CN104558148B - Ciliary nerve trophic factor mutant and its modification type mutant and purposes - Google Patents
Ciliary nerve trophic factor mutant and its modification type mutant and purposes Download PDFInfo
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- CN104558148B CN104558148B CN201310491131.XA CN201310491131A CN104558148B CN 104558148 B CN104558148 B CN 104558148B CN 201310491131 A CN201310491131 A CN 201310491131A CN 104558148 B CN104558148 B CN 104558148B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
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- A—HUMAN NECESSITIES
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Abstract
The present invention provides a kind of ciliary nerve trophic factor mutant and its modification type mutant and purposes.Wherein, the ciliary nerve trophic factor mutant is by the basis of the amino acid sequence of ciliary neurotrophic factor, 63rd glutamine is sported arginine, and eliminated obtained from 15 amino acid of C-terminal by the cysteine for remaining the 17th.Ciliary nerve trophic factor mutant through the invention can obtain the high modification type ciliary neurotrophic factor of modification specificity, to overcome brought by modification as modified outcome is inhomogenous, the series of problems such as unstable result, and keep recombinant C NTF protein structures more stable, there is higher biological activity and solubility compared with native protein, and modification type ciliary neurotrophic factor has long-acting compared with unmodified type ciliary neurotrophic factor.
Description
Technical field
The invention belongs to biomedicine field, in particular to a kind of modification type ciliary nerve trophic factor mutant,
DNA, recombinant expression carrier, genetically engineered host cell, modification type ciliary nerve trophic factor mutant, ciliary nerves battalion
Support purposes, the pharmaceutical composition of factor mutant and modification type ciliary nerve trophic factor mutant.
Background technology
Ciliary neurotrophic factor (Ciliary neurotrophic factor, referred to as CNTF) is a kind of small molecule
Albumen, overall length are made of 200 amino acid, and molecular weight is in 23KD or so.CNTF is stored with two dimeric forms in vivo,
Developmental biology acts on after enzymolysis is at monomer.CNTF can promote to include motor neuron, sensory neuron, sympathetic nerve
The growth and differentiation of a variety of nerve cells such as member, hippocampal neuron and Deiter's cells.
CNTF is because be initially to be extracted from the ciliary body of chicken, and can maintain the survival of chicken parasympathetic ganglion and obtain
Name.It is found in the experimental study that Jiang Renyuan CNTF are used to treat muscular dystrophy lateral sclerosis of spinal cord (ALS) nineties,
Clinical research is carried out to one group of ALS patient and finds that the weight of patient substantially reduces, it is apparent using medicine group weight ratio placebo
It reduces.CNTF is once used to treatment amyotrophic lateral sclerosis (ALS), but is terminated due to side reaction is too big and uncertain therapeutic efficacy is cut
Clinical trial, but it is surprised to find that the weight of drug user is all decreased obviously in an experiment.Regeneron companies are according to this hair afterwards
It is existing, it develops recombined human CNTF drugs and the three phases clinical trial of obesity is completed, and achieve statistically with notable
The result of sex differernce.Research shows that CNTF can adjust the weight set point of body positioned at the receptor of hypothalamus by activating
Section ingests to play the role of reduction, loses weight.For this result tested in obesity field, as a result
Show that CNTF can be combined in vivo with the specific receptor of hypothalamus, activate the key signal access of appetite-suppressing, inhibits feed
Desire and body is made not will produce hunger, to make weight loss.The study found that CNTF has multiple functions, can promote more
The survival of kind nerve cell, nutrition muscle promote glucose-lipid metabolism and adjust energy balance and feeding behaviour.In nervous system,
Muscle systems is of great significance and wide clinical landscapes in the treatment of obesity extremely relevant disease.
And since there are half-life shorts in treating or preventing obesity and its relevant disease by CNTF, it is active lower scarce
It falls into, the mutant (or variant) of various CNTF, therefore the routine of CNTF mutant has been had developed in currently available technology
What preparation method was known to those skilled in the art.
Such as Chinese patent application discloses No.CN1687413A and discloses a kind of CNTF mutant (hCNTF), the hCNTF
The 17th cysteine replace with alanine；63rd glutamine replaces with arginine；64th histidine replaces
It is changed to alanine；Remove 1-30 amino acid of C-terminal of hCNTF.In addition, CN1687413A also discloses a kind of CNTF mutation
Body, the 17th cysteine become serine, and remove 16 amino acid of C-terminal.
Chinese patent application discloses No.CN1387568A and discloses a kind of CNTF mutant --- Ax15 of commercialization,
15 amino acid that 63 glutamine acid is sported arginine and C-terminal are substituted and the 17th cysteine
It is replaced.
Although and above-mentioned CNTF mutant treat or prevent relevant disease activity increase, there are still partly decline
Phase is short, and immunogenicity height and the lower defect of activity need multiple injection that could obtain satisfactory curative effect in clinical, this
Huge economic pressures, stress and somatic reaction are brought to patient.Therefore, it is badly in need of developing at present a kind of with higher work
The property and longer CNTF mutant drug of curative effect time is imperative.
And the method known in the state of the art for modifying albumen using polyethylene glycol (PEG) has following benefit：Molecular weight increase,
Increased Plasma Half-life, immunogenicity reduce.PEG possesses unique parent-hydrophobic balance.When being coupled to drug molecule surface, by it
Advantageous property assigns the drug after modification, changes their biological distribution behaviors and dissolubility in aqueous solution, in its modification
Physical barrier is produced around drug, reduces the enzymolysis of drug.The antigenic determinant for covering protein surface, reduces the immunogene of albumen
Property.The molecular weight and volume for increasing albumen, reduce the filtration of glomerulus, making albumen, action time is longer in vivo.PEG in
Commodity production for the first time in 1940.The use of PEG has passed through U.S. FDA certification, meets United States Pharmacopeia (USP), national formulary
(NF), it is raw to be widely used in food, feed, personal care articles, chemistry, medication chemistry etc. for Food Chemical Codex (FCC) standard
Produce sphere of life.Accordingly it is desirable to modified CNTF using PEG or PEG Type of Collective objects, the effect of to improve drug,
Extend the time that drug acts in vivo, and reduces access times.
For example, Chinese patent application, which discloses No.CN101185762A, discloses a kind of use PEG Type of Collective objects modification CNTF
The method of mutant.But since in CNTF mutant, the side-chain radical with polar amino acid residue can be changed
Modification is learned, i.e., at least lysine, cysteine, histidine, arginine, aspartic acid, paddy in CNTF or CNTF mutant
Propylhomoserin, serine, threonine, the N- Amino End Groups of tyrosine and peptide chain and C- end carboxyls etc..Therefore, it finds to use in practice
PEG Type of Collective object is unstable to the decorating site of CNTF or CNTF mutant, as a result causes it is difficult to which character is uniform after being modified
Modified outcome.
Invention content
To solve above-mentioned problems of the prior art, the present invention provides a kind of modification type ciliary neurotrophic factor
Mutant, DNA, recombinant expression carrier, genetically engineered host cell, modification type ciliary nerve trophic factor mutant, the eyelash
Purposes, the pharmaceutical composition of shape neurotrophic factor mutant and modification type ciliary nerve trophic factor mutant.
Specifically, the present invention provides：
(1) a kind of ciliary nerve trophic factor mutant, wherein the ciliary nerve trophic factor mutant is to pass through
On the basis of the amino acid sequence of ciliary neurotrophic factor, the 17th cysteine is remained, by the 63rd paddy ammonia
Amide sports arginine, and eliminates obtained from 15 amino acid of C-terminal.
(2) according to the ciliary nerve trophic factor mutant described in (1), wherein the ciliary neurotrophic factor is prominent
Variant has SEQ ID NO：Amino acid sequence shown in 3.
(3) a kind of DNA, wherein the nucleotides sequence of the DNA is classified as coding according to the ciliary nerves battalion described in (1) or (2)
Support the nucleotide sequence of factor mutant.
(4) according to the DNA described in (3), wherein the nucleotides sequence of the DNA is classified as SEQ ID NO：Nucleotide shown in 4
Sequence.
(5) a kind of recombinant expression carrier, wherein the recombinant expression carrier contains the nucleotide sequence of (3) or (4).
(6) according to the recombinant expression carrier described in (5), wherein the recombinant expression carrier is PET24a as shown in Figure 1
+/CNTFnew/CC22 expression vectors.
(7) a kind of genetically engineered host cell, wherein the host cell contains the load of the recombinant expression described in (5) or (6)
Body.
(8) according to the genetically engineered host cell described in (7), wherein the host cell is CNTFnew/CC22 engineerings
Bacterium.
(9) a kind of modification type ciliary nerve trophic factor mutant, wherein the modification type ciliary neurotrophic factor
It is by carrying out pointed decoration according to the 17th cysteine of the ciliary nerve trophic factor mutant described in (1) or (2)
Obtained from, the pointed decoration is by being realized in conjunction with chemical modifier on the 17th cysteine.
(10) according to the modification type ciliary nerve trophic factor mutant described in (9), wherein the chemical modifier is poly-
Ethylene glycol or polyglycol polymer.
(11) according to the modification type ciliary nerve trophic factor mutant described in (10), wherein the chemical modifier
It is selected from：The poly- second of polyethylene glycol, methoxy poly (ethylene glycol), methoxy poly (ethylene glycol)-succinimidyl succinate, methoxyl group two
Alcohol-succinimdyl carbonate, methoxy poly (ethylene glycol) tresylate, methoxy poly (ethylene glycol)-propionic aldehyde, methoxyl group are poly-
Ethylene glycol-succinimidyl propionate, methoxy poly (ethylene glycol)-succinimide α methylbutyrates, methoxy poly (ethylene glycol)-
N-hydroxysuccinimide, methoxy poly (ethylene glycol)-maleimide or methoxy poly (ethylene glycol)-acetaldehyde；Preferably methoxyl group
Polyethylene glycol-maleimide.
(12) it is being prepared according to the ciliary nerve trophic factor mutant described in (1) or (2) for treating and/or preventing fertilizer
Purposes in the drug of fat disease and fat related type-2 diabetes mellitus.
(13) the modification type ciliary nerve trophic factor mutant according to any one of (9)-(11) is preparing use
Purposes in the drug for treating and/or preventing obesity and fat related type-2 diabetes mellitus.
(14) a kind of pharmaceutical composition for treating and/or preventing obesity and fat related type-2 diabetes mellitus, wherein
The pharmaceutical composition include according to described in (1) or (2) ciliary nerve trophic factor mutant and/or according to (9)-(11)
Any one of described in modification type ciliary nerve trophic factor mutant.
Compared with the prior art, the present invention has the following advantages and good effect：
1. the present invention has selected the relatively small number of cysteine of quantity in the amino acid sequence of albumen, and is surprised to find that
By being retained in the cysteine of CNTF the 17th, it can obtain and modify the high modification type ciliary neurotrophic factor of specificity,
Amido modified type ciliary neurotrophic factor that modification specificity is used higher than this field (such as Chinese patent
No.CN101185762A), to overcome that is brought by modification such as to modify unstable result series of problems.
2. the present invention has selected the cysteine of CNTF the 17th as pointed decoration site, make recombination engineering CNTF
It is closest with structure with original CNTF protein sequences, so that recombinant C NTF mutant proteins stability, solubility and biology is lived
Property be higher than native protein.
3. although the present invention is retained in the cysteine of CNTF the 17th, for being sought by the ciliary nerves of rite-directed mutagenesis
Supporting the biological activity of factor mutant does not influence, and therefore, can be used for modifying proves effective ciliary god in the prior art
Through nutrition factor mutant, uniform modification type ciliary neurotrophic factor is modified to obtain.
4. the chemical modifier (such as mPEG-MAL) of the present invention is securely and reliably, the FDA certifications in the U.S. are had passed through, it can be with
For in the extensive field such as food, medicine, cosmetics.
5. the ciliary nerve trophic factor mutant of the present invention and the tool of modification type ciliary nerve trophic factor mutant
There is the significant effect for inhibiting body weight increase, is the most significantly modification type ciliary nerve trophic factor mutant and unmodified type
Ciliary nerve trophic factor mutant compare with apparent long-acting, so as to reach treat or prevent obesity and
The remarkable result of its relevant disease.
Description of the drawings
Fig. 1 is the structure schematic diagram that CNTF is mutated body expression vector；
Fig. 2 is the coded sequence PCR qualification figures of CNTFnew/CC22, wherein M is DNA molecular amount standard (DNA
Marker)；1 is negative control；The result of the bacterium colony for 8 CNTFnew/CC22 engineering bacterias that 2-9 is respectively selected at random；
Fig. 3 is the result figure of NedI and EcorI double digestions identification, wherein M is DNA molecular amount standard；1-8 be respectively with
The result for 8 CNTFnew/CC22 engineering bacteria bacterium colonies that machine is selected；9 be negative control；
Fig. 4 is the complete genome sequence measurement result figure of CNTFnewCC22；
Fig. 5 is the expression of results figure of purpose PROTEIN C NTFnew/CC22, wherein M is protein molecular weight standard
(Protein Marker)；1 is negative control；The knot for 8 CNTFnew/CC22 engineering bacteria bacterium colonies that 2-9 is respectively selected at random
Fruit；
Fig. 6 is CNTFnew/CC22 protein SDS-PAGE electrophoretograms, wherein left road is protein molecular weight standard, right road is
CNTFnew/CC22 albumen；
Fig. 7 is the sequence alignment figure of 15 amino acid sequences of N-terminal of CNTFnew/CC22 albumen；
Fig. 8 is CNTFnew/CC22 protein immunoblot electrophoretograms, wherein left road is protein molecular weight standard, right road is
The result of CNTFnew/CC22 albumen；
Fig. 9 is the schematic diagram that CNTFnew/CC22 albumen is combined with MAL-mPEG molecules；
Figure 10 is the MAL-mPEG modification CNTFnew/CC22 protein electrophoresis figures of straight chain and branch, wherein M is protein point
Sub- amount standard；1 is the CNTFnew/CC22 albumen after Y-MAL-40K modifications；2 be the CNTFnew/ after M-MAL-20K modifications
CC22 albumen；
Figure 11 is that the MAL-mPEG of straight chain after purification and branch modifies CNTFnew/CC22 protein electrophoresis figures, wherein M is
Protein molecular weight standard；1 is purification result after the M-MAL-20K modifications of CNTFnew/CC22 albumen；2 be CNTFnew/CC22 eggs
Purification result after white Y-MAL-40K modifications；
Figure 12 is that polyethyleneglycol modified rear product purifies chromatography result figure through QFF；
Figure 13 is gel-filtration analysis (Superdex200, GEHealthcare) figure of QFF eluting peaks；Wherein, QFF is pure
Change product elution peak gleanings P1, P2, P3 after modifying, first peak of linear elution is known as P1, is mono-modified product, linear elution
Second peak is known as P2, and linear elution third peak is known as P3, refers to Figure 13.
Figure 14 is the analysed by reverse phase HPLC result figure of former albumen and mono-modified product (using the C18 reverse phases of Shiseido
Column, gradient 5%-95%, elution time are 60 minutes.Detection wavelength is 214nm).Wherein, left figure is former albumen；It is right
Figure is modification sample；
Figure 15 is that the SDS-PAGE of mono-modified product analyzes (6% refers to the concentration of polyacrylamide in separation gel),
In, left side swimming lane is protein molecular weight standard；Right lanes are product Y-MAL-40K-CC22 after CNTF modifications after purification；
Figure 16 is the analysed by reverse phase HPLC collection of illustrative plates of former PROTEIN C NTFnew/CC22 tryptic digestion samples；
Figure 17 is the analysed by reverse phase HPLC collection of illustrative plates of Y-MAL-40K tryptic digestion samples；
Figure 18 is respectively the extraction ion flow graph (above two of m/z593 in CNTFnew/CC22 and Y-MAL-40K-CC22
Figure) and first mass spectrometric figure (following two figure).
Specific implementation mode
Description below by way of specific implementation mode and the invention will be further described with reference to attached drawing, but it is pair that this, which is not,
The limitation of the present invention, those skilled in the art's basic thought according to the present invention can make various modifications or improvements, but only
The basic thought for not departing from the present invention, is all within the scope of the present invention.
The purpose of the present invention is to provide a kind of ciliary nerve trophic factor mutants and a kind of modification type ciliary nerves to seek
Support the factor, with improve ciliary neurotrophic factor therapeutic effect under the premise of, the uniform modification of character after the modification of acquisition
The ciliary neurotrophic factor of type.
All the time it is believed that the cysteine of the amino acid sequence of CNTF or CNTF mutant the 17th is easy and it
His CNTF or CNTF mutant forms disulfide bond, and coupling forms dimer and influences its biological activity；Therefore, people are in order to obtain
Obtaining stable CNTF mutant can select the cysteine by the 17th to replace with the alanine for being less susceptible to be formed disulfide bond first
Or serine.But by CNTF amino acid sequences (SEQ ID NO：1) Design & Analysis of System and a large amount of experiment
In, present inventors have surprisingly found that when being modified CNTF or CNTF mutant using PEG or PEG Type of Collective object, intentionally select
The cysteine for selecting the amino acid sequence the 17th for retaining CNTF or CNTF mutant, can not only improve the same of therapeutic effect
When, and after the modification that can be obtained the uniform modification type of character ciliary neurotrophic factor.
The present inventor is by experiment it has furthermore been found that even if remaining the half of the amino acid sequence the 17th of CNTF mutant
In the case of cystine, using the method for mutation CNTF, the 63rd glutamine such as is mutated into arginine, removes C-terminal the
1-30 amino acid etc. will not influence stabilization of the CNTF mutant in further chemical modification, i.e., with chemical modifier
In cohesive process, the 17th cysteine of CNTF mutant will not be combined with CNTF or CNTF mutant and form copolymerization
Object or dimer form on the contrary, it can be combined with chemical modifier such as PEG and its PEG Type of Collective object and modify uniform repair
Decorations type CNTF mutant.
Specifically：
First aspect present invention provides a kind of ciliary nerve trophic factor mutant, wherein the ciliary nerves battalion
Foster factor mutant is by the basis of the amino acid sequence of ciliary neurotrophic factor, remaining the 17th half Guang ammonia
63rd glutamine is sported arginine, and eliminated obtained from 15 amino acid of C-terminal by acid.
Preferably, the ciliary nerve trophic factor mutant has SEQ ID NO：Amino acid sequence shown in 3.
Second aspect of the present invention provides a kind of DNA, wherein the nucleotides sequence of the DNA, which is classified as, encodes above-mentioned ciliary
The nucleotide sequence of neurotrophic factor mutant.
Preferably, the nucleotides sequence of the DNA is classified as SEQ ID NO：Nucleotide sequence shown in 4.
Third aspect present invention provides a kind of recombinant expression carrier, wherein the recombinant expression carrier contains the DNA
Nucleotide sequence.
Preferably, the recombinant expression carrier is PET24a+/CNTFnew/CC22 expression vectors as shown in Figure 1.
Fourth aspect present invention provides a kind of genetically engineered host cell, wherein the host cell contains described
Recombinant expression carrier.
Preferably, the host cell is CNTFnew/CC22 engineering bacterias.
Fifth aspect present invention provides a kind of modification type ciliary nerve trophic factor mutant, wherein the modification
Type ciliary neurotrophic factor is determined by the 17th cysteine to above-mentioned ciliary nerve trophic factor mutant
Obtained from point modification, the pointed decoration is by being realized in conjunction with chemical modifier on the 17th cysteine
's.
Methods known in the art, such as Zhang Linlin etc., modified by polyethyleneglycol recombination can be used in the method for pointed decoration
The research of Filgrastim, bioengineering journal, method disclosed in 2005,21 (6), under the conditions of 4 DEG C
Albumen to be finished is placed in the buffer solution of different pH value, to ensure its biological activity.Lowry methods measure albumen concentration, will
Albumen is diluted to each concentration needed for experiment, by mol ratio (PEG：Protein) dressing agent is added, it is uniformly mixed, by orthogonal
The temperature of experimental design is reacted, reaction time 2-16h.
The chemical modifier can use in protein engineering field common chemical modifier (including high score
Sub, polymer-modified dose etc.), as long as can be combined with the sulfydryl of cysteine, obtain product after uniform modification.?
Herein, " uniform " refers to no isomer, and purity is 95% or more.
