reaction solution mistake containing coupled product
SourceQ pillar finally carries out gradient elution with the 5mM Tris hydrochloride buffer (pH8.0) containing 500mM sodium chloride, thus
Collect the eluent containing unit point modified outcome.Swimming lane 1: protein molecular weight standard；Swimming lane 2-10 is coupled product process
The eluent of SourceQ purifying.
Fig. 6 be in embodiment 8 40kD polyethylene glycol with there is 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
The result of the SDS electrophoresis detection of the eluent purified.Purifying refer to by embodiment 7 obtain containing coupled product
Reaction solution crosses SourceQ pillar, finally carries out gradient with the 5mM Tris hydrochloride buffer (pH8.0) containing 500mM sodium chloride
Elution, 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 of SourceQ purifying.
Fig. 7 is in embodiment 9, Examples 1 and 2 preparation after modifying growth and differentiation factor (i.e. chemical compounds I or
His-tag-GDF11) promote the value-added activity figure of primary hippocampal cells.
Fig. 8 is in embodiment 12, embodiment 3 and 4 prepare after modifying growth and differentiation factor (i.e. compound ii or
GST-tag-GDF11) promote the value-added activity figure of primary hippocampal cells.
Fig. 9 is in embodiment 15, embodiment 5 and 6 prepare after modifying growth and differentiation factor (i.e. compound III or
20KD polyethylene glycol-GDF11) promote the value-added activity figure of primary hippocampal cells.
Figure 10 is in embodiment 18, embodiment 7 and 8 prepare after modifying growth and differentiation factor (i.e. compounds Ⅳ or
40KD polyethylene glycol-GDF11) promote the value-added activity figure of primary hippocampal cells.
Figure 11 is in embodiment 10, and chemical compounds I promotes the activity figure of primary hippocampal cells Synaptic formation.
Figure 12 is in embodiment 13, and compound ii promotes the activity figure of primary hippocampal cells Synaptic formation.
Figure 13 is in embodiment 16, and compound III promotes the activity figure of primary hippocampal cells Synaptic formation.
Figure 14 is in embodiment 19, and compounds Ⅳ promotes the activity figure of primary hippocampal cells Synaptic formation.
Figure 15 is in embodiment 20, the promotion activity figure of chemical compounds I, II, III and IV pair of old mouse human-subject test.(a)
Finding the time used in hiding platform changes over time curve；(b) in target quadrant the time it takes.
Figure 16 is in embodiment 21, the promotion activity figure of chemical compounds I, II, III and IV pair of senile dementia mouse human-subject test.
(a) finding the time used in hiding platform changes over time curve；(b) in target quadrant the time it takes.
Figure 17 is the image of the primary hippocampal cells in embodiment 10, after different disposal.Toward the training of primary hippocampal cells
It supports and is 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, that is, GDF11 active fragment is as experimental group.Control group hippocampal cell only adds DMEM culture medium.Micro- after one day
Hippocampal cell is imaged under mirror.In figure, (a) is (b) (c) chemical compounds I, GDF11 active fragment and DMEM processing respectively
Primary hippocampal cells afterwards.
Figure 18 is the content pair of GDF11 in normal population and senile dementia human albumin's extract in embodiment 22
Than.
Figure 19 is the changes of contents contrast table of normal population and 10 kinds of senile dementia patients metabolism small molecules in embodiment 23
Lattice.
Specific embodiment
In order to be preferably illustrated to technical characteristic and effect of the invention, below using specific 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 modified growth and differentiation factor (alternatively referred to as modified compound), which is
Compound after growth and differentiation factor is modified, is coupled with growth and differentiation factor by modifier and is formed.The Growth and Differentiation because
Son is shown for following (a) or (b) or (c): (a) deriving from the spontaneous growth differentiation 11 of mammal；It (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 deletion and/or addition and have promote neuron regeneration ability by natural
Protein derived from growth and differentiation factor 11 or polypeptide；(c) with (a) or (b) shown in amino acid sequence have at least 50% (ratio
Such as 50%, 60%, 70%, 80%, 90%, 95%, 99%) homology and having promote neuron regeneration ability by (a) or
(b) protein or polypeptide derived from amino acid sequence shown in.Naturally refer in the spontaneous growth differentiation 11 all with life
It is coming or self-assembling formation.
Heretofore described growth and differentiation factor can be the natural growth point of source of people, source of mouse or other mammals
Change the factor 11, the growth and differentiation factor homology of separate sources can reach 83.5%；It is also possible to spontaneous growth differentiation factor
11 amino acid sequence is by the substitution and/or deletion and/or addition of one or more amino acid residues and with promotion neuron
The protein as derived from spontaneous growth differentiation 11 or polypeptide of power of regeneration, such as: the work of these growth and differentiation factors 11
Property segment；With 50% or more homology and either there is promotion neuron regeneration energy with these growth factors or active fragment
The protein or polypeptide of power derived by these growth and differentiation factor 11 or active fragments.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 source of mouse
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 the peptide fragment for promoting neuron regeneration ability from growth and differentiation factor 11 and with growth and differentiation factor 11
(Katsimpardi L.Science 2014；344,630-634) SEQ ID NO.6, the SEQ ID, such as in sequence table
The amino acid sequence source of people growth and differentiation factor 11 of NO.6 and the common ground of source of mouse growth and differentiation factor 11, implementation of the invention
Example, which also further demonstrates amino acid sequence polypeptide shown in SEQ ID NO.6, has promotion neuron regeneration ability (referring to Fig. 7-
14)；The growth and differentiation factor can also be with source of people growth and differentiation factor 11 with 70% or more homology and with promotion
Protein shown in SEQ ID NO.7, amino acid sequence are in neuron regeneration ability protein or polypeptide, such as sequence table
The common ground of source of people Growth differentiation factor 8 and source of mouse Growth differentiation factor 8.
The modified growth and differentiation factor is even by an above-mentioned growth and differentiation factor and one or more modifiers
What connection was formed, coupling can be covalent bond and be also possible to non-covalent bond；It can be repaired by chemical modification or amalgamation and expression to realize
The coupling of jewelry and growth and differentiation factor.
The modifier can be tag polypeptide, plasma protein, the plasma protein fractions, macromolecule or small molecule.
In the present invention, the polypeptide refers to the ol cpds as made of 2~100 amino acid molecular dehydrating condensations,
Molecular weight is lower than 10,000Da.The protein is to be formed between the alpha amino acid by nucleic acid encode by α amino and α carboxyl
The peptide chain that peptide bond is formed by connecting, translated post-processing and generate with specific stereochemical structure, active macromolecular.High score
Son refer to other than isolating protein as numerous atoms or atomic group mainly with relative molecular weight made of Covalent bonding together 10,000
Above compound.Small molecule refers to molecule of the molecular weight less than 500.
When the modifier is tag polypeptide, it is preferable that the tag polypeptide is His-tag, and (it has Flag-tag
Amino acid sequence shown in SEQ ID NO.12 in sequence table), (it is with shown in SEQ ID NO.13 in sequence table by HA-tag
Amino acid sequence), c-Myc-tag (it is with amino acid sequence shown in SEQ ID NO.14 in sequence table), AVi-tag (its
With amino acid sequence shown in SEQ ID NO.15 in sequence table), (it is with SEQ ID NO.16 in sequence table by MBP-tag
Shown in amino acid sequence), (it is with amino acid sequence shown in SEQ ID NO.17 in sequence table by GST-tag, SNAP-tag
Column), Halo-tag (it is with amino acid sequence shown in SEQ ID NO.18 in sequence table), (it is with sequence by SUMO-tag
Amino acid sequence shown in SEQ ID NO.19 in table).It is highly preferred that the tag polypeptide is His-tag, amino acid sequence
As shown in SEQ ID NO.1 in sequence table or SEQ ID NO.2, label H is-tag by covalent bond, that is, peptide bond with
The N-terminal and/or C-terminal of the growth and differentiation factor are coupled.It is highly preferred that the tag polypeptide is GST-tag, amino acid sequence
As shown in SEQ ID NO.3 in sequence table, a tag polypeptide GST-tag passes through covalent bond, that is, peptide bond and the growth point
Change N-terminal and/or the C-terminal coupling of the factor.In addition above-mentioned label can increase the molecular weight of albumen itself, 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 modifier is the segment of plasma protein or plasma protein, it is preferable that the plasma protein be albumin,
Fibrinogen, immunoglobulin, turns one of thyroprotein and Thyroid binding protein or a variety of at transferrins；Institute
The segment for stating plasma protein is the region Fc of immunoglobulin.The segment of one plasma protein or plasma protein passes through covalent
The N-terminal and/or C-terminal of key, that is, peptide bond and the growth and differentiation factor are coupled.
When the modifier is the segment of tag polypeptide or plasma protein or plasma protein, can by chemical modification or
The mode of amalgamation and expression realizes the coupling of modifier Yu the growth and differentiation factor, that is, prepares the modified growth point of the present invention
Change the factor.
The method that the amalgamation and expression mode prepares the modified growth and differentiation factor successively includes:
Encode the synthesis step of the gene order of the modified growth and differentiation factor；The modified Growth and Differentiation
The gene order of the factor connect 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 modifier is macromolecule, macromolecule is pharmaceutically common macromolecule, it is preferable that the macromolecule is
Polyethylene glycol (PEG)；The polyethylene glycol is linear or bifurcated molecule, molecular weight 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.Modifier and life can be realized by way of chemical modification
The coupling of long differentiation factor prepares the modified growth and differentiation factor of the present invention.The chemical modification mode prepares the warp
The method of the growth and differentiation factor of modification includes: mixing step: by the growth and differentiation factor and modifier with appropriate mole
Than mixing；Reaction step: being added reaction reagent into mixed solution, chemically react, to obtain reaction solution；Purifying
Step: the reaction solution is purified, to obtain the modified growth and differentiation factor.Preferably, 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 modifier carries out unit point modification to growth and differentiation factor, then need to select special unit point modification polyethylene glycol
(the polyethylene glycol title of single-point modification are as follows: mPEG-butyrALD, purchase come real from the triumphant positive biology in Beijing, article No. KZ-SC)
Existing.
When the modifier 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 free amino, sulfydryl or the carboxyl in covalent bond and growth and differentiation factor, and different is small
Molecule coupling labeled group may be different.I.e. growth and differentiation factor be glycosylated, phosphorylation, acylation, ethylization or methylation.Specifically
Preparation method be enzymic catalytic reaction method: growth and differentiation factor is mixed with small numerator modified object, it is anti-that corresponding enzymatic is added
It should.
When modifier is the segment of tag polypeptide, plasma protein or plasma protein, the present invention also provides encode 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, which can be, passes through full genome
Synthetic method obtains, and the nucleic acid molecules of modifier can also will be encoded, with coding blood plasma by designing identical restriction enzyme site
The nucleic acid molecules of the segment of albumen or plasma protein link together.
When modifier is the segment of tag polypeptide, plasma protein or plasma protein, the present invention also provides include 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 modifier is the segment of tag polypeptide, plasma protein or plasma protein, the present invention also provides include above-mentioned core
Acid molecule or host cell comprising above-mentioned recombinant expression carrier.Preferably, the host cell is Escherichia coli
(Escherichiacoli)。
The present invention also provides a kind of pharmaceutical compositions, include: above-mentioned modified growth and differentiation factor, above-mentioned nucleic acid point
Sub, above-mentioned recombinant expression carrier or above-mentioned host cell；And pharmaceutically acceptable carrier.The pharmaceutically acceptable carrier
Are as follows: pharmaceutically acceptable various common buffers (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, monosaccharide, polysaccharide, amino acid, chela
One in mixture (such as EDTA), sugar alcohol (such as mannitol), polyethylene glycol and surfactant (such as TWEEN and PLURONICS)
Kind is a variety of.
Preferably, described pharmaceutical composition is including following component: 0.01-0.2M phosphoric acid in terms of 100 microlitres by total volume
Buffer (pH 7.0), 0.01-0.1M glucose, 0.001-0.05M mannitol, modified Growth and Differentiation described in 1-5 microgram
The factor.It is highly preferred that described pharmaceutical composition, it is in terms of 100 microlitres by total volume, including following component: 0.1M phosphate buffer
(pH 7.0), 0.05M glucose, 0.01M mannitol, 2 microgram growth and differentiation factors.
The dosage form of described pharmaceutical composition may is that tablet, capsule, oral solution, syrup, particle, dripping pill；Water needle, freeze-drying
Powder needle, aseptic powder injection, injection liquor, spray formulation.Preferably parenteral solution formulation or spray formulation.
Described pharmaceutical composition can be administered as follows: oral, schneiderian membrance administration, intravenous injection, intramuscular injection,
Intravenous drip, intraperitoneal injection, arteria hepatica injection and subcutaneous embedding.Unit dosage form are as follows: modified growth point described in 1 microgram
Change the factor/kg body weight.
The present invention also provides a kind of sustained release preparations, include: above-mentioned modified growth and differentiation factor, 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 dosage form of the sustained release preparation is liposome, microballoon, hydrogel, Osmotic minipumps or microcapsules.
It include above-mentioned modified growth and differentiation factor, above-mentioned nucleic acid molecules, above-mentioned recombination the present invention also provides one kind
Expression vector, above-mentioned host cell, aforementioned pharmaceutical compositions or above-mentioned sustained release preparation kit.It can also be wrapped in kit
Include other substances, such as negative control or positive control etc..
Above-mentioned modified growth and differentiation factor, 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.
Above-mentioned modified growth and differentiation factor, 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 improving or are treating answering in cognitive function arrhythmic agents
With.The cognitive function is not normal to refer to failure of memory relevant to aging.
A method of prevention improves or treats senile dementia, and this method is given always by certain administration route
The above-mentioned modified growth and differentiation factor 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 method of improve or treatment cognitive function is not normal, this method is to give cognition function by certain administration route
The not normal above-mentioned modified growth and differentiation factor 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 administration route is oral, schneiderian membrance administration, intravenous injection, intramuscular injection, intravenous drip, abdominal cavity
Injection, arteria hepatica injection or subcutaneous embedding.
A method of extending above-mentioned growth and differentiation factor Half-life in vivo, growth and differentiation factor modification is prepared into
Above-mentioned modified growth and differentiation factor, above-mentioned nucleic acid molecules, above-mentioned recombinant expression carrier, above-mentioned host cell, said medicine
Composition or above-mentioned sustained release preparation.
It below by embodiment prepared by the compound in the present invention and application is described in detail.
Various culture mediums and common agents used in the present invention are all made of conventional method preparation, divide involved in embodiment
Specific experimental condition and method is such as not specified in sub- biologic operation, please refers to SambrookJ etc. and edits, Science Press, and 2002,
The specification of Molecular Cloning:A Laboratory guide (third edition) or corresponding product.
What the N-terminal of sub 11 active fragments of embodiment 1:His-tag and human growth and differentiation factor 7 carried out that coupling is prepared contains
The liquid of His-tag-GDF11 (chemical compounds I)
His-tag-GDF11 manufactured in the present embodiment, amino acid sequence referring to sequence table SEQ ID NO.8, wherein
His-tag has amino acid sequence shown in SEQ ID NO.2, and the active fragment of human growth and differentiation factor 7 11 has SEQ ID
Amino acid sequence shown in NO.6, the free carboxy in His-tag sequence on C-terminal, that is, histidine are coupled to human growth and differentiation factor 7
On the alpha- amino of N-terminal, peptide bond is formed.
The present embodiment chemical compounds I specific the preparation method is as follows:
(1) pass through the gene of full genome synthetic method composite coding chemical compounds I: the gene has SEQ ID in sequence table
Base sequence shown in NO.10, the gene successively contain (being detailed in Fig. 1 and SEQ ID NO.10) from 5 ' ends to 3 ' ends: Nco I enzyme
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 1 active fragment of differentiation 1, Xho I restriction enzyme site (the 363rd base-the
368 bases)；This step is completed by Sangon Biotech (Shanghai) Co., Ltd., which is connected to pUC57 plasmid vector
On.
