CN101555284A - Truncated-type human ciliary nerve nutrition factor active segment and fusion protein thereof - Google Patents

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, and the like.

Description

Truncated-type human ciliary nerve nutrition factor active segment and fusion rotein thereof

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 proteic prokaryotic expression preparation method.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 CNTF) is found in 1976 by Helfand etc. ciliary neurotrophic factor at first, and Barbin in 1984 etc. extract from chicken ciliary nerves unit and obtain, because of the survival that can keep the chicken parasympathetic ganglion is gained the name.CNTF can impel multiple neuron survival, is that first foundly can maintain the survival of body and stripped dynamoneure 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 a nonactive essential cysteine residues is arranged at the 17th.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 with after its receptor alpha subunit combines 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, combine 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 effective object is a neural system, multiple neurocyte such as the Sensory neurone of ciliary nerves ganglion cell, sympathetic neuron, spinal ganglion, dynamoneure, cholinergic neuron, dopamine neuron all 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, influences work, life and doings, and the state of an illness is carrying out property and increases the weight of, lose independent living ability gradually, the morbidity back is many because of complication death.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, Chi Dai sickness rate and morbidity all are 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 of treatment 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 catecholamine energy, 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 contract disease and the Duchenne-Arandisease (SMA) etc. of Heng Yandun (Huntington) chorea, amyotrophic lateral sclerosis (ALS), parkinsonism, black substance degeneration, multisystem committee.

1.2CNTF with the retina degenerative disease

CNTF has provide protection to retinal ganglial cells.In numerous neurotrophic factors, CNTF uniquely can effectively promote retinal ganglial cells regenerated nutritional factor, 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 a 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 makes the mouse weight loss.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, and body weight reduces; For the fat model mice (DIO mice) of diet induced, CNTF also can make it weight loss, and this mouse model more can be represented the practical situation of human obesity.CNTF may make the body weight point of adjustment downward modulation of thalamus, thereby inhibition food intake, fat-reducing and other methods of treatment are compared with keeping on a diet by strictness, excessive food intake after the drug withdrawal can not take place and cause the body weight bounce-back in CNTF, therefore, CNTF is expected to exploitation becomes a kind of novel, medicine of losing weight efficiently, 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 that nerve injury is repaired with the solubility CNTFRA that exists in the muscle.Peripheral nerve cuts off can cause the death of corresponding spinal cord CM, 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 nascent rat 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.The semi-permeable membranes micro-capsule that the viable cell that can secrete CNTF is housed is implanted the pathology part, can improve Local C NTF concentration, reduce Plasma Concentration, alleviate 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.Under 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, kind surplus neurotrophic factor (NTFs) has 20.But since half a century, because the effect of hemato encephalic barrier, the clinical application of NTFs is still very limited.Still lack a rational and effective central administration approach at present clinically.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 subjected 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 a protein macromolecule, be difficult to see through hemato encephalic barrier, thereby having limited its treatment at maincenter uses, though some Transplanted cellss carried out at present and the research of gene therapy aspect have obtained some progress in experimentation on animals, because these treatment plans are all taked traumatic means such as operation and intracerebroventricular, from clinical application very big 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 has merged the protein transduction peptide simultaneously for 14 amino acid of Human Ciliary Neurotrophic Factor N end disappearance, 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 nucleic acids encoding said proteins sequence, 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 proteic method of preparation CNTFt, described method comprises: by to the CNTF molecule and with the analysis of the key position of receptors bind, the proteic solvability of while comprehensive comparative analysis CNTF, stability and immunogenicity, the cDNA of design and acquisition CNTF truncation type active fragments; The powerful film advantage of conjugated protein transduction peptide, TAT protein transduction peptide and CNTF truncation type active fragments are merged, obtain the gene order of CNTFt of the present invention, made up prokaryotic expression carrier, transformed into escherichia coli, 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 by any described carrier transformed host cells, and by its deutero-new strain system or clone.

The present invention has also showed saturating film 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 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 the 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 many kinds of treatment of diseases purposes of CNTFt.CNTFt can be used for 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 the pharmaceutical composition as 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.

Contain the proteic pharmaceutical composition of CNTFt can be mixed with by comprise in intravenously, subcutaneous, muscle, the sheath, 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.

After swimming lane 1,2 and 3:pBV220-CNTFt enzyme are cut; Swimming lane 4:DNA Marker.

The expression of Fig. 3 .CNTFt.

1: albumen Marker; 2: full bacterium; 3: supernatant; 4: precipitation; 5: inclusion body washings I washes postprecipitation; 6: inclusion body washings I washes the back supernatant; 7: the inclusion body cleaning solution II is washed the back supernatant; 8: the inclusion body cleaning solution II is washed postprecipitation.

Fig. 4. the SDS-PAGE electrophorogram of inclusion body after 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 measures the TF-1 cell activity.

Fig. 7 fluorescence immunoassay group demonstration CNTFt sees through the SH-SY5Y cytolemma.

A:CNTFt; The B:rhCNTF reference substance.