Preferably, the chemical modifier is polyethylene glycol (PEG) or polyglycol polymer.
Herein, " polyglycol polymer " refers to polyethyleneglycol derivative, in answering for polyethylene glycol
In, end group plays conclusive effect, and the polyethylene glycol of different end group has different purposes.Poly- second two in practical applications
The alcohol macromolecule end of the chain be not limited solely to band terminal hydroxy group, other stronger functionalization groups of reactivity, as p-methyl benzenesulfonic acid ester group,
Amino, carboxyl, aldehyde radical etc. can also be introduced in the both ends of polyglycol chain.The introducing of these functionalization groups expands poly- second two
The application range of alcohol keeps it more in sustained-release and controlled release, the targeting dispenser of organic synthesis, Peptide systhesis, Polymer Synthesizing and drug etc.
Aspect all has wide application prospect.
It is further preferred that the present invention is 1kD for the polyethylene glycol of modification or the molecular weight of polyglycol polymer
~100kD.
Preferably, the chemical modifier is selected from：Polyethylene glycol, methoxy poly (ethylene glycol), methoxy poly (ethylene glycol)-
Succinimidyl succinate, mPEG-SC, methoxy poly (ethylene glycol) trifluoroethyl sulfonic acid
Ester, methoxy poly (ethylene glycol)-propionic aldehyde, methoxy poly (ethylene glycol)-succinimidyl propionate, methoxy poly (ethylene glycol)-succinyl
Imines α methylbutyrates, methoxy poly (ethylene glycol)-n-hydroxysuccinimide, methoxy poly (ethylene glycol)-maleimide or first
Oxygroup polyethylene glycol-acetaldehyde；More preferably methoxy poly (ethylene glycol)-maleimide.
Sixth aspect present invention provides the ciliary nerve trophic factor mutant or modification type ciliary nerve nutrition
Purposes of the factor in preparing treatment and/or preventing the drug of obesity and its relevant disease (such as type-2 diabetes mellitus).
Seventh aspect present invention provides a kind of for treating and/or preventing obesity and its relevant disease (such as II types sugar
Urine disease) pharmaceutical composition, wherein the pharmaceutical composition include above-mentioned ciliary nerve trophic factor mutant and/or
Above-mentioned modification type ciliary nerve trophic factor mutant.Preferably, the pharmaceutical composition also includes pharmaceutic adjuvant.
The content of present invention is further explained and described in mode by the following examples, but these embodiments are not understood that
For limiting the scope of the invention.
The complete genome sequence of embodiment 1CNTF mutant synthesizes
According to ciliary neurotrophic factor (CNTF) DNA sequence dna (SEQ ID NO：And ciliary neurotrophic factor 2)
(CNTF) amino acid sequence (SEQ ID NO：1), the DNA sequence dna SEQ of complete genome sequence synthesis effects of recombinant ciliary neurotrophic factor
ID NO：4, the amino acid sequence of coding is SEQ ID NO：3.Wherein, effects of recombinant ciliary neurotrophic factor is that reservation is original
17th Cys of CNTF amino acid sequences, and the 63rd glutamine in ciliary neurotrophic factor is mutated into arginine, it goes
Except 15 amino acid of C-terminal form new CNTF mutant.The complete genome sequence synthesis of CNTF mutant can be by Shanghai JaRa
Bioengineering Co., Ltd completes.
The identification of the structure and engineering bacteria of embodiment 2CNTF mutant protein CNTFnew/CC22 expression vectors
(1) CNTF is mutated the structure of body expression vector
After complete genome sequence synthesis, synthesis 5 μ L of segment (20ng/ μ L) are taken, restriction enzyme NedI1 μ L (10U/ μ are added
) and EcorI1 μ L (15U/ μ L) (above-mentioned restriction enzyme is purchased from TAKARA companies) L.2 μ of restriction enzyme enzyme buffer liquid is added
L adds 11 μ L of distilled water, becomes 20 μ L systems.37 DEG C of water-baths, digestion 1 hour.Meanwhile pET24a+ plasmids being taken (to be purchased from Novagen
Company) 5 μ L (100ng/ μ L), identical restriction enzyme NedI1 μ L (10U/ μ L) and EcorI1 μ L (15U/ μ L) (limits is added
Property restriction endonuclease processed is purchased from TAKARA companies), add 11 μ L of distilled water, becomes 20 μ L systems.37 DEG C of water-baths, digestion 1 hour, with digestion
Carrier pET24a+.
After the completion of digestion, 1% agarose gel electrophoresis is carried out.Cutting the purposeful band of lower band, (CNTF bands are (about
Glue 500bp)), using Ago-Gel QIAquick Gel Extraction Kit (being purchased from Tiangeng biochemical technology Co., Ltd) to the purpose after digestion
Segment and carrier segments are recycled.
The carrier pET24a+5 μ L (50ng/ μ L) after 5 μ L of the synthesis target fragment after digestion (10ng/ μ L), digestion are taken, are added
Enter 1 μ L (350U/ μ L) T4DNA ligases (being purchased from TAKARA companies), be added 10 × ligase buffer solution, 2 μ L (for mixed liquor,
The formula of TAKARA companies, Tris-HCl (pH7.6) 660mM, MgCl266mM, DTT100mM, ATP1mM), 7 μ of distilled water is added
L becomes the coupled reaction system of 20 μ L.16 DEG C of connections are overnight.Expression vector is connected into, pET24a+/CNTFnew/ is named as
CC22.Fig. 1 schematically shows the building process of CNTF mutation body expression vectors.
(2) expression vector pET24a+/CNTFnew/CC22 is transformed into competent cell BL21 (DE3), is prepared
CNTFnew/CC22 engineering bacterias
Not antibiotic LB liquid medium formula：Tryptone (Tryptone) 10g/L, yeast extract (Yeast
Extract) 5g/L, sodium chloride (NaCl) 10g/L.
LB solid mediums containing 50 μ g/ml kanamycins：Tryptone (Tryptone) 10g/L, yeast extract
(Yeast extract) 5g/L, sodium chloride (NaCl) 10g/L, kanamycins 50mg/L.
Specific steps：The totally 2 μ L of the expression vector pET24a+/CNTFnew/CC22 after connection are taken, (100 μ are added to
L/ branch) in competent cell BL21 (DE3) (being purchased from Tiangeng biochemical technology Co., Ltd), it is positioned over after tapping 30 minutes on ice,
After 42 DEG C of water-bath heat shocks 60 seconds, rapidly as 2 minutes on ice.Not antibiotic 300 μ L of LB liquid medium are added, as shaking
In bed, 37 DEG C, 200 revs/min are cultivated 1 hour.Bacterium solution after culture is applied to the training of the LB solids containing 50 μ g/ml kanamycins
It supports on base tablet, is incubated overnight in 37 DEG C of incubators, obtain the single bacterium colony with pET24a+/CNTFnew/CC22 plasmids.
Single bacterium colony is taken, is inoculated into 5ml LB liquid mediums (containing 50 μ g/ml kanamycins), 37 DEG C, 200 revs/min of trainings overnight
It supports.Engineering bacteria is obtained, CNTFnew/CC22 engineering bacterias are named as.
(3) pass through PCR identifications, double digestion is identified, complete genome sequence measures three kinds of methods and determines CNTFnew/CC22 engineerings
Include coded sequence (the SEQ ID NO of destination protein CNTFnew/CC22 in bacterium：4).
PCR qualification process：The 1 μ L (OD3-4) of bacterium solution for the CNTFnew/CC22 engineering bacterias being incubated overnight are taken, primer 1 is added
(sequence is：ATG GCT TTC ACA GAG CAT TC) it is 1 μ L (10 μM), (sequence is primer 2：GTC ATG GAT GGA
CCT TAC TG) be 1 μ L (10 μM), archaeal dna polymerase be 1 μ L (2.5U/ μ L), 2 × PCR buffer solutions, 10 μ L, 6 μ L of distilled water, at
For the PCR reaction systems of 20 μ L.It is put into PCR instrument, temperature cycles are：The first step：94 DEG C 5 minutes.Second step：94 DEG C (30 seconds,
55 DEG C 30 seconds, 72 DEG C 1 minute, totally 30 cycle.Third walks：72 DEG C 10 minutes.4th step：It is taken out after 4 DEG C of constant temperature.PCR terminates
The agarose gel electrophoresis for carrying out 1% to sample afterwards, observes result.
The result of PCR identifications is referring to Fig. 2, wherein M is DNA Marker；1 is negative control；2-9 is respectively selected at random
8 CNTFnew/CC22 engineering bacterias bacterium colony result.
PCR qualification results：8 engineering bacteria bacterium colonies of random picking are positive bacterium colony.
Double digestion qualification process：The bacterium solution 5ml (OD3-3.5) for the CNTFnew/CC22 engineering bacterias being incubated overnight is taken, is utilized
Small amount plasmid extraction kit (being purchased from Axeygen companies) extracts plasmid.The 5 μ L of plasmid (100ng/ μ L) of extraction are taken, it is restricted
Each 1 μ L of restriction endonuclease NedI10U/ μ L and EcorI (15U/ μ L), 2 μ L of restriction enzyme enzyme buffer liquid, 11 μ L of distilled water become 20 μ
The double digestion system of L, in 37 DEG C of water-baths, digestion 1 hour carries out 1% agarose gel electrophoresis, observes result later.Double enzymes
The result of identification is cut referring to Fig. 3, wherein M is DNA Marker；8 CNTFnew/CC22 engineerings that 1-8 is respectively selected at random
The result of bacterium bacterium colony；9 be negative control.
Double digestion qualification result：8 engineering bacteria bacterium colonies of random picking are positive bacterium colony.
The complete genome sequence of CNTFnewCC22 is measured to be completed by Beijing promise match genome research center.CNTFnewCC22's
Complete genome sequence measurement result is shown in Fig. 4.
Complete genome sequence measurement result：It is consistent with the sequence designed and synthesized in advance.
The expression of 3 destination protein CNTFnew/CC22 of embodiment
1) LB agar plates are prepared：Tryptone 10g, yeast extract 5g, NaCl5g, agar 10g, Jia Shui are settled to
Kanamycins is added when temperature drops to 50 DEG C or so in 1L, autoclave sterilization, until final concentration of 50 μ g/ml, take 20ml to fall to train
Support ware, after solidification LB agar plates.
2) CNTFnew/CC22 engineering bacterias are cultivated：By the streak inoculation of CNTFnew/CC22 engineering bacterias in LB agar plates, 37
DEG C overnight incubation.The good single bacterium colony of growth conditions is selected on the LB tablets overnight from culture medium, is inoculated in the test tube containing LB
In, 37 DEG C of culture 10h.Then transferred species to containing 200ml LB liquid mediums (tryptone 10g, yeast extract 5g,
NaCl5g adds water to be settled to 1L, and high pressure sterilization dispenses triangular flask, every bottle of 200ml) triangular flask in, 37 DEG C of overnight incubations are made
Seed liquor.Seed liquor is inoculated in 1% ratio in the 50L fermentation tanks of the culture solution containing 30L2YT, fermentation medium is 2YT (pancreases
Distillation water dissolution is added in peptone 480g, yeast extract 300g, NaCl50g, and 5N NaOH adjust PH to 7.0, and constant volume arrives
30L, 0.12Mpa, 121 DEG C, 30min, high pressure sterilization).Control 37 DEG C of fermentation temperature, rotating speed 250-450r/min, ventilatory capacity 30-
After culture to A600=1.2 or so, isopropylthio-β-D-galactoside (IPTG) is added to final concentration in 50L/min, pH7.3
It is induced for 1.0mM.Continue culture 4.5 hours, terminate fermentation, collects thalline, be stored in spare in -30 DEG C of refrigerators.Take table
Up to the thalline after destination protein, SDS-PAGE electrophoresis is carried out, the expression of results of observation destination protein CNTFnew/CC22 is shown in Fig. 5,
Wherein, M is protein marker；1 is negative control；8 CNTFnew/CC22 engineering bacteria bacterium colonies that 2-9 is respectively selected at random
Result.
The purifying of 4 destination protein CNTFnew/CC22 of embodiment
The fermentation thalli that will be obtained by 3 method of embodiment, by 1：10 ratios add 20mMTris-HCl, pH8.0, high-pressure homogenization
It is broken, operating condition：1000bar, temperature are controlled at 8 DEG C or less.3 cycles, 10,000rpm, 4 DEG C centrifuge 30 minutes, collect
Supernatant is isolated and purified.It is that octyl-agarose medium is (public purchased from U.S. GE to use hydrophobic chromatography, selected medium first
Department, Octyl Sepharose4fast flow, chromatographic column XK26/20).Select 50mM Tris-HCl, pH8.0,1M (NH4)2SO4Buffer solution balance chromatographic column, be crushed supernatant in be added 1M ammonium sulfate after loading, with equilibration buffer elute 1 column volume,
It then uses gradient elution, by buffer exchange is 50mM Tris-HCl, pH8.0 in 5 column volumes.Eluting peak is collected, is adjusted
PH5.0, with 50mM acetic acid-sodium acetate, pH5.0 buffer solution dialysis desaltings, sample uses cation exchange medium CM FF after desalination
Polishing purification is carried out, gained CNTF purity is up to 95% or more.Purification result is shown in Fig. 6：CNTFnew/CC22 protein SDS-PAGEs
Electrophoretogram, wherein left road is protein molecular weight standard, right road is CNTFnew/CC22 albumen.
15 determined amino acid sequences of N-terminal of embodiment 5CNTFnew/CC22 albumen
Sequence alignment be by the N-terminal sequencing result of 15 amino acid sequences of N-terminal of CNTFnew/CC22 albumen and CNTF into
Row compares, and measurement result is shown in Fig. 7, as a result illustrate the sequencing results of 15 amino acid sequences of N-terminal of CNTFnew/CC22 albumen with
It is pre-designed identical.
The external activity of embodiment 6CNTFnew/CC22 albumen is identified
TF-1CN5a1 cells are the cell line for depending on GM-CSF, and cell surface has rhCNTF high-affinities
Receptor, so CNTF can promote the proliferation of TF-1CN5a1 cells.TF-1CN5a1 is added in the CNTFnew/CC22 of various concentration
In cell culture, the growing state of cell is observed, to measure CNTFnew/CC22 external activities, the specific method is as follows：
By TF-1CN5a1 cells (being purchased from U.S. ATCC, article No. CRL-2512TM), routine culture is in GM-CSF conditions
In the culture solution of (GM-CSF containing 500ng in 100ml RPMI1640), the cell of exponential phase is collected when experiment, with nothing
RPMI1640 (being purchased from invitrogen companies) wash liquid of serum 3 times, then with 10% calf serum RPMI1640 cultures
It is 1.0 × 106 that liquid, which adjusts cell number, adds 50 μ l cell suspensions per hole, uses the CNTFnew/CC22 albumen obtained in embodiment 4
After 10 μ g/ml of initial concentration are serially diluted by 4 times, 50 μ l are added per hole.Using 50 μ l serum-free RPMI1640 culture mediums as
Negative control (is purchased from United Kingdom National biological products assay institute NIBSC. article No.s with CNTF standard items：94/684) it is positive control,
Initial concentration is 200ng/ml, and after 4 times of progress is serially diluted, 50 μ l are added per hole.Culture plate is in 37 DEG C, 5%CO2Under the conditions of train
After supporting 48h, the WST-8 that 50mg/ml is added per hole (is purchased from green skies biotechnology research institute.Article No.：NIBSC94/684) solution
20 μ l continue after cultivating 4h, and colorimetric in microplate reader, measurements wavelength are 570nm, reference wavelength 630nm again after mixing.It is each real
It tests result to be repeated 3 times, finally takes the average result of 3 experiments.
As a result：The external activity for the CNTFnew/CC22 albumen that embodiment 4 obtains：8.37×106IU/mg。
Unmutated ciliary neurotrophic factor control group (being purchased from sigma, C3710-10UG) is tested while being provided with, it is real
Testing result confirms that the external activity of unmutated ciliary neurotrophic factor is about 8.53 × 104IU/mg, by rite-directed mutagenesis
The biological activity of ciliary nerve trophic factor mutant is higher than unmutated natural molecule.
Embodiment 7CNTFnew/CC22 active measurement in Mice Body
Laboratory mice uses male Kunming mouse (deriving from Beijing Vital River Experimental Animals Technology Co., Ltd.), weight
18-22g.It is divided into positive controls, negative control group and experimental group, every group 6.With (the purchase of physiological saline solution CNTF standard items
From United Kingdom National biological products assay institute.Article No.：NIBSC94/684), sample is that CNTFnew/CC22 sterlings freeze dried powder is (real
Apply 4 gained albumen of example and gained be further lyophilized), equally with physiological saline solution and it is diluted to 100 μ g/ml.With every mouse 125
The amount of μ g/kg/ days is injected.Negative control, that is, physiological saline.The weight of every group of every mouse is weighed before injection respectively.Continuously
It is administered within five days, weighs the weight of mouse and observe its variation.
(1) flow such as the following table 1：
Table 1.CNTF acts on mouse loss of weight and measures flow
Note：Experimental period is 0 day on the day of first administration so that the previous day is administered as -1 day.
△：According to previous operation performance, it can shift to an earlier date or postpone, but keep as possible daily in the same time.
(2) measurement index：Changes of weight rate
Weight × 100% before changes of weight rate=(weight before certain group administration of weight-after certain group administration)/administration
(tested group of certain daily weight-should for body weight increase inhibiting rate (%)=(control group daily weight-control group d0 weight)-
Group d0 weight)/(control group daily weight-control group d0 weight) × 100%
Mouse weight result of variations is shown in Table 2：
2 mouse weight of table changes table
As shown in Table 2：Mouse after three groups of hypodermic injections, weight are increased, but the mouse weight of sample sets increases
It is long to be less than negative control group (p < 0.05), while being less than positive control (p < 0.05) (CNTF standard items) group.
(2) mouse weight growth inhibition rate see the table below 3：
3 mouse weight growth inhibition rate of table
CNTFnew/CC22 body weight increases inhibiting rate is higher than the body weight increase of positive control (CNTF standard items) as shown in Table 3
Inhibiting rate.
The immunoreactivity of embodiment 8CNTFnew/CC22 albumen
Protein immunoblotting experiment is carried out using the monoclonal antibody of CNTF as specific antibody, studies immune response
Property.
Use the monoclonal antibody (being purchased from Abcam companies) of CNTF as primary antibody, with the goat-anti of horseradish peroxidase-labeled
Mouse IgG (being purchased from abcam companies) is used as ELIAS secondary antibody.The CNTFnew/CC22 albumen that embodiment 4 obtains is done into SDS- first
PAGE electrophoresis, resolving gel concentration 12%, concentration gum concentration be 4%, applied sample amount be 15 μ l, constant pressure 110V, electrophoresis 1.5 hours,
Electrophoresis is terminated, gel is transferred on nitrocellulose membrane (NC films), is developed the color after being incubated respectively with primary antibody and secondary antibody.It is shown in Fig. 8
CNTFnew/CC22 protein immunoblot electrophoretogram results show that CNTFnew/CC22 albumen can be with the monoclonal antibody of CNTF
Specific binding, illustrate the present invention CNTFnew/CC22 albumen still with natural CNTF with similar immunoreactivity.
The modification type CNTF albumen of the present invention does not have to change the ability that albumen is combined with specific antibody after site mutation.
The modification of embodiment 9CNTFnew/CC22 albumen
The CNTFnew/CC22 albumen that the MAL-mPEG of straight chain and branch obtains embodiment 4 is utilized respectively in the present embodiment
It is modified.
1) preparation of the CNTFnew/CC22 albumen of the MAL-mPEG modifications of straight chain 20KD：
The MAL-mPEG of straight chain 20KD is purchased from Jiankai Science and Technology Co., Ltd., Beijing, commercial disignation：M-MAL-20K.Molecular formula
For：
The schematic diagram that CNTFnew/CC22 albumen is combined with MAL-mPEG molecules can be with reference chart 11, and the specific method is as follows：
The CNTFnew/CC22 albumen concentration of purifying is adjusted to 1mg/ml, by 1：1~1：Molecular weight is added in 10 ratio
For 20KD polyethyleneglycol modified dose of straight chain maleimide-(i.e. the MAL-mPEG of straight chain 20KD, 4 DEG C react 12 hours, be added
5mM DTT terminate reaction.
2) preparation of the CNTFnew/CC22 albumen of the MAL-mPEG modifications of branch 40KD
The MAL-mPEG of branch 40KD is purchased from Jiankai Science and Technology Co., Ltd., Beijing.Commercial disignation：Y-MAL-40K.Molecular formula
It sees below：
The CNTFnew/CC22 albumen concentration of purifying is adjusted to 1mg/ml, by 1：1~1：Molecular weight is added in 10 ratios
Polyethyleneglycol modified dose of the branch maleimide-(i.e. the MAL-mPEG of branch 40KD) of 40KD, 4 DEG C are reacted 12 hours, are added
5mM DTT terminate reaction.
3) MAL-mPEG of straight chain and branch is to the protein modified results of CNTFnewCC22
The modification type CNTFnew/CC22 albumen that the above method is obtained to the MAL-mPEG modifications of straight chain and branch carries out SDS
Electrophoresis, the result is shown in Figure 10, wherein M is protein marker；1 is the CNTFnew/CC22 albumen after Y-MAL-40K modifications；2 are
CNTFnew/CC22 albumen after M-MAL-20K modifications.
The purifying of albumen after the MAL-mPEG of 10 straight chain of embodiment and branch modifies CNTFnew/CC22 albumen
GE companies of the U.S., model (are purchased from using anionic exchange medium Q FF：Hiprep16/10Q FF) it is modified
Agent, the separation of unmodified protein and modified outcome.Used equilibration buffer is Tris-HCl, pH8.0,5 cylinders of 50mM
The interior buffer solution B (50mMTris-HCl, pH8.0,1M NaCl) of product carries out gradient elution, collects eluting peak and carries out SEC (gels
Filtration chromatography) it checks, determine that P1 is modified outcome.
The result is shown in Figure 1 1-15, wherein in fig. 11, M marker；1 repaiies for CNTFnew/CC22 albumen M-MAL-20K
Purification result after decorations；2 be purification result after the-MAL-40K modifications of CNTFnew/CC22 protein Ys.
The decorating site that the MAL-mPEG of 11 straight chain of embodiment and branch modifies CNTFnew/CC22 albumen is really
It is fixed
By the former albumen (i.e. unmodified protein) obtained by 4 method of embodiment and the 40K-Y- obtained by 10 method of embodiment
The mono-modified product purified after MAL-PEG modifications carries out tryptic digestion, determines the decorating site of modified outcome.It is de- with 5mL
Former albumen and modified outcome sample are distinguished desalination to containing by salt plug (be purchased from GE companies of the U.S., model Hitrap Desalting)
The 50mM NH of 2M ureas4HCO3In buffer solution, collect peak point, in mass ratio 1：50 are added trypsase (purchased from U.S. sigma public affairs
Department), 37 DEG C of reactions overnight (12 hours), are added 1%TFA (trifluoroacetic acid) and terminate.Digestion sample uses Shiseido C18 reverse phases
Column, gradient 5%-95%, 60 minutes.Detection wavelength：214nm.The reverse phase collection of illustrative plates of digestion sample is referring to Figure 16-18.
As can be seen that the reverse phase collection of illustrative plates of former albumen is almost the same from figure, former albumen disappearance peak and modification sample newly occur
It is the peptide fragment containing C17 that the first mass spectrometric figure at peak, which not can confirm that,.Therefore the position for determining that modification occurs further is analyzed by ion stream
Point.According to the amino acid sequence of former albumen, obtain theoretically the tryptic digestion segment containing C17 be DLCSR (for protein molecular from
15th to 19 amino acid peptide fragments), theoretical m/z=593 extracts former albumen and modification respectively using mass spectral analysis software
The m/z593 ions flow graph and corresponding first mass spectrometric of sample, as shown in figure 18：
The extraction ion flow graph (two figure above) of m/z593 and first mass spectrometric figure are (following in respectively PEGCC22 and CC22
Two figures).It can be seen that in the digestion sample of former albumen, the eluting peak retention time corresponding to m/z593 peptide fragment ion streams is
8.25 minutes, the mass spectrographic molecular weight of level-one was 593.2, consistent with the theoretical molecular weight of DLCSR, illustrates to contain in former albumen
The segment of DLCSR.In modifying sample, without 8.25 minutes eluting peaks, it is although retention time occur in corresponding m/z593 peptide fragments
29.7 minutes eluting peaks, but its first mass spectrometric cannot obtain clear molecular weight (charge-mass ratio), and it is m/ to show the eluting peak not
Z593 segments.It compares accordingly, show that modification reaction is happened on peptide fragment containing DLCSR, according to maleimide dressing agent and half Guang
The feature of specific reaction occurs for propylhomoserin sulfydryl, determines that modification reaction is happened on the C17 of DLCSR peptide fragments.
Active measurement outside proteosome after embodiment 12 is modified
TF-1CN5a1 cells are the cell line for depending on GM-CSF, and cell surface has rhCNTF high-affinities
Receptor, so CNTF can promote the proliferation of TF-1CN5a1 cells.TF- is added in CNTFnew/CC22 after the modification of various concentration
In 1CN5a1 cell cultures, the growing state of cell is observed, to measure CNTFnew/CC22 external activities after modification, specific side
Method is as follows：
By TF-1CN5a1 cells (being purchased from U.S. ATCC, article No. CRL-2512TM), routine culture is in GM-CSF conditions
In the culture solution of (GM-CSF containing 500ng in 100mlRPMI1640), the cell of exponential phase is collected when experiment, with nothing
RPMI1640 (being purchased from invitrogen companies) wash liquid of serum 3 times, then with 10% calf serum RPMI1640 cultures
It is 1.0 × 10 that liquid, which adjusts cell number,6, add 50 μ l cell suspensions per hole, using being purified after the modification obtained by 10 method of embodiment
Mono-modified product (straight chain 20KD MAL-mPEG modification CNTFnew/CC22 albumen and branch 40KD MAL-mPEG modification
CNTFnew/CC22 albumen) after 10 μ g/ml are serially diluted by 4 times, per hole be added 50 μ l.With 50 μ l serum-frees RPMI1640
Culture medium (is purchased from United Kingdom National biological products assay institute NIB SC. article No.s as negative control, with CNTF standard items：94/684)
For positive control, after 4 times of progress is serially diluted, 50 μ l are added per hole by initial concentration 200ng/ml.Culture plate 37 DEG C, 5%
CO2Under the conditions of culture 48h after, per hole be added 50mg/ml WST-8 (be purchased from green skies biotechnology research institute.Article No.：NIB
SC94/684) 20 μ l of solution continue after cultivating 4h, and colorimetric in microplate reader, measurements wavelength are 570nm, reference wavelength again after mixing
For 630nm.Each experimental result is repeated 3 times, and final result takes the average result of 3 experiments.