(2) by the pUC57 plasmid of the gene (with base sequence shown in SEQ ID NO.10) containing coded compounds I
Carrier carries out restriction enzyme cutting: 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 carried out 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 ligase, condition of contact be 16 DEG C overnight, gene order A and plasmid in connection reaction
The molar ratio of Frame sequence B is 4:1, obtains connection product；
(5) connection product is converted into e. coli bl21 (DE3), the condition of conversion be 42 DEG C thermal shock 90 seconds；It (6) will conversion
Escherichia coli afterwards are uniformly applied to the solid LB plate containing ammonia benzyl chloramphenicol resistance, and (wherein the content of ammonia benzyl mycin is 50 micro-
Grams per milliliter) on, 37 DEG C are incubated overnight, and can grow many bacterium colonies on LB plate at this time；(7) 4 to 5 bacterium colonies are chosen to 1 liter with toothpick
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
It supports, the revolving speed rocked is set as 220rpm.Culture to bacterium solution absorbance is between 0.4-0.6 600nm at when, addition IPTG lure
Protein expression is led, the IPTG ultimate density of addition is 0.6mM；The condition of inducible protein expression is 37 DEG C, 220rpm；
(8) Escherichia coli are collected by centrifugation after inducing 5 hours, centrifugal condition 1500g, 15 minutes；
(9) Escherichia coli of collection 5mM Tris hydrochloride buffer is suspended, then carries out ultrasonication, finally by
12000g is centrifuged 15min precipitating residue, lives in the supernatant obtained at this time containing a large amount of His-tag and human growth and differentiation factor 7 11
Property the compound i.e. His-tag-GDF11 (compound I) that is coupled in N-terminal of segment, supernatant can directly upper nickel column into
The purifying of row embodiment 2.
Purified after the N-terminal of 11 active fragment of embodiment 2:His-tag modified human growth and differentiation factor with nickel column
His-tag inside the present embodiment has amino acid sequence shown in SEQ ID NO.2, human growth and differentiation factor 7 tool
There is amino acid sequence shown in SEQ ID NO.6, His-tag is coupled to the N-terminal of 11 active fragment of growth and differentiation factor.Specifically
Ground, the growth and differentiation factor of the His-tag modification of the present embodiment are the His-tag-GDF11 that embodiment 1 obtains.
The present embodiment specific implementation method is successively are as follows:
(1) nickel column is balanced: containing 2 milliliters of glucan pearls in nickel column, with 20 milliliters of 5mM Tris hydrochloric acid
(pH8.0) pillar balance was carried out；
(2) supernatant containing coupled product for obtaining embodiment 1 crosses nickel column, flow velocity 2ml/min, coupling here
Product is the growth and differentiation factor of N-terminal and His-tag coupling；
(3) pillar is rinsed with the 5mM Tris hydrochloride buffer (pH8.0) containing 20mM imidazoles, flow velocity 2ml/min is gone
Except foreign protein；
(4) pillar, flow velocity 2ml/ are eluted with the 5mM Tris hydrochloride buffer (pH8.0) containing 400mM imidazoles again
Min, collects eluent, and the growth and differentiation factor containing a large amount of N-terminals and His-tag coupling in eluent is even as a result referring to Fig. 3
The size of co-product is 13kDa.
Sub 11 active fragments of embodiment 3:GST-tag and human growth and differentiation factor 7 N-terminal carry out coupling be prepared containing
The liquid of GST-tag-GDF11 (compound ii)
GST-tag inside the present embodiment has amino acid sequence shown in SEQ ID NO.3, human growth and differentiation factor 7 11
Active fragment has amino acid sequence shown in SEQ ID NO.6, and the carboxyl of GST-tag C-terminal passes through covalent bond, that is, peptide bond coupling
To the N-terminal of sub 11 active fragments of human growth and differentiation factor 7, coupled product, that is, GST-tag-GDF11, the amino acid sequence of the modification albumen
Column are as shown in SEQ ID NO.9.
The present embodiment specific implementation method is as follows:
(1) pass through the gene of full genome synthetic method composite coding GST-tag-GDF11: the gene has in sequence table
Base sequence shown in SEQ ID NO.11, the gene successively contain (being detailed in Fig. 2 and SEQ ID NO.11) from 5 ' ends to 3 ' ends:
Nco I restriction enzyme site (the 1st the-the 6 base of base), is compiled the nucleotide sequence (the 7th the-the 660 base of base) for encoding GST-tag
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 site (the
991 the-the 996 bases of base)；This step is completed by Sangon Biotech (Shanghai) Co., Ltd., which is connected to
On pUC57 plasmid vector.
(2) (will there is base sequence shown in SEQ ID NO.11 in sequence table comprising the gene for encoding GST-tag-GDF11
Column) pUC57 plasmid vector carry out restriction enzyme cutting: restriction enzyme be Nco I and Xho I, cleavage reaction temperature
Degree is 37 DEG C, and the time is 2 hours, and the gene content of cleavage reaction is 50ng；Obtain small fragment: gene order A (coding GST-
The gene of tag-GDF11)；
(3) pET28a (+) plasmid vector is carried out 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 is that 50ng obtains large fragment: the Frame sequence B of plasmid；
(4) purifying and connection of the gene and plasmid after restriction enzyme cutting: respectively to the frame of said gene sequence A and plasmid
Frame sequence B carries out agarose electrophoresis purifying, and the Frame sequence B of gene order A and plasmid after purification are carried out with T4 ligase
Connection, condition of contact are 16 DEG C and stay overnight that the molar ratio of the Frame sequence B of gene order A and plasmid is 4:1 in connection reaction, obtain
To connection product；
(5) connection product is converted into e. coli bl21 (DE3), the condition of conversion be 42 DEG C thermal shock 90 seconds；
(6) Escherichia coli after conversion are uniformly applied to solid LB plate (the wherein ammonia benzyl containing ammonia benzyl chloramphenicol resistance
The content of mycin is 50 mcg/mls) on, 37 DEG C are incubated overnight, and can grow many bacterium colonies on LB plate at this time；
(7) 4 to 5 bacterium colonies are chosen to 1 liter of LB liquid medium (wherein ammonia benzyl mycin containing ammonia benzyl chloramphenicol resistance with toothpick
Content be 50 mcg/mls) it is inner, 37 DEG C are rocked culture, and the revolving speed rocked is set as 220rpm.Culture is to bacterium solution in 600nm
When locating absorbance between 0.4-0.6, the expression of IPTG inducible protein is added, the IPTG ultimate density of addition is 0.6mM；Induce egg
The condition of white expression is 37 DEG C, 220rpm；
(8) Escherichia coli are collected by centrifugation after inducing 5 hours, centrifugal condition 1500g, 15 minutes；
(9) Escherichia coli of collection 5mM Tris hydrochloride buffer is suspended, then carries out ultrasonication, finally by
12000g is centrifuged 15min precipitating residue, contains a large amount of GST-tag and sub 11 active tablets of human growth and differentiation factor 7 in supernatant at this time
The compound i.e. GST-tag-GDF11 (compound ii) that section is coupled in N-terminal, supernatant are used directly for purifying.
With containing the affine of glutathione after the N-terminal of 11 active fragment of embodiment 4:GST-tag modified human growth and differentiation factor
Column is purified.
GST-tag inside the present embodiment has SEQ ID NO.3 sequence, and human growth and differentiation factor 7 11 active fragments of son have
SEQ ID NO.6 sequence, GST-tag are 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 obtains.
The present embodiment specific implementation method is successively are as follows:
(1) affinity column containing mating type glutathione is balanced: the affinity column of QIAGEN mating type glutathione
In contain 2 milliliters of glucan pearls, with 20 milliliters of 5mM Tris hydrochloric acid (pH8.0) carried out pillar balance；
(2) supernatant containing coupled product for preparing embodiment 3 crosses pillar, flow velocity 2ml/min, coupling here
Product is the growth and differentiation factor of N-terminal and GST-tag coupling；
(3) with containing 200mM sodium chloride 5mMTris hydrochloride buffer (pH8.0) rinse pillar, flow velocity 2ml/min,
Remove foreign protein；
(4) pillar is eluted with the 5mM Tris hydrochloride buffer (pH8.0) of the glutathione to dissociate containing 25mM, flow velocity is
2ml/min collects eluent, the growth and differentiation factor containing a large amount of N-terminals and GST-tag coupling in eluent, 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 is prepared
Liquid containing 20kDa polyethylene glycol-GDF11 (compound III)
Sub 11 active fragments of the human growth and differentiation factor 7 that the present embodiment uses have amino acid sequence shown in SEQ ID NO.6
Column.
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 (there is amino acid sequence shown in SEQ ID NO.6,
Purchased from PeproTech, 120-11), protein concentration is measured at a wavelength of 280 nm, then adjusts concentration to 5mg/ml；
(2) sub 11 active fragments of human growth and differentiation factor 7 that 10ml concentration is 5mg/ml are taken, 20kDa is added thereto and 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 coupled of end dissociative) solid 100mg, is stirred in room temperature condition to being completely dissolved, polyethylene glycol here is for spy
The N-terminal of different 11 active fragment of modified human growth and differentiation factor, the polyethylene glycol and sub 11 active fragments of above-mentioned human growth and differentiation factor 7
Mixed molar ratio is 5:1, obtains 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.At this time 90% or more
Sub 11 active fragments of human growth and differentiation factor 7 by single polyethyleneglycol modified, i.e. a polyethylene glycol and a growth and differentiation factor
11 molecules are coupled, and conjugation sites are the alpha-amidos at the end growth and differentiation factor 11N.At this moment, reaction solution is used directly for
Anion exchange column purification.
Anion column is used after the N-terminal of sub 11 active fragments of the polyethyleneglycol modified human growth and differentiation factor 7 of embodiment 6:20kDa
SourceQ purifying
The reaction solution containing 20kDa polyethylene glycol-GDF11 (compound III) that embodiment 5 is obtained is chromatographed with SourceQ
Column (GE Healthcare) purifying.The specific method is as follows:
(1) chromatographic column is balanced with containing 5mM Tris hydrochloride buffer (pH8.0)；
(2) reaction solution containing 20kDa polyethylene glycol-GDF11 for obtaining embodiment 5 after balancing carries out loading；
(3) after loading, gradient elution is carried out with A liquid and B liquid mixed liquor, wherein A liquid is 5mM Tris hydrochloride buffer
(pH8.0), B liquid is the 5mM Tris hydrochloride buffer (pH8.0) containing 500mM sodium chloride, specific gradient elution method are as follows:
Pillar 5min only is crossed with A liquid at the beginning, gradually decreases the ratio of A liquid after this, simultaneously gradually promotes the ratio of B liquid,
So that by the unit volume eluent of pillar, the ratio of B liquid in 50min in, from 0% it is gradually linear promoted to 100%,
To effectively establish the NaCl concentration gradient for crossing pillar, carrying out gradient elution, (this step can pass through Akta egg
White purification system software set, GE Healthcare).Timing when since gradient elution, unreacted polyethylene glycol substantially on
Neutral, so being eluted out at first after 10min, the sequence that protein peak occurs in elution is that the life that multidigit point is modified is long
Sub 11 active fragments of the human growth and differentiation factor 7 that 1 active fragment of differentiation 1 (elution starts rear 18min appearance), unit point are modified
(elution starts rear 30min appearance) and unmodified human growth and differentiation factor 7 11 active fragments of son (elution starts rear 45min appearance).
Different elution protein peaks can be collected by the ultraviolet detection of 280nm, the eluent for collecting second protein peak obtains only
The alpha-amido of 11 active fragment N-terminal of growth and differentiation factor is by polyethyleneglycol modified compound, to isolate and purify out unit point
Sub 11 active fragments of the human growth and differentiation factor 7 of modification.For purification result referring to 5, 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 is prepared
Liquid containing 40kDa polyethylene glycol-GDF11 (compounds Ⅳ)
Sub 11 active fragments of the human growth and differentiation factor 7 that the present embodiment uses have amino acid sequence shown in SEQ ID NO.6
Column.The present embodiment specific implementation method is as follows:
(1) 2 microlitres of 11 active fragments of human growth factor is taken (to there is amino acid sequence shown in SEQ ID NO.6, be purchased from
PeproTech, 120-11), protein concentration is measured at a wavelength of 280 nm, then adjusts concentration to 5mg/ml；
(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 in room temperature condition to being completely dissolved, to obtain sub 11 active fragments of human growth and differentiation factor 7 and 40kDa
The mixture of polyethylene glycol；Here polyethylene glycol is polyethylene glycol (mPEG-ButyrALD, the molecule of special modification albumen n end
Measure 40kDa, Nektar, the alpha amino coupled which can only dissociate with albumen n end), polyethylene glycol and people's Growth and Differentiation
The molar ratio of 11 active fragment of factor mixing is 5:1；
(3) it is reducing agent sodium cyanoborohydride (the CH3BNNa) (concentration of reducing agent in the mixture after addition that concentration, which is added,
It is stored at room temperature 24 hours for 50mM) with salt acid for adjusting pH to 5.The obtained reaction solution containing 40kDa polyethylene glycol-GDF11.
At this time in the reaction solution 11 active fragments of 90% or more human growth and differentiation factor 7 by single polyethyleneglycol modified, an i.e. poly- second
Glycol is coupled with a 11 active fragment molecule of growth and differentiation factor, and conjugation sites are the α-at the end growth and differentiation factor 11N
Amino.At this moment, reaction solution is used directly for anion exchange column purification.
Anion column is used after the N-terminal of sub 11 active fragments of the polyethyleneglycol modified human growth and differentiation factor 7 of embodiment 8:40kDa
SourceQ purifying
The reaction solution containing 40kDa polyethylene glycol-GDF11 (compounds Ⅳ) that embodiment 7 is obtained is chromatographed with SourceQ
Column (GE Healthcare) purifying.The specific method is as follows:
(1) chromatographic column is balanced with containing 5mM Tris hydrochloride buffer (pH8.0)；
(2) reaction solution containing 40kDa polyethylene glycol-GDF11 for obtaining embodiment 7 after balancing carries out loading；
(3) after loading, gradient elution is carried out with A liquid and B liquid mixed liquor, wherein A liquid is 5mMTris hydrochloride buffer
(pH8.0), B liquid is the 5mM Tris hydrochloride buffer (pH8.0) containing 500mM sodium chloride, the method for specific gradient elution
Are as follows: pillar 5min only is crossed with A liquid at the beginning, gradually decreases the ratio of A liquid after this, simultaneously gradually promotes the ratio of B liquid
Example so that by the unit volume eluent of pillar, the ratio of B liquid in 50min in, from 0% gradually it is linear promoted to
100%, to effectively establish the NaCl concentration gradient for crossing pillar, carrying out gradient elution, (this step can pass through
Akta protein purification system software set, GE Healthcare).Timing when since gradient elution, unreacted polyethylene glycol
Substantially without charge, so being eluted out at first after 6min.The sequence that protein peak occurs in elution is the modification of multidigit point
Human growth and differentiation factor 7 11 active fragments of son (elution starts rear 15min appearance), human growth and differentiation factor 7 11 of unit point modification are living
Property segment (elution starts rear 22min appearance) 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 the ultraviolet detection of 280nm, collect the eluent of second protein peak i.e.
The alpha-amido of only 11 active fragment N-terminal of growth and differentiation factor is obtained by polyethyleneglycol modified compound, to isolate and purify out
Sub 11 active fragments of the human growth and differentiation factor 7 of unit point modification.Purification result is 53kDa referring to Fig. 6, the size of coupled product.
Sub 11 active fragments of the human growth and differentiation factor 7 of the end embodiment 9:N and His-tag coupling, i.e. Examples 1 and 2 are prepared into
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 product after purification for taking out 2 microlitres, measures its absorbance at ultraviolet 280nm, from
And calculate the protein concentration of coupled product.With the primary hippocampal cells (original used in the present invention in 24 orifice plate culture rat sources
It is equal referring to the method recorded in following documents for the cultural method of hippocampal cell: Guo, W., Y.Ji, et al. (2014) "
Neuronal activity alters BDNF-TrkB signaling kinetics and downstream
Functions. " J Cell Sci127 (Pt 10): 2249-2260.), culture used medium is DMEM, condition of culture 37
DEG C, 5% carbon dioxide.Until cell density reaches every hole 4 × 105When a cell, toward a some holes of 24 orifice plates in (6 holes) plus
Enter coupled product, the coupled product being added in each hole 500 microgram containing albumen, as experiment A group-His-tag-GDF11.Simultaneously
(it is with SEQ for sub 11 active fragments of human growth and differentiation factor 7 of 500 micrograms of (6 holes) addition in toward the other hole of 24 orifice plates
The amino acid sequence of IDNO.6), as experiment B group-GDF11.Finally, a remaining some holes (6 holes) is not done and is located in 24 orifice plates
Reason, as blank control group.
Continue culture cell 24 hours under conditions of original, then under an optical microscope to experiment A group, experiment B group
Cell count is carried out respectively with blank control group, calculates separately experiment A group, the experiment white cellular control unit of B group cell number duty
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 increment, the activity of coupled product is the active more than 4 times of sub 11 active fragments of human growth and differentiation factor 7, specific to join
See Fig. 7.
Product, that is, Examples 1 and 2 that embodiment 10:His-tag and sub 11 active fragments of human growth and differentiation factor 7 are coupled in N-terminal
The His-tag-GDF11 being prepared promotes the activity of primary hippocampal cells Synaptic formation
The present embodiment specific implementation method is as follows:
The His-tag-GDF11 coupled product after purification for taking out 2 microlitres, measures its absorbance at 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 item
Part is 37 DEG C, 5% carbon dioxide.Until cell density reaches every hole 4 × 105When a cell, toward the partial hole of 24 orifice plates in plus
Enter coupled product, the coupled product being added in each hole 500 microgram containing albumen, as experiment A group-His-tag-GDF11 (A group
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
(its amino acid sequence with SEQ ID NO.6), as experiment B group (6 holes of B group coprocessing).Finally, remaining in 24 orifice plates
A some holes be not processed, as blank control group (blank control group totally 6 holes).
Continue culture cell 24 hours under conditions of original, then under an optical microscope to experiment A group, experiment B group
Cynapse counting is carried out respectively with blank control group, calculates separately experiment A group, the experiment white control group cynapse of B group cynapse number duty
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 of His-tag coupling) compares life
Long 1 active fragment of differentiation 1 can more promote the formation of primary hippocampal cynapse, and the activity of coupled product is human growth and differentiation factor 7
11 active fragments it is active more than 5 times, referring specifically to Figure 11.Different groups of hippocampal cell is imaged under the microscope, is joined
Figure 17 is seen, from Figure 17 as it can be seen that experiment B group (i.e. 11 activity of human growth and differentiation factor 7 of the amino acid sequence with SEQ ID NO.6
Segment processing) more cynapses obviously are formed than blank control group, experiment A group (His-tag-GDF11 processing) is obvious again than real
It tests B and forms more cynapses.
Product, that is, embodiment 3 and 4 that embodiment 11:GST-tag and sub 11 active fragments of human growth and differentiation factor 7 are coupled in N-terminal
The long-acting benefit of the GST-tag-GDF11 being prepared in blood
GST-tag inside the present embodiment has SEQ ID NO.3 sequence, and human growth and differentiation factor 7 11 active fragments of son have
Amino acid sequence shown in SEQ ID NO.6, GST-tag are coupled to the N-terminal of growth and differentiation factor.
The present embodiment passes through measurement GST-tag-GDF11 coupled product and sub 11 active tablets of unmodified human growth and differentiation factor 7
Section (its amino acid sequence with SEQ ID NO.6) is in mouse intracorporal half-life period, thus life after examining GST-tag to modify
The long-acting benefit of long 1 active fragment of differentiation 1.It is specific as follows: to select 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 product, the dosage of injection are 20mg/kg weight.After injection, point is used at the following time
Take blood needle 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.After taking 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 product respectively with ELISA
Concentration.Measurement result discovery: human growth and differentiation factor 7 11 active fragments of son are 5 hours average, GST- in mouse intracorporal half-life period
Tag-GDF11 coupled product is 24 hours average in mouse intracorporal half-life period.This shows the coupled product energy of GST-tag modification
Sub 11 active fragments of human growth and differentiation factor 7 are significantly improved in the half-life period of Mice Body intracellular metabolite, thus the treatment with the longer time
Effect.
Product, that is, embodiment 3 and 4 that embodiment 12:GST-tag and sub 11 active fragments of human growth and differentiation factor 7 are coupled in N-terminal
The GST-tag-GDF11 being prepared promotes the value-added activity of primary hippocampal cells
GST-tag inside the present embodiment has amino acid sequence shown in SEQ ID NO.3, human growth and differentiation factor 7 11
Active fragment has 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: 2 microlitres of GST-tag-GDF11 coupled product is taken out, in ultraviolet 280nm
Lower its absorbance of measurement, to calculate the protein concentration of coupled product.With 24 orifice plate culture primary hippocampal cells, training used is cultivated
Supporting base is DMEM, and condition of culture is 37 DEG C, 5% carbon dioxide.Until cell density reaches every hole 4 × 105When a cell, toward 24
GST-tag-GDF11 coupled product is added in one some holes of orifice plate, coupled product 500 microgram containing albumen being added in each hole,
As experiment A group.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
(its amino acid sequence with SEQ ID NO.6), as experiment B group.Finally, a remaining some holes is not done and is located in 24 orifice plates
Reason, as blank control group.
Continue culture cell 24 hours under conditions of original, then under an optical microscope to experiment A group, experiment B group
Cell count is carried out respectively with blank control group, calculates separately experiment A group, the experiment white cellular control unit of B group cell number duty
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 increment, the activity of coupled product are active 2 times of the sub 11 active fragment active fragments of human growth and differentiation factor 7
It is more, referring specifically to Fig. 8.
Product, that is, embodiment 3 and 4 that embodiment 13:GST-tag and sub 11 active fragments of human growth and differentiation factor 7 are coupled in N-terminal
The GST-tag-GDF11 being prepared promotes the activity of primary hippocampal cells Synaptic formation
GST-tag inside the present embodiment has amino acid sequence shown in SEQ ID NO.3, human growth and differentiation factor 7 11
Active fragment has 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: 2 microlitres of GST-tag-GDF11 coupled product is taken out, in ultraviolet 280nm
Lower its absorbance of measurement, to calculate the protein concentration of coupled product.With 24 orifice plate culture primary hippocampal cells, training used is cultivated
Supporting base is DMEM, and condition of culture is 37 DEG C, 5% carbon dioxide.Until cell density reaches every hole 4 × 105When a cell, toward 24
GST-tag-GDF11 coupled product is added in one some holes of orifice plate, coupled product 500 microgram containing albumen being added in each hole,
As experiment A group.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
(its amino acid sequence with SEQ ID NO.6), as experiment B group.
Finally, a remaining some holes is not processed in 24 orifice plates, as blank control group.Continue to train under conditions of original
It supports cell 24 hours, cynapse counting is then carried out respectively to experiment A group, experiment B group and blank control group under an optical microscope,
Calculate separately experiment A group, the experiment white control group number of synapses purpose ratio of B group cynapse number duty.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 that sub 11 active fragments of human growth and differentiation factor 7 are active more than 3 times, referring specifically to Figure 12.
Embodiment 14:20kDa polyethylene glycol and sub 11 active fragments of human growth and differentiation factor 7 are implemented in the product that N-terminal is coupled
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 conjugation
Section, sub 11 active fragments of the human growth and differentiation factor 7 being related to have amino acid sequence shown in SEQ ID NO.6.
The present embodiment passes through measurement 20kDa polyethylene glycol-GDF11 coupled product and unmodified human growth and differentiation factor 7 11
Active fragment (its amino acid sequence with SEQ ID NO.6) is in mouse intracorporal half-life period, to examine the poly- second two of 20kD
The long-acting benefit of GDF11 after alcohol modification.The adult mice specific as follows for selecting 12 C75BL/6 strains, average weight are 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 product, the dosage of injection is 20mg/kg weight.After injection, point is with taking at the following time
Blood needle takes blood in mice eye: 5 minutes, 10 minutes, and 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.After taking blood, blood plasma is collected by centrifugation in room temperature, and centrifugal rotational speed is
1000g, centrifugation time are 30 minutes.
Survey human growth and differentiation factor 7 11 active fragments of son and 20kDa polyethylene glycol-GDF11 coupling in blood plasma respectively with ELISA
The concentration of product.Measurement result discovery: human growth and differentiation factor 7 11 active fragments of son are average 5 small in mouse intracorporal half-life period
When, coupled product is 18 hours average in mouse intracorporal half-life period.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, thus the treatment with the longer time
Effect.
Embodiment 15:20kDa polyethylene glycol and sub 11 active fragments of human growth and differentiation factor 7 are implemented in the product that N-terminal is coupled
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 conjugation
Section, sub 11 active fragments of the human growth and differentiation factor 7 being related to have amino acid sequence shown in SEQ ID NO.6.
The present embodiment specific implementation method is as follows: taking out 2 microlitres of coupled product, its extinction is measured at ultraviolet 280nm
Degree, 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.Until cell density reaches every hole 4 × 105When a cell, toward some of 24 orifice plates
Coupled product, the coupled product being added in each hole 500 microgram containing albumen, as experiment A group are added in hole.Simultaneously toward 24 orifice plates
Other hole in sub 11 active fragments (its amino acid with SEQ ID NO.6 of human growth and differentiation factor 7 of 500 micrograms is added
Sequence), as experiment B group.Finally, a remaining some holes is not processed in 24 orifice plates, as blank control group.
Continue culture cell 24 hours under conditions of original, then under an optical microscope to experiment A group, experiment B group
Cell count is carried out respectively with blank control group, calculates separately experiment A group, the experiment white cellular control unit of B group cell number duty
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 the active 1.5 times of left sides of sub 11 active fragments of human growth and differentiation factor 7
The right side, referring specifically to Fig. 9.
Embodiment 16:20kDa polyethylene glycol and sub 11 active fragments of human growth and differentiation factor 7 are implemented in the product that N-terminal is coupled
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 conjugation
Section, sub 11 active fragments of the human growth and differentiation factor 7 being related to have amino acid sequence shown in SEQ ID NO.6.
The present embodiment specific implementation method is as follows: 2 microlitres of 20kDa polyethylene glycol-GDF11 coupled product is taken out, in purple
Its absorbance is measured under outer 280nm, to calculate the protein concentration of coupled product.With 24 orifice plate culture primary hippocampal cells, training
Supporting used medium is DMEM, and condition of culture is 37 DEG C, 5% carbon dioxide.Until cell density reaches every hole 4 × 105It is a thin
When born of the same parents, toward a some holes of 24 orifice plates in be added coupled product, the coupled product being added in each hole 500 microgram containing albumen, as
Test A group.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 the amino acid sequence of SEQ ID NO.6), as experiment B group.Finally, a remaining some holes is not processed in 24 orifice plates, as
Blank control group.
Continue culture cell 24 hours under conditions of original, then under an optical microscope to experiment A group, experiment B group
Cynapse counting is carried out respectively with blank control group, calculates separately experiment A group, the experiment white control group cynapse of B group cynapse number duty
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
It can promote the formation of primary hippocampal cynapse, the activity of coupled product is sub active 1.2 times of 11 active fragments of human growth and differentiation factor 7
Left and right, referring specifically to Figure 13.
Embodiment 17:40kDa polyethylene glycol and sub 11 active fragments of human growth and differentiation factor 7 are implemented in the product that N-terminal is coupled
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 conjugation
Section, sub 11 active fragments of the human growth and differentiation factor 7 being related to have amino acid sequence shown in SEQ ID NO.6.
Pass through measurement 40kDa polyethylene glycol-GDF11 coupled product and sub 11 active fragments of unmodified human growth and differentiation factor 7
(its amino acid sequence with SEQ ID NO.6) in mouse intracorporal half-life period, thus after examining 40kD polyethyleneglycol modified
The long-acting benefit of GDF11.It is specific as follows: to select the adult mice of 12 C75BL/6 strains, average weight is 30g or so.It 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 product, the dosage of injection are 20mg/kg weight.After injection, point is with taking blood needle to exist at the following time
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.After taking blood, blood plasma is collected by centrifugation in room temperature, and centrifugal rotational speed is
1000g, centrifugation time are 30 minutes.
Survey human growth and differentiation factor 7 11 active fragments of son and 40kDa polyethylene glycol-GDF11 coupling in blood plasma respectively with ELISA
The concentration of product.Measurement result discovery: human growth and differentiation factor 7 11 active fragments of son are average 5 small in mouse intracorporal half-life period
When, coupled product is 16 hours average in mouse intracorporal half-life period.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, thus the treatment with the longer time
Effect.
Embodiment 18:40kDa polyethylene glycol and sub 11 active fragments of human growth and differentiation factor 7 are implemented in the product that N-terminal is coupled
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 conjugation
Section, sub 11 active fragments of the human growth and differentiation factor 7 being related to have amino acid sequence shown in SEQ ID NO.6.
The present embodiment specific implementation method is as follows: 2 microlitres of 40kDa polyethylene glycol-GDF11 coupled product is taken out, in purple
Its absorbance is measured under outer 280nm, to calculate the protein concentration of coupled product.With 24 orifice plate culture primary hippocampal cells, training
Supporting used medium is DMEM, and condition of culture is 37 DEG C, 5% carbon dioxide.Until cell density reaches every hole 4 × 105It is a thin
When born of the same parents, toward a some holes of 24 orifice plates in coupled product is added, the 40kDa polyethylene glycol-GDF11 coupled product being added in each hole
Containing 500 microgram of albumen, as experiment A group.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 (its amino acid sequence with SEQ ID NO.6), as experiment B group.Finally, remaining one in 24 orifice plates
Some holes is not processed, as blank control group.
Continue culture cell 24 hours under conditions of original, then under an optical microscope to experiment A group, experiment B group
Cell count is carried out respectively with blank control group, calculates separately experiment A group, the experiment white cellular control unit of B group cell number duty
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 the active 1.8 times of left sides of sub 11 active fragments of human growth and differentiation factor 7
The right side, referring specifically to Figure 10.
Embodiment 19:40kDa polyethylene glycol and sub 11 active fragments of human growth and differentiation factor 7 are implemented in the product that N-terminal is coupled
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 conjugation
Section, sub 11 active fragments of the human growth and differentiation factor 7 being related to have amino acid sequence shown in SEQ ID NO.6.
The present embodiment specific implementation method is as follows: 2 microlitres of 40kDa polyethylene glycol-GDF11 coupled product is taken out, in purple
Its absorbance is measured under outer 280nm, to calculate the protein concentration of coupled product.With 24 orifice plate culture primary hippocampal cells, training
Supporting used medium is DMEM, and condition of culture is 37 DEG C, 5% carbon dioxide.Until cell density reaches every hole 4 × 105It is a thin
When born of the same parents, toward a some holes of 24 orifice plates in be added coupled product, the coupled product being added in each hole 500 microgram containing albumen, as
Test A group.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 the amino acid sequence of SEQ ID NO.6), as experiment B group.Finally, a remaining some holes is not processed in 24 orifice plates, as
Blank control group.
Continue culture cell 24 hours under conditions of original, then under an optical microscope to experiment A group, experiment B group
Cynapse counting is carried out respectively with blank control group, calculates separately experiment A group, the experiment white control group cynapse of B group cynapse number duty
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
It can promote the formation of primary hippocampal cynapse, the activity of coupled product is sub active 1.4 times of 11 active fragments of human growth and differentiation factor 7
Left and right, referring specifically to Figure 14.
Embodiment 20: chemical compounds I (His-tag-GDF11 of Examples 1 and 2 preparation), compound ii (make by 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 promoted at aged mice (18 monthly ages) cognitive ability.
The 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 guarantees 25 micro- grams/times of dosage, administration 8 times (average to be administered once every three days) in 24 days,
Administration group 1-4 is 20 18 months C57BL/6 hero mouse, and chemical compounds I-IV, which is sequentially allocated, gives 1-4 administration group.No administration group is 20
Only 18 months C57BL/6 hero mouse receive 8 Saline injections for 24 days before Behaviors survey.Negative control group is 20 3
A month C57BL/6 hero mouse receives 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 carries 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
The position of platform of group, water maze is probabilistically assigned, and is remained unchanged in entire training.In training, mouse is released from any position
It is put into water maze, and it is allowed to search for hiding platform in 120 seconds.If mouse does 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 automatically recorded by intelligent video camera head
Get off.Water maze test carries out after last time training 48 hours, and every mouse is released into the water fan of not placement platform
Gong Zhong, and it is allowed to move freely 60 seconds.Its route that moves about is automatically recorded by intelligent video camera head.When the test phase is than training period
Between it is one times short, to avoid mouse generate 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 mouse time used in target quadrant in water maze test, can be with from figure
Find out that the Gdf11 after 20kD is polyethyleneglycol modified can significantly improve the spatial memory of mouse.
Embodiment 21: chemical compounds I (His-tag-GDF11 of Examples 1 and 2 preparation), compound ii (make by 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
It is bred and is raised.Each experimental mouse model all passes through rat-tail and carries out identified for genes, it is ensured that app gene and PS1 gene
Stablize mutation.Chemical compounds I-IV is injected into mouse body respectively by caudal vein.Administration group guarantees 25 micro- grams/times of administration
Dosage, the administration before Behaviors survey in 24 days 8 times, administration group 1-4 are 20 14 week old 5XFAD mouse, chemical compounds I-
IV is sequentially allocated and gives 1-4 administration group.No administration group is the 5XFAD mouse of 20 14 week old, is received within 24 days before Behaviors survey
8 Saline injections.Negative control group is the C57BL/6 hero mouse of 20 14 week old, equally receives Saline injection.Behaviouristics
Experiment is the learning ability and memory that water maze laboratory is used to detect mouse.Water maze laboratory is the same as embodiment 20.Test result
Referring to Figure 16, wherein figure (a) is spatial memory learning ability curve graph, after as can be seen from the figure 20kD is polyethyleneglycol modified
Gdf11 can significantly improve the Spatial learning abilities of Elderly dementia patients；Scheming (b) is Senlie dementia model mouse spatial memory
Assessment figure, as can be seen from the figure His-tag modification 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.
Firstly, with heparin tube 3 milliliters of everyone blood plasma of vein of acquisition of sodium citrate, 1 milliliter of centrifuging and taking supernatant, up and down
4 DEG C of transports are to use for laboratory in protein extracting after being mixed by inversion.The blood plasma isolated averagely is dispensed into the lab
In 1.5mlEP pipe, -80 DEG C of refrigerators are saved.
Secondly, blood plasma is taken out from -80 DEG C of refrigerators, it is inserted in and thaws on ice, concussion is mixed and is inserted in ice after defrosting.It is transferring
Buffer (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).It is checked under same volume normal
The variation of the protein expression level of people's group and old dementia patients group, one-dimensional native gel electrophoresis separates target protein laggard
Row Western blot.The antibody of GDF11 is incubated for and the goat-anti rabbit-anti of horseradish peroxidase-labeled jointly in 5% skimmed milk power
Body combination.Concentrations versus's result of GDF11 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, having for detecting is bright
The small molecule and its variation tendency such as Figure 19 of aobvious changes of contents.
One, it extracts
1) blood plasma is taken out from -80 DEG C of refrigerators, is inserted in and thaws on ice, concussion is mixed and is inserted in ice after defrosting；
2) filter paper that filter paper breaks into 6mm is laid in 96 orifice plates with punch；
3) 5 μ L blood plasma are drawn with liquid-transfering gun to add on filter paper, 100ul100% methanol is added；
4) sample is stored at room temperature 40min；
5) in the static whole supernatants to EP pipe of rear absorption, and 4 DEG C are centrifuged, 12000rpm, 20min；
6) it is drawn in the dedicated specimen bottle of the supreme machine of 80ul supernatant after being centrifuged with rifle, machine in waiting.
Two, 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 pillar (2.1 millimeters of height, 50 millimeters of internal diameters) (GE Healthcare), mobility is used
The mixture (volume ratio 2:1) of acetonitrile and isopropanol；Mass spectrum uses following parameter: air pressure inside 25psi, ionizer are
ESI, ionizer voltage are set as 5000V, and temperature setting is 300 degree, the room Q2 collision energy 40eV.
Testing result shows in the metabolite of GDF11, propionyl Propionyl AC (C3), lysophosphatidyl choline
LysoPC a C18:2, phosphatidyl PC aa C36:6, phosphatidyl C16:1-OH, phosphatidyl PC aa C38:0, phosphatidyl PC aa
C38:6, phosphatidyl PC aa C40:1, phosphatidyl PC aa C40:2, phosphatidyl PC aa C40:6, phosphatidyl PC aeC40:6
Concentration in normal person and old dementia patients there are significant difference, referring specifically to Figure 19, it is possible thereby to determine, above-mentioned metabolism
Product can be used for diagnosing and screening senile dementia disease, i.e., using above-mentioned metabolite as the marker of senile dementia.When
The concentration of above-mentioned one or more metabolites meets or exceeds preset threshold value 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
The measurement of metabolism group method.
Sequence table
<110>Jiangsu Hao Si Biotechnology Co., Ltd
<120>a kind of modified growth and differentiation factor and its preparation method and application
<130> M1CNCN180676
<150> 2016104959971
<151> 2016-06-28
<160> 19
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
His His His His His His
1 5
<210> 2
<211> 10
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Met Gly Ser Ser His His His His His His
1 5 10
<210> 3
<211> 218
<212> PRT
<213>artificial sequence (Artificial Sequence)
<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>mankind (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>mouse (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>mankind (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>mankind (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 (Artificial Sequence)
<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 (Artificial Sequence)
<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 (Artificial Sequence)
<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 (Artificial Sequence)
<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 (Artificial Sequence)
<400> 12
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
<210> 13
<211> 9
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 13
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
1 5
<210> 14
<211> 10
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 14
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
<210> 15
<211> 15
<212> PRT
<213>artificial sequence (Artificial Sequence)
<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 (Artificial Sequence)
<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 (Artificial Sequence)
<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 (Artificial Sequence)
<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 (Artificial Sequence)
<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 (12)
1. a kind of preparation method of pegylated growth differentiation factor, the preparation method include the following steps:
(i) protein modified polyethylene glycol will be used for and growth and differentiation factor solution is added, form the first mixture；
(ii) reducing agent is added to first mixture, obtains the crude product containing the pegylated growth differentiation factor
Second mixture.