Fig. 8. fluorescence immunoassay group demonstration 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

With health adult's white corpuscle genomic dna is template, overlapping extension PCR (SOE-PCR) amplifying target genes.Amplify total length CNTF gene with primer P1 and P4, reaction conditions is 95 ℃ of pre-sex change 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 the full fragment of CNTF band intron; Reclaim; With gained CNTF is 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); P3 (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 pre-sex change 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 pre-sex change 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 the amplified production of above-mentioned exons 1 of purifying and exon 2 respectively, respectively get 1 μ L as template, P1 and P4 respectively get 1 μ L as primer, adopt 2 * Pfu-MasterMix, 10 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 pre-sex change 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 tangible amplified band (Fig. 1) at the 600bp place meets with required purpose clip size.The recovery test kit reclaims.

After reclaiming fragment,, reclaim the back and connect, make up pBV220-CNTFt, be transformed in the intestinal bacteria with pBV220 carrier while double digestion.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 the correct competent cell of pBV220-CNTFt recombinant plasmid transformed e. coli bl21/pLysS (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, get activation bacterium liquid 10% and be inoculated in the 10mL LB nutrient solution (containing penbritin 100mg/L), be cultured to OD ₅₅₀Be 0.6 o'clock, temperature is transferred to 37-47 ℃ of abduction delivering.

Collect 4-8 hour thalline of abduction delivering, 10000g collected and expresses 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, pH 7.0-9.0;

Inclusion body cleaning solution II: 4mol/L urea, 50mmol/L NaCl, 50mmol/LTris-HCl, 5mmol/L EDTA, pH 7.0-9.0;

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 of inclusion body dialysis renaturation

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 10000g slowly adds the volume ratio of supernatant with 1: 20 in the renaturation solution, hatches a week in 4 ℃.Centrifugal 15 minutes of 10 000g get supernatant liquor and slowly dialyse, and store after the filter membrane degerming.

The sedimentary major part of dilution refolding is a foreign protein, and the supernatant after centrifugal can reclaim the target protein (Fig. 4) of the overwhelming majority, so dilution refolding has been removed foreign protein in renaturation, is a kind of convenience, simple and good method that suitable amplification easily row is 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 " Chinese biological goods rules " appendix method in 2000.The electrophoretic apparent molecular weight of CNTFt is 22kD, and theoretical molecular is 21.4kD, meets the requirements.

5.CNTFt immunoblotting detects

Expressed proteins is carried out protein immunoblot and is detected, and the result shows that anti-CNTF monoclonal anti physical efficiency combines (Fig. 5) with expressed protein-specific, illustrates that structure, expression and purifying are successful.

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 earlier 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 is in 37 ℃, 5%CO ₂Cultivated under the condition about 48 hours, every hole adds 20 μ lMTT, cultivate 5 hours again after, add 20%SDS 80 μ l, on enzyme connection instrument, survey D570 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 goes into brain

For conclusive evidence CNTFt penetrating cytolemma whether, in the SH-SY5Y of logarithmic phase cell, add CNTFt and commercially available rhCNTF effect respectively after 1 hour, carry out the evaluation of fluorescence immunoassay group, establish PBS simultaneously and contrast.The result shows that CNTFt handles and occurs bright green fluorescence in the cell, and the cell green fluorescence that rhCNTF handles 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 abdominal cavity injection mouse, be that an anti-immunohistochemistry of doing is detected with anti-CNTF.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.Sealing back 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 a 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, as the target of searching for after the mouse entry, is the mouse place of entry with first and second quadrant, and 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 last 15 second, training finishes, and stops once more 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.All external interference are avoided in water maze laboratory environmental requirement peace and quiet.With mouse from entry to the time of finding security platform (being latent period) weigh its spatial memory ability for quantitative criterion.

Model mice is divided into AD model physiological saline control group, rhCNTF abdominal cavity treatment group and rectal treatment group and CNTFt abdominal cavity treatment 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 15 at random, give normal diet as the normal control group, all the other are fed with high energy diet (cholesterol 1%, yolk powder 10%, lard 10%, basal feed 79%), cause DIO model (diet induced type fat model), give 8 gram nutrient fodders the 1st every of every day in week, increases by 2 grams later on weekly, freely drink water, altogether 8 weeks of modeling.During the modeling, survey 1 modeling situation of body weight with electronic balance weekly with the observation mouse.The 8th weekend, surpassing 20% of normal group mouse body weight with model group mouse body weight is the success of DIO model, from the mouse of modeling success, select 50, be divided into 5 groups at random, 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%, calculate the weight increase rate, with mouse with etherization after, accurately measuring the distance of 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 no abnormal, and experiment finishes to put to death animal, 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 body weight change sees Table 1.

Table 1. various dose CNTFt under different way of administration to the influence of mouse body weight

*, p＜0.01, *, p＜0.05vs DIO-contrast.

Embodiment seven: preparation is the injection of active ingredient with CNTFt

Prescription:

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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accccaaccg gaggtgactt ccatcaagct atacataccc ttcttctcca agtcgctgcc 360

tttgcatacc agatagagga gttaatgata ctcctggaat acaagatccc ccgcaatgag 420

gctgatggga tgcctattaa tgttggagat ggtggtctct ttgagaaaaa gctgtggggc 480

ctaaaggtgc tgcaggagct ttcacagtgg acagtaaggt ccatccatga ccttcgtttc 540

atttcttctc atcagactgg gtaa 564

<210>4

<211>519

<212>DNA

＜213〉artificial sequence

<400>4

atggacctct gtagccgctc tatctggcta gcaaggaaga ttcgttcaga cctgactgct 60

cttacggaat cctatgtