CN101555284B - Truncated-type human ciliary nerve nutrition factor active segment and fusion protein thereof - Google Patents
Truncated-type human ciliary nerve nutrition factor active segment and fusion protein thereof Download PDFInfo
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- CN101555284B CN101555284B CN 200810088976 CN200810088976A CN101555284B CN 101555284 B CN101555284 B CN 101555284B CN 200810088976 CN200810088976 CN 200810088976 CN 200810088976 A CN200810088976 A CN 200810088976A CN 101555284 B CN101555284 B CN 101555284B
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Abstract
The invention relates to a protein fusing truncated-type human ciliary nerve nutrition factor active segment of protein transduction peptide and a nucleic acid molecule encoding the ribonucleotide of the truncated-type human ciliary-nerve nutrition factor active segment. The invention relates to an expression vector containing the nucleic acid molecule and also relates to the application of the protein or the nucleic acid molecule to the preparation of the medicine used for treating regression neurosis including Alzheimer's disease, Parkinson's disease, substantia nigra degenerative change, multiple sclerosis, Huntington, spinal muscular atrophy, amyotrophic lateralizing sclerosis and other motor neuron diseases and the medicine for treating retina degenerative disease, glaucoma, obesity or diseases relating to obesity and diseases relating to nerve damage. The invention particularly relates to the medicine composition including the protein or the nucleic acid molecule and the application of the medicine composition by multiple modes, such as injection, hypoglossal buccal administration, lung inhalation, nasal cavity mucosa and anus pill or skin absorption, eye drop absorption, andthe like.
Description
Technical field:
The present invention relates to recombination fusion protein and its preparation method, be specifically related to the protein transduction peptide and merge the truncated-type human ciliary nerve nutrition factor active segment coding nucleic acid, and the prokaryotic expression preparation method of albumen.The invention still further relates to described transduction peptide and merge the albumen of truncated-type human ciliary nerve nutrition factor active segment or nucleic acid molecule separately and pharmaceutical composition, can be used for the treatment of nerve degenerative diseases, retina degenerative disease, obesity or the disease relevant with obesity, and the purposes in the nerve injury relative disease.
Technical background:
Ciliary neurotrophic factor (Ciliary neurotrophic factor, CNTF) is found in 1976 by Helfand etc. that at first Barbin in 1984 etc. extract from chicken ciliary nerves unit and obtain, because the survival that can keep the chicken parasympathetic ganglion is gained the name.CNTF can impel the survival of multiple neurocyte, is that first foundly can maintain the survival of body and Isolated Spinal Cord motor neuron and the neurotrophic factor of enation.
People CNTF gene is positioned at the long-armed near-end 11q12 of people's o.11 karyomit(e), is single copy gene.Do not have signal peptide, do not have glycosylated universal sequence, only at the 17th a nonactive essential cysteine residues is arranged.With the molecular structure of leukaemia inhibitory factor (LIF), IL-6, OSM and granulocyte colony-stimulating factor (G-CSF) etc. certain similarity is arranged, belong to the nervosa cytokine family.The classical signals pathway of CNTF be CNTF after its receptor alpha subunit is combined again with cytolemma on subunit gp130 and LIFR β polymerization, form receptor complex, the jak kinase of activated membrane inboard, make the STAT phosphorylation, enter in the nuclear, be combined with specific dna sequence dna, genetic transcription and albumen are synthetic in the regulating cell nuclear.CNTF has the various biological function in vivo, Main Function is to liking neural system, the multiple neurocyte such as the Sensory neurone of ciliary nerves ganglion cell, sympathetic neuron, spinal ganglion, dynamoneure, cholinergic neuron, dopamine neuron there is effect, can promote differentiation, the growth of these neurocyte, support its existence, regeneration and reparation after the promotion nerve injury.