As a result as follows：
Straight chain 20KD MAL-mPEG modification CNTFnew/CC22 albumen external activities be：4.9×106IU/mg.(for
Average result)
Branch 40KD MAL-mPEG modification CNTFnew/CC22 albumen external activities be：7.35×106IU/mg.It is (same
On)
The above results illustrate after the PEG of different molecular weight modifications, prominent by the ciliary neurotrophic factor of rite-directed mutagenesis
It is 87% that the In vitro biological activity of variant, which has different degrees of reduction, branch 40KD modified outcome activity preservation rates, activity
Change smaller；Straight chain 20KD modified outcome activity preservation rates are 59%, are decreased, but external activity is far above positive control
Group (p < 0.05).
Embodiment 13PEG-CNTFnew/CC22 active measurement in Mice Body
Laboratory mice uses male Kunming mouse (deriving from Beijing Vital River Experimental Animals Technology Co., Ltd.), weight
18-22g.It is divided into positive controls, negative control group and experimental group, every group 8.With (the purchase of physiological saline solution CNTF standard items
From United Kingdom National biological products assay institute article No.s：NIBSC94/684), experimental group sample Y40K-PEG-CNTFnew/CC22
It is water-soluble with physiology salt with M20K-PEG-CNTFnew/CC22 sterlings freeze dried powder (10 gained albumen of embodiment is further lyophilized)
It solves and is diluted to 0.5mg/ml.It is injected with the amount of every 100 μ g/kg/ days of mouse.Negative control, that is, physiological saline.Injection
The preceding weight for weighing every group of every mouse respectively.It is administered within continuous five days, weighs the weight of mouse, observe the changes of weight of mouse,
It is another that injection group weekly is set simultaneously, it is divided into modification sample sets, unmodified sample sets, positive controls and negative control group, with every
The amount of 200 μ g/kg/ days of mouse is injected.Negative control, that is, physiological saline.Every group of every mouse is weighed before injection respectively
Weight.Weekly administration 1 time, weighs the weight of mouse, observes the changes of weight of mouse.
(1) flow such as the following table 4：
Table 4.CNTF acts on mouse loss of weight and measures flow (daily)
Note：Experimental period is 0 day on the day of first administration so that the previous day is administered as -1 day.
△：According to previous operation performance, it can shift to an earlier date or postpone, but keep as possible daily in the same time.
Table 5.CNTF acts on mouse loss of weight and measures flow (weekly)
Note：Experimental period is 0 day on the day of first administration so that the previous day is administered as -1 day.
△：According to previous operation performance, it can shift to an earlier date or postpone, but keep as possible daily in the same time.
(2) measurement index：Changes of weight rate
Weight × 100% before changes of weight rate=(weight before certain group administration of weight-after certain group administration)/administration：
(tested group of certain daily weight-should for body weight increase inhibiting rate (%)=(control group daily weight-control group d0 weight)-
Group d0 weight)/(control group daily weight-control group d0 weight) × 100%
Mouse weight result of variations is shown in Table 6 and table 7：
6 mouse weight of table changes table (being administered for continuous 5 days)
As shown in Table 6：Mouse after three groups of injections, weight are increased, but the increasing of the mouse weight of sample sets is long
Less than negative control group (p < 0.05), while being less than positive control (CNTF standard items) group (p < 0.05).
7 mouse weight of table changes table (weekly administration 1 time)
As shown in Table 7：For administration group 1 times a week, the mouse after four groups of injections, weight is increased, but is modified
The mouse weight of sample sets, which increases, is respectively less than negative control group (p < 0.05), while being less than positive control (CNTF standard items) group
(p < 0.05), research confirm PEG modified medicaments compared with unmodified drug with long-acting.
(2) mouse weight growth inhibition rate see the table below 8：
8 mouse weight growth inhibition rate of table (is administered) for continuous 5 days
Y40K-PEG-CNTFnew/CC22 and M20K-PEG-CNTFnew/CC as shown in Table 8
22 samples are higher than the body weight increase inhibiting rate of mouse the body weight increase inhibiting rate of positive control (CNTF standard items)
(p < 0.05).
9 mouse weight growth inhibition rate of table (weekly administration 1 time)
Weight of the Y40K-PEG-CNTFnew/CC22 and M20K-PEG-CNTFnew/CC22 samples to mouse as shown in Table 9
There were significant differences compared with the control group for growth inhibition rate (p < 0.01), especially Y40K-PEG-CNTFnew/CC22 and M20K-
PEG-CNTFnew/CC22 samples are administered can reach CNTFnew/CC22 daily administration effects once a week, and CNTFnew/
CC22 weekly administrations cannot effectively inhibit the growth of mouse weight.
Claims (6)
1. a kind of modification type ciliary nerve trophic factor mutant, wherein the modification type ciliary neurotrophic factor is logical
It crosses obtained from carrying out pointed decoration to the 17th cysteine of ciliary nerve trophic factor mutant, the pointed decoration is
By being realized in conjunction with chemical modifier on the 17th cysteine, and wherein, ciliary nerves battalion
Foster factor mutant is by the basis of the amino acid sequence of ciliary neurotrophic factor, remaining the 17th half Guang ammonia
63rd glutamine is sported arginine, and eliminated obtained from 15 amino acid of C-terminal by acid；Wherein, institute
The amino acid sequence for the ciliary nerve trophic factor mutant stated such as SEQ ID NO：Shown in 3；And the wherein described chemical modification
Agent is polyethylene glycol or polyglycol polymer.
2. modification type ciliary nerve trophic factor mutant according to claim 1, wherein the chemical modifier choosing
From：Polyethylene glycol, methoxy poly (ethylene glycol), methoxy poly (ethylene glycol)-succinimidyl succinate, methoxy poly (ethylene glycol)-
Succinimdyl carbonate, methoxy poly (ethylene glycol) tresylate, methoxy poly (ethylene glycol)-propionic aldehyde, the poly- second of methoxyl group
Glycol-succinimidyl propionate, methoxy poly (ethylene glycol)-succinimide α methylbutyrates, methoxy poly (ethylene glycol)-N-
HOSu NHS, methoxy poly (ethylene glycol)-maleimide or methoxy poly (ethylene glycol)-acetaldehyde.
3. modification type ciliary nerve trophic factor mutant according to claim 2, wherein the chemical modifier is
Methoxy poly (ethylene glycol)-maleimide.
4. modification type ciliary nerve trophic factor mutant according to claim 1, wherein the chemical modifier is
Polyethyleneglycol modified dose of the branch maleimide-of 40KD.
5. the modification type ciliary nerve trophic factor mutant according to any one of claim 1-4 is being prepared for controlling
Treat and/or prevent the purposes in the drug of obesity and fat related type-2 diabetes mellitus.
6. a kind of pharmaceutical composition for treating and/or preventing obesity and fat related type-2 diabetes mellitus, wherein described
Pharmaceutical composition includes the modification type ciliary nerve trophic factor mutant according to any one of claim 1-4.
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