2. the preparation method of pegylated growth differentiation factor as described in claim 1, the preparation method further include as
Lower step:
(iii) second mixture is purified, the sterling of the pegylated growth differentiation factor is obtained；
Optional, the growth and differentiation factor is as follows (a) or (b) or (c) shown: (a) deriving from the spontaneous growth of mammal
Differentiation 11；(b) by the amino acid sequence of the spontaneous growth differentiation 11 by 1,2, or more amino acid
The substitution and/or deletion and/or addition of residue and being spread out by spontaneous growth differentiation 11 with promotion neuron regeneration ability
Raw protein or polypeptide；(c) with (a) or (b) shown in amino acid sequence have at least 50%, 60%, 70%, 80%,
83.5%, 90%, 95% or 99% homology and having promote neuron regeneration ability as (a) or (b) shown in amino acid
Protein or polypeptide derived from sequence；
Optional, the growth and differentiation factor is source of people growth and differentiation factor 11, SEQ ID NO.4 in amino acid sequence such as sequence table
It is shown；Or the growth and differentiation factor is source of mouse growth and differentiation factor 11, SEQ ID NO.5 in amino acid sequence such as sequence table
It is shown；Or the growth and differentiation factor is the active fragment of source of people growth and differentiation factor 11, in amino acid sequence such as sequence table
Shown in SEQ ID NO.6；Or the growth and differentiation factor is with amino acid sequence shown in SEQ ID NO.7 in sequence table
Column；
It is optional, the N-terminal that the growth and differentiation factor is connected to for protein modified polyethylene glycol；
It is optional, it is described for protein modified polyethylene glycol be linear or bifurcated polyethylene glycol；
Optional, the molecular weight for protein modified polyethylene glycol is 1-100KD, 10-80KD or 20-40KD；
It is optional, it is described for protein modified polyethylene glycol be monomethyl polyethylene glycol mPEG-ButyrALD；
Optional, described for the molar ratio of protein modified polyethylene glycol and the growth and differentiation factor is 1:5-5:1, preferably 1:
3-3:1,1:2-2:1, more preferable 1:1；
Optional, the reducing agent is sodium cyanoborohydride；
Optional, the molar ratio for protein modified polyethylene glycol and the reducing agent is 1:1-200, preferably 1:20-
150, more preferable 1:50-100；
Optional, step (ii) is carried out under the conditions of pH value is 3-10；
It is optional, in step (iii), the second mixture described in anion exchange column purification；
It is optional, in step (iii), the second mixture described in SourceQ column chromatography.
3. a kind of pegylated growth differentiation factor, the pegylated growth differentiation factor is according to claim 1 or 2
What the preparation method was prepared.
4. a kind of preparation method of pegylated growth differentiation factor, the preparation method is that: protein modified gather will be used for
The N-terminal or C-terminal or pendant reactive group of ethylene glycol and growth and differentiation factor connect.
5. the preparation method of pegylated growth differentiation factor as claimed in claim 4, the preparation method further include as
Lower step:
The trip for passing through covalent bond and the growth and differentiation factor for protein modified polyethylene glycol and growth and differentiation factor
From amino, sulfydryl or carboxyl coupling；
Optional, the growth and differentiation factor is as follows (a) or (b) or (c) shown: (a) deriving from the spontaneous growth of mammal
Differentiation 11；(b) by the amino acid sequence of the spontaneous growth differentiation 11 by 1,2, or more amino acid
The substitution and/or deletion and/or addition of residue and being spread out by spontaneous growth differentiation 11 with promotion neuron regeneration ability
Raw protein or polypeptide；(c) with (a) or (b) shown in amino acid sequence have at least 50%, 60%, 70%, 80%,
83.5%, 90%, 95% or 99% homology and having promote neuron regeneration ability as (a) or (b) shown in amino acid
Protein or polypeptide derived from sequence；
Optional, the growth and differentiation factor is source of people growth and differentiation factor 11, SEQ ID NO.4 in amino acid sequence such as sequence table
It is shown；Or the growth and differentiation factor is source of mouse growth and differentiation factor 11, SEQ ID NO.5 in amino acid sequence such as sequence table
It is shown；Or the growth and differentiation factor is the active fragment of source of people growth and differentiation factor 11, in amino acid sequence such as sequence table
Shown in SEQ ID NO.6；Or the growth and differentiation factor is with amino acid sequence shown in SEQ ID NO.7 in sequence table
Column；
It is optional, it is described for protein modified polyethylene glycol be linear or bifurcated polyethylene glycol；
It is optional, the molecular weight for protein modified polyethylene glycol be 1-100KD, 10-80KD or 20-40KD,
Optional, described for the molar ratio of protein modified polyethylene glycol and the growth and differentiation factor is 1:5-5:1, preferably 1:
3-3:1,1:2-2:1, more preferable 1:1.
6. a kind of pegylated growth differentiation factor, the pegylated growth differentiation factor is according to claim 4 or 5
What the preparation method was prepared.
7. a kind of pharmaceutical composition, includes: pegylated growth differentiation factor described in claim 3 or 6 and pharmaceutically may be used
The carrier of receiving；
Optionally, the pharmaceutically acceptable carrier are as follows: pharmaceutically acceptable buffer, protein, gelatin, monosaccharide, more
One of sugar, amino acid, chelating agent, sugar alcohol, polyethylene glycol and surfactant are a variety of.
8. a kind of sustained release preparation, includes: pegylated growth differentiation factor or claim described in claim 3 or 6
Pharmaceutical composition described in 7 and pharmaceutically acceptable biocompatible substance；
Preferably, the dosage form of the sustained release preparation is liposome, microballoon, hydrogel, Osmotic minipumps or microcapsules.
9. the preparation method of the pegylated growth differentiation factor as described in claim 1,2,4 or 5, such as claim 3 or 6
The pegylated growth differentiation factor, pharmaceutical composition as claimed in claim 7, or it is as claimed in claim 8
Sustained release preparation in prevention, improve, slow down or/and treat senile dementia, prevent, improve, slow down or/and treat cognitive function mistake
Often, improve and learn energy or/and memory, promote hippocampal cell increment or/and promote hippocampal cell Synaptic formation, or prevent, change
Be apt to, slow down or/and treat can by promote hippocampal cell increment or/and promote hippocampal cell Synaptic formation prevent, improve,
Slow down or/and application or purposes in the disease treated.
10. the preparation method of the pegylated growth differentiation factor as described in claim 1,2,4 or 5, such as claim 3 or
Pegylated growth differentiation factor described in 6, pharmaceutical composition as claimed in claim 7, or it is as claimed in claim 8
Sustained release preparation, for preventing, improving, slow down or/and treating senile dementia, prevents, improves, slows down or/and treats and recognize in preparation
Know malfunction, improve learning ability or/and memory, promote hippocampal cell increment or/and promotes hippocampal cell Synaptic formation,
Or prevent, improve, slow down or/and treat can by promote hippocampal cell increment or/and promote hippocampal cell Synaptic formation
Application or purposes in the drug of the disease prevented, improve, slow down or/and treated, therapeutic agent or diagnostic reagent.
11. a kind of improvement learning ability or/and memory, or promote hippocampal cell increment or/and promote hippocampal cell cynapse shape
At method, the method is non-treatment purpose, and the method is gathering as described in claim 3 or 6 to subject's application
Glycation growth and differentiation factor, pharmaceutical composition as claimed in claim 7 or sustained release preparation as claimed in claim 8.
12. a kind of kit, the kit includes the pegylated growth differentiation factor as described in claim 3 or 6, such as
Pharmaceutical composition as claimed in claim 7 or sustained release preparation as claimed in claim 8.
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| CN108129572B (en) * | 2018-01-04 | 2021-01-29 | 河南大学 | Application of human GDF11-Fc fusion protein in treating ulcerative colitis |
| 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 |
| 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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| WO2015038938A1 (en) * | 2013-09-13 | 2015-03-19 | The California Institute For Biomedical Research | Modified therapeutic agents and compositions thereof |
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| CN113845583A (en) * | 2020-06-28 | 2021-12-28 | 江苏中新医药有限公司 | Modified recombinant human nerve growth factor and preparation method thereof |
| 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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| CN107325168A (en) | 2017-11-07 |
| CN109134664B (en) | 2022-08-26 |
| CN109111517B (en) | 2022-12-20 |
| CN107325168B (en) | 2018-10-30 |
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