1.CNTF with disease
1.1CNTF with nerve retrograde affection
Alzheimer's disease (Alzheimer ' s disease, AD) be the fatefulue central nervous system degenerative disease of a kind of carrying out property.The main brain cell that destroys causes that gradual memory and cognitive power go down, aphasis, and thinking and capacity descend, mental act is unusual, affects work, life and doings, and the state of an illness is carrying out property and increases the weight of, lose gradually independent living ability, many because of complication death after the morbidity.The number of whole world trouble alzheimer's disease has surpassed 1,800 ten thousand at present, and number of patients is still constantly increasing.China's alzheimer's disease number of patients has surpassed 5,000,000, and along with the quickening of China's aging population process, this numeral will be more huge.With advancing age, dull-witted sickness rate and morbidity all is the trend that increases gradually.In the elderly population more than 60 years old, every increase of age 5 years old, the ill danger of alzheimer's disease just can increase by 1.85 times, and alzheimer's disease has become 21 century and has threatened one of human serious disease.Although patient's number is not still cured measure at present increasing, all hasten one's steps and seek better methods for the treatment of in the whole world, delays morbidity or stop progression of disease.Some type neurocyte of specific region is carrying out property sex change death in the alzheimer's disease people brain, and area of liability comprises amygdala, hippocampus and hippocampus peripheral region, and the cell colony that is contaminted comprises catecholaminergic, five hydroxytryptamine energy and cholinergic nerve cell.CNTF can stop these neuronic degeneration forfeitures in the body, can promote to suffer the axoneuron of neurological damage or neurodegenerative disease damage to survive.CNTF also has therapeutic action to Heng Yandun (Huntington) chorea, amyotrophic lateral sclerosis (ALS), parkinsonism, black substance degeneration, multisystem committee's contracting disease and Duchenne-Arandisease (SMA) etc.
1.2CNTF with the retina degenerative disease
CNTF has provide protection to retinal ganglial cells.In numerous neurotrophic factors, CNTF is unique nutritional factor that can effectively promote retinal ganglial cells regeneration, and CNTF can induce the retinal neuronal cell differentiation, reduces nerve cell apoptosis, promotes cell regeneration.Experiment in vivo and vitro research proves that all CNTF is necessary nutritional factor in photosensory cell (cone cell and the rod photoreceptor cell) growth course.Intravitreal injection CNTF can produce significant protective effect to photosensory cell.CNTF also can treat glaucoma.
1.3CNTF with obesity or with obesity relevant disease
Obesity is a kind of chronic disease of serious harm HUMAN HEALTH, and obesity can improve the risk of diseases such as suffering from diabetes, heart trouble, hypertension, apoplexy and cancer greatly.Experiment finds that CNTF reduces the body fat of mouse, thereby Mouse Weight is descended.CNTF and the receptors bind that is positioned at the thalamus position by the STAT3 signal transduction path, can make obesity mice (ob/obmice) fat that lacks leptin reduce weight loss; For the obese model mouse (DIO mice) of diet induced, CNTF also can make it weight loss, and this mouse model more can represent the practical situation of human obesity.CNTF may make the body weight point of adjustment downward modulation of thalamus, thereby inhibition food intake, with compare by strictly keep on a diet fat-reducing and other methods for the treatment of, excessive food intake after the drug withdrawal can not occur and cause the body weight bounce-back in CNTF, therefore, CNTF is expected to exploitation becomes a kind of novel, efficient medicine of losing weight, and has wide clinical application DEVELOPMENT PROSPECT.
1.4CNTF with the nerve injury relative disease
There is a large amount of CNTF in the peripheral nerve schwann cell, when peripheral nerve is impaired, CNTF is released and enters the intercellular substance, these CNTF can play a role alone to cell space by the aixs cylinder antiport, also can form mixture and bring into play the effect that promotes repairing of neural injury with the solubility CNTFRA that exists in the muscle.Peripheral nerve injury can cause that corresponding spinal cord CM is dead, uses CNTF and can support section divide the survival of spinal cord CM.CNTF promotes motor neuron aixs cylinder branch in normal muscle to germinate.CNTF can prevent that new born rats from cutting off the death of the motor neuron that causes because of aixs cylinder, and the experiment of cut surface nerve shows that CNTF can effectively prevent the regression of the most of neurocyte of nucleus of facial nerve.
2. the present Research of recombinant human CNTF
CNTF is mainly used in treatment motor neurone disease such as amyotrophic lateral sclerosis (ALS) at present.But the effect of clinical application is also unsatisfactory.It is local that the semi-permeable membranes micro-capsule that the viable cell that can secrete CNTF is housed is implanted pathology, can improve Local C NTF concentration, reduces Plasma Concentration, alleviates side effect.It is effective to the treatment retinitis pigmentosa that coated cell carries the first phase experiment of CNTF therapy to demonstrate CNTF.It is less that CNTF treats the research of other CNS diseases.CNTF is all lower in the level in the most of zone of CNS, and natural production is extremely low in the human body, and genetically engineered is the reliable means that obtains a large amount of high-purity C NTF.
3.CNTF with hemato encephalic barrier
Hemato encephalic barrier is the barrier system that is present between blood and the cerebral tissue, and capillary endothelial cell is the basic comprising composition of hemato encephalic barrier with tight connection between neurocyte.In the normal physiological situation, only allow gas molecule and relative mass to pass through less than the fat-soluble small molecules of 0.6kD.Since the nerve growth factor discovery, neurotrophic factor (NTFs) existing more than 20 is planted.But since half a century, because the effect of hemato encephalic barrier, the clinical application of NTFs is still very limited.Still lack clinically at present a rationally effective central administration approach.The route of administration of having reported comprises that mainly peripheral-system administration, brain essence direct injection, tricorn administration, intrathecal injection and nose administration are several.Peripherally administered, traumatic little, but be subject to having a strong impact on of hemato encephalic barrier, the amount that enters brain essence is very limited.Brain essence direct injection, tricorn administration, sheath inner injecting medicine-feeding amount are controlled easily, and site of action is direct, but traumatic administration has a big risk application difficult.CNTF is protein macromolecule, be difficult to see through hemato encephalic barrier, thereby having limited its treatment at maincenter uses, although some Transplanted cellss of carrying out at present and the research of gene therapy aspect have made some progress in experimentation on animals, because these treatment plans are all taked the traumatic means such as operation and intracerebroventricular, from clinical application very large distance are arranged still.
It is the CNTF vehicle that the present invention selects the protein transduction peptide, adopt genetic engineering technique that the CNTF gene is changed structure, make 14 amino acid of CNTF albumen n end disappearance of clonal expression, to increase its activity, and remove 15 amino acid of C end, to increase stability and solubility.Detect by activity, prove that the CNTF protein active fragment of brachymemma demonstrates higher biological activity than commercially available rhCNTF.
Summary of the invention:
One aspect of the present invention provides a kind of to have the spanning transduction membrane function and has the more truncated-type human ciliary nerve nutrition factor active segment of high biological activity, described protein specificity is that 14 amino acid of Human Ciliary Neurotrophic Factor N end disappearance have merged the protein transduction peptide simultaneously, and has removed 15 amino acid of C end.Described protein transduction peptide merges truncated-type human ciliary nerve nutrition factor active segment (hereinafter being abbreviated as CNTFt), and its aminoacid sequence is shown in SEQ ID NO.1, and wherein the aminoacid sequence of truncation type active fragments is shown in SEQ ID NO.2.
Another aspect of the present invention provides the nucleotide sequence of encoding said proteins, wherein the protein transduction peptide merge truncated-type human ciliary nerve nutrition factor active segment nucleotide sequence shown in SEQ IDNO.3, and the nucleotide sequence of truncation type active fragments is shown in SEQ ID NO.4.
The present invention further provides the method for preparing CNTFt albumen, described method comprises: by to the CNTF molecule and with the analysis of the key position of receptors bind, solvability, stability and the immunogenicity of while comprehensive comparative analysis CNTF albumen, the cDNA of design and acquisition CNTF truncation type active fragments; Powerful permeable membrane advantage in conjunction with the protein transduction peptide, TAT protein transduction peptide and CNTF truncation type active fragments are merged, obtain the gene order of CNTF t of the present invention, made up prokaryotic expression carrier, transform intestinal bacteria, carry out abduction delivering, inclusion body obtains highly purified CNTFt activated protein behind the washing dilution refolding.Thereby the recombinant expression vector of the nucleic acid molecule that contains CNTFt is provided, comprise the host cell that is transformed by any described carrier, and by its new strain or clone of deriving.
The present invention has also showed permeable membrane ability and the biological activity of CNTFt.CNTFt can permeate through cell membranes enter the SH-SY5Y cell, and can enter brain essence by hemato encephalic barrier, can be used for passing through all cytolemma and the biomembranous therepic use such as skin, mucous membrane, cornea, serous coat, sarolemma, blood vessel and lymph film, choroid, neu, hemato encephalic barrier; Can promote the Growth and survival of TF-1 cell, promote various neuronic growths and survival, comprise hippocampal neuron, cortical neuron etc., and promote neural the reparation and regeneration, reduce the decline of neuronic natural death and ability of learning and memory.
The invention provides the therepic use of CNTFt various diseases.CNTFt can be used for the motor neurone diseases such as alzheimer's disease, parkinsonism, black substance degeneration, multiple sclerosis, Heng Yandun chorea, Duchenne-Arandisease and amyotrophic lateral sclerosis, and the nerve injury relative disease etc.; CNTFt can reduce body weight, can be used for obesity or the disease relevant with obesity; CNTFt can be used for the treatment of retina degenerative disease and glaucoma.
The present invention further provides and contain CNTFt and nucleic acid molecule thereof as the pharmaceutical composition of activeconstituents, described pharmaceutical composition comprises pharmaceutically acceptable carrier, and the patient is safe from danger, and does not make the effective constituent inactivation or does not disturb the effect of effective constituent.
The pharmaceutical composition that contains CNTFt albumen can be mixed with by comprise in intravenously, subcutaneous, muscle, the sheath, the mode administration such as nasal membrane, oral mucosa, sublingual administration or mucous membrane of rectum, preferred pharmaceutical composition of the present invention is used by parenteral formulation, and its effective ways of using comprise the agent of suction mist, skin basting agent, eye drip liquor, injection or its sustained-release preparation.
Description of drawings:
Fig. 1 .SOE-PCR amplifies 1.2% agarose gel electrophoresis figure of purpose CNTF gene.
Swimming lane 1:DNA marker; Swimming lane 2:CNTFt.
Fig. 2. double digestion is identified the pBV220-CNTFt plasmid.
The expression of Fig. 3 .CNTFt.
1: albumen Marke r; 2: full bacterium; 3: supernatant; 4: precipitation; 5: inclusion body washings I washes postprecipitation; 6: inclusion body washings I washes rear supernatant; 7: the inclusion body cleaning solution II is washed rear supernatant; 8: the inclusion body cleaning solution II is washed postprecipitation.
Fig. 4. the SDS-PAGE electrophorogram of inclusion body behind the dilution dialysis renaturation.
Swimming lane 1: albumen Marker; Swimming lane 2-4:CNTFt.
Fig. 5 .Western blot analyzes the result of CNTFt.
Fig. 6 .CNTFt is to the determination of activity of TF-1 cell.
Fig. 7 immunohistochemistry shows that CNTFt sees through the SH-SY5Y cytolemma.
A:CNTF t; The B:rhCNTF reference substance.
Fig. 8. immunohistochemistry shows that CNTFt sees through blood-brain barrier of mice.
The hippocampal dentate of A:CNTFt administration group; The hippocampus position of B:rhCNTF reference substance group.
Fig. 9 .Morris determined with Morris water CNTFt is to the therapeutic action of the beta induced Alzheimer disease model mouse of A.
*，p＜0.01。
Embodiment:
The present invention will be more clearly described by the following example, and these embodiment are illustrative, not should be understood to limit the scope of the invention.
Embodiment one: the structure of CNTFt
Take health adult's white corpuscle genomic dna as template, overlapping extension PCR (SOE-PCR) amplifying target genes.Amplify total length CNTF gene with primer P1 and P4, reaction conditions is 95 ℃ of denaturations 5 minutes; 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1 minute, 72 ℃ were extended 4 minutes, and carried out 30 circulations; Extend in 72 ℃ at last and finished reaction in 10 minutes.1% agarose gel electrophoresis is cut glue and is reclaimed, and obtains CNTF with the full fragment of intron; Reclaim; Take gained CNTF as template, P1 and the P2 primer amplification exons 1 that partners, P3 and P4 amplification exon 2.Wherein each primer sequence is: P1 (CG GGT ACC ATG GAC CTC TGTAGC CGC TCT ATC TG); P2 (GCC CTG ATG CTT CAC ATA GGA TTC CGTAAG AGC AG); P 3 (CTC TTA CGG AAT CCT ATG TGA AGC ATC AGG GCCTG); P4 (CGC GTC GAC TTA CCC AGT CTG ATG AGA AG)
The PCR condition of EXON 1 is: 95 ℃ of denaturations 5 minutes; 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1 minute, 72 ℃ were extended 15 seconds, and carried out 30 circulations; Extend in 72 ℃ at last and finished reaction in 10 minutes.2% agarose gel electrophoresis detects amplified production, cuts glue and reclaims;
The PCR condition of EXON 2 is: 95 ℃ of denaturations 5 minutes; 94 ℃ of sex change 1 minute, 60 ℃ of annealing 1 minute, 72 ℃ were extended 30 seconds, and carried out 30 circulations; Extend in 72 ℃ at last and finished reaction in 10 minutes.1.2% agarose gel electrophoresis detects amplified production, cuts glue and reclaims.
Reclaim respectively the amplified production of the above-mentioned exons 1 of purifying and exon 2, respectively get 1 μ L as template, P1 and P4 respectively get 1 μ L as primer, adopt 2 * Pfu-MasterMix10 μ L, adding water 6 μ L, to make reaction system be 20 μ L, and performing PCR amplifies required goal gene.The PCR condition is: 95 ℃ of denaturations 5 minutes; 94 ℃ of sex change 1 minute, 48 ℃ of annealing 1 minute, 72 ℃ were extended 80 seconds, and carried out 5 circulations; 94 ℃ of sex change 1 minute, 58 ℃ of annealing 1 minute, 72 ℃ were extended 80 seconds, and carried out 25 circulations; Extend in 72 ℃ at last and finished reaction in 10 minutes.1.2% agarose gel electrophoresis detects amplified production, and the visible obvious amplified band (Fig. 1) at the 600bp place meets with required purpose clip size.The recovery test kit reclaims.
After reclaiming fragment, with pBV220 carrier while double digestion, connect after reclaiming, make up pBV220-CNTFt, be transformed in the intestinal bacteria.Enzyme is cut and is identified acquisition pBV220-CNTFt plasmid (Fig. 2).The nucleotide sequencing proof is consistent with theoretical value.
Embodiment two: the expression of CNTFt and renaturation
1. the abduction delivering of recombination in intestinal bacteria
Make up correct pBV220-CNTFt recombinant plasmid transformed e. coli bl21/competent cell of pLys S (DE3) through identifying, the positive bacterium colony that picking transforms is put in the LB nutrient solution that 3mL contains penbritin 100mg/L, 30 ℃ of shaking table overnight incubation, getting activation bacterium liquid 10% is inoculated in the 10mL LB nutrient solution (containing penbritin 100mg/L), being cultured to OD550 is 0.6 o'clock, and temperature is transferred to 37-47 ℃ of abduction delivering.
Collect 4-8 hour thalline of abduction delivering, 10 000g collected and express thalline in centrifugal 10 minutes, ultrasonication, and centrifugation supernatant and precipitation are carried out SDS-PAGE and are analyzed (Fig. 3).
2. inclusion body treatment solution
Inclusion body washings I:2mol/L urea, 50mmol/L NaCl, 50mmol/LTris-HCl, 5mmol/L EDTA, pH7.0-9.0;
Inclusion body cleaning solution II: 4mol/L urea, 50mmol/L NaCl, 50mmol/LTris-HCl, 5mmol/L EDTA, pH7.0-9.0;
Solubilization of inclusion bodies liquid: 10mol/L urea, 10mmol/L citrate buffer solution pH3.0-5.0;
Renaturing inclusion bodies liquid: 10mmol/L Tris-HCl, 0.2mol/L L-arginine, 0.1mmol/L reduced glutathion, 0.01mmol/L Sleep-promoting factor B pH8.0-9.0;
3. the dilution dialysis renaturation of inclusion body
With inclusion body washings I and II washing, 4 ℃ are spent the night precipitation inclusion body after the ultrasonication respectively.Inclusion body after the washing is dissolved in the solubilization of inclusion bodies liquid, and centrifugal 10 minutes of 10 000g slowly add the volume ratio of supernatant with 1: 20 in the renaturation solution, hatch a week in 4 ℃.Centrifugal 15 minutes of 10 000g get supernatant liquor and slowly dialyse, and store after the filter membrane degerming.
The major part of dilution refolding precipitation is foreign protein, supernatant after centrifugal can reclaim the target protein (Fig. 4) of the overwhelming majority, so dilution refolding has been removed foreign protein in renaturation, it is the good method that a kind of convenience, simple and easy suitable amplification of going are produced.The method of dilution refolding has increased quantity of sample handling greatly, therefore more saves time, and is more conducive to amplify and rate of recovery height, is fit to carry out the needs of study on the industrialization.
4.CNTFt the mensuration of molecular weight
Carry out the SDS-PAGE electrophoresis according to version " Products in China rules " appendix method in 2000.The apparent molecular weight of CNTFt electrophoresis is 22kD, and theoretical molecular is 21.4kD, meets the requirements.
5.CNTFt immunoblotting detects
The albumen of expressing carries out protein immunoblot and detects, and the result shows that anti-CNTF monoclonal anti physical efficiency is combined (Fig. 5) with expressed protein-specific, illustrates that structure, expression and purifying are successfully.
Embodiment three: the biologic activity of CNTFt detects
Human erythroleukemia cell TF-1 cell cultures before the use, is washed cell 4 times with serum free medium DMEM first in 1640 substratum that contain 10% new-born calf serum and 10ng/mlGM-CSF, being diluted to concentration with the DMEM substratum is 4 * 10
5/ ml adds above-mentioned 96 orifice plates with every hole 100 μ l cells, CNTFt sample serum free medium DMEM doubling dilution, and every hole application of sample 100 μ l, making final concentration is 0.10,0.50,1,5,10,50,100,500,1000,2000ng/ml, in 37 ℃, 5%CO
2Cultivated under the condition about 48 hours, every hole adds 20 μ lMTT, cultivate again 5 hours after, add 20%SDS 80 μ l, survey D570 at enzyme connection instrument behind the abundant dissolving crystallized particle.The activity that similarity condition is measured with reference to product rhCNTF compares.
Embodiment four: CNTFt crosses cytolemma, passes through hemato encephalic barrier and enters brain
For conclusive evidence CNTFt penetrating cytolemma whether, in the SH-SY5Y of logarithmic phase cell, add respectively CNTFt and commercially available rhCNTF effect after 1 hour, carry out immunohistochemistry and identify, establish simultaneously PBS and contrast.The result shows that CNTFt processes and occurs bright green fluorescence in the cell, and the cell green fluorescence that rhCNTF processes weak (Fig. 7) illustrates that CNTFt can cross over cytolemma and enter kytoplasm.
For whether conclusive evidence CNTFt passes through hemato encephalic barrier, the capable frozen section of cerebral tissue is got in execution in 1 hour behind the CNTFt Intraperitoneal injection mouse, do immunohistochemistry take anti-CNTF as primary antibodie and detect.The pallium of CNTFt group and hippocampal slices show the green fluorescence issue, and the rhCNTF group is not seen fluorescence (Fig. 8) in brain essence, and the result shows that CNTFt can see through hemato encephalic barrier and enter in the brain.
Embodiment five: CNTFt is to the curative effect of Alzheimer disease model mouse
The making of Alzheimer disease animal model: A β (25-35) is dissolved in the 1ml sterile saline, and concentration is 1.0mg/ml.After the sealing in 37 ℃ of incubators 96 hours, make it become virose condensed state after.Carry out A β injection in the mouse tricorn, injected dose is every mouse 3 μ l, the physiological saline of sham operated rats injection equal volume.Inject and mouse was carried out the Morris determined with Morris water in the 11st day, continuous 5 days, water maze laboratory latent period was modeling success mouse greater than 120 seconds mouse.The mouse of modeling success is used for experiment.
The Morris water maze: water maze auto-controller device is by institute of Materia Medica,Chinese Academy of Medical Sciences development, pool diameter 100cm, and depth of water 35cm, water temperature is controlled at (23 ± 2) ℃.Security platform is placed the third quadrant of Morris water maze, and as the target of searching for after the mouse entry, take first and second quadrant as the mouse place of entry, the water surface exceeds the about 1cm of security platform.The sky, pond links to each other with computer by pick up camera, the computer-automatic collection data.Experiment is trained 2 times every day, trains 3 minutes at every turn.Before each entry mouse placed security platform upper 15 second, training finishes, and again stops 15 seconds, to strengthen memory effect.If mouse entry 120 seconds with the interior security platform of failing to find in the pond, it can be positioned over had a rest 30 seconds on the security platform after, train again next time, trained altogether 5 days.The water maze laboratory environmental requirement is quiet, avoids all external interference.With mouse from entry to the time of finding security platform (being latent period) weigh its spatial memory capacity for quantitative criterion.
Model mice is divided into AD model physiological saline control group, rhCNTF abdominal cavity treating group and rectal treatment group and CNTFt abdominal cavity treating group and rectal treatment group at random.Every group of 1 mouse.Dosage 100 μ g/kg/day, successive administration 10 days.Morris water maze study of behaviour experiments experiment result shows that no matter the AD model mice is that abdominal injection or rectum give CNTFt and all can significantly shorten its latent period, with AD model physiological saline group comparing difference very significantly (p＜0.01); And compare there was no significant difference (p＞0.05) (Fig. 9) with AD model physiological saline group the latent period that the rhCNTF group fails to improve the AD model mice.
Embodiment six: the anti-obesic action of CNTFt
Get some mouse (ablactation KM mouse, male and female half and half, the 10-15 gram), selects at random 15 as Normal group, give normal diet, all the other are fed with high energy diet (cholesterol 1%, yolk powder 10%, lard 10%, basal feed 79%), cause DI O model (diet induced type obese model), give 8 gram nutrient fodders the 1st every of every day in week, increases weekly later on by 2 grams, freely drink water, altogether 8 weeks of modeling.During the modeling, survey 1 modeling situation with the observation mouse of body weight with electronic balance weekly.The 8th weekend, take the model group Mouse Weight surpass the normal group Mouse Weight 20% for the DIO model successfully, from the mouse of modeling success, select 50, be divided at random 5 groups, be respectively model control group, the high low dose group of intraperitoneal administration and the high low dose group of rectal administration, from normal mouse, select 10 as normal control.Low dosage CNTFt administration is 100 μ g/kg/day, high dosage administration 1000 μ g/kg/day, totally 10 days.Control group gives the physiological saline of isodose.
Experimental session notices that at any time animal behavior changes, and observes the mouse diet situation of every day, and cerebration and ight soil are done rare degree.Last administration and fasting are after 24 hours, take by weighing the weight of animals, and by (whole opisthosoma weighs-initial body weight)/initial body weight * 100%, the calculating body weight increment rate, with mouse with etherization after, the distance of Measurement accuracy mouse from nose to anus is that body is long, and calculates Lee ' s index: Lee ' s exponential formula=(body weight g) according to following formula
1/3The long cm of * 1000/ body.
Find experimental session in the experiment, the every behavior of animal comprises drinking-water, and cerebration is normal, and ight soil is tested and finished to put to death animal without unusually, checks each internal organs, and the heart of animal, liver, spleen, lung, kidney etc. are all normal, and the liver color and luster is scarlet, and is smooth.The Mouse Weight variation sees Table 1.
Table 1. various dose CNTF t under different way of administration on the impact of Mouse Weight
*, p＜0.01, *, p＜0.05 vs DIO-contrast.
Embodiment seven: the injection prescription of preparation take CNTFt as active ingredient:
| CNTFt | 100mg |
| NaCl | 8g |
| KCl | 0.20g |
| Na 2HPO 4 | 1.15g |
| KH 2PO 4 | 0.20g |
| Water for injection | 1000ml |
| The pH value | 7.4 |
Packing 1ml/ props up after the sterile filtration, and freeze-dried powder is made in freezing preservation, or freeze-drying.
Embodiment eight: preparation treatment retinal degenerative disease, optic nerve injury and glaucomatous eye drops prescription:
| CNTFt | 100mg |
| Hyaluronic acid | 2.5g |
| Vitamin B6 | 2.5g |
| N.F,USP MANNITOL | 15g |
| Water for injection | 1000ml |
| The pH value | 7.0 |
Packing 10ml/ props up after the sterile filtration, 4 ℃ of preservations.
Sequence table
＜110〉truncated-type human ciliary nerve nutrition factor active segment and fusion rotein thereof
＜120〉Inst of Toxic Medicinal Materials, P.L.A. Academy of Military Medical Sciences
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His Thr Leu Leu Leu Gln Val Ala Ala Phe Ala Tyr Gln Ile Glu Glu
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His Asp Leu Arg Phe Ile Ser Ser His Gln Thr Gly
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35
Claims (15)
1. the fusion rotein of protein transduction peptide and truncated-type human ciliary nerve nutrition factor active segment is characterized in that this albumen is the aminoacid sequence shown in the SEQ ID NO.1.
2. truncated-type human ciliary nerve nutrition factor active segment is characterized in that this fragment is the aminoacid sequence shown in the SEQ ID NO.2.
3. the nucleic acid molecule shown in the SEQ ID NO.3 of albumen shown in the coding claim 1.
4. the nucleic acid molecule shown in the SEQ ID NO.4 of albumen shown in the coding claim 2.
5. nucleic acid molecule that is formed by the nucleotide sequence of coding claim 1 described albumen.
6. expression vector that contains the nucleotide sequence of coding claim 1 described albumen.
7. the recombinant expression vector that contains the described nucleic acid molecule of claim 4.
8. claim 1 or 2 described albumen are in the purposes for the preparation of the medicine for the treatment of nerve degenerative diseases, and described disease comprises alzheimer's disease, parkinsonism and amyotrophic lateral sclerosis.
9. claim 1 or 2 described albumen are for the preparation of the purposes in the medicine for the treatment of of obesity.
10. claim 1 or 2 described albumen are for the preparation of the purposes in treatment retina degenerative disease and the glaucomatous medicine.
11. a pharmaceutical composition, it contains claim 1 or 2 described albumen as activeconstituents and pharmaceutically acceptable carrier or excipient.
12. pharmaceutical composition according to claim 11, the administering mode of described pharmaceutical composition are selected from intravenously administrable, subcutaneous administration, muscle administration, intrathecal drug delivery or mucosa delivery.
13. pharmaceutical composition according to claim 12, described mucosa delivery are selected from nasal administration, mouth mucosa drug administration or mucous membrane of rectum administration.
14. pharmaceutical composition according to claim 11, the dosage form of described pharmaceutical composition are selected from the corresponding sustained-release preparation of oral preparations or parenteral formulation or its.
15. pharmaceutical composition according to claim 14, the parenteral formulation form of described pharmaceutical composition are selected from injection, freeze-dried preparation, the agent of suction mist, skin basting agent, eye drip liquor or its sustained-release preparation.
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| CN 200810088976 CN101555284B (en) | 2008-04-10 | 2008-04-10 | Truncated-type human ciliary nerve nutrition factor active segment and fusion protein thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200810088976 CN101555284B (en) | 2008-04-10 | 2008-04-10 | Truncated-type human ciliary nerve nutrition factor active segment and fusion protein thereof |
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| CN1563087A (en) * | 2004-04-02 | 2005-01-12 | 西南生物工程产业化中试基地有限公司 | Polypeptide of ramification of ciliary nerves nutrilite for curing obesity |
| CN101144082A (en) * | 2007-06-12 | 2008-03-19 | 兰州生物制品研究所 | Recombination human ciliary neurotrophy factor, mutant and application thereof |
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| CN1563087A (en) * | 2004-04-02 | 2005-01-12 | 西南生物工程产业化中试基地有限公司 | Polypeptide of ramification of ciliary nerves nutrilite for curing obesity |
| CN101144082A (en) * | 2007-06-12 | 2008-03-19 | 兰州生物制品研究所 | Recombination human ciliary neurotrophy factor, mutant and application thereof |
Non-Patent Citations (2)
| Title |
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| 袁松涛等人.小鼠睫状神经营养因子基因在视网膜色素上皮细胞系ARPE-19细胞中表达的研究.《中华眼科杂志》.2006,第42卷(第11期),1017-1022. * |
| 袁松涛等人.小鼠野生型和截短型睫状神经营养因子基因的克隆及真核表达.《眼科新进展》.2006,第26卷(第4期),241-244. * |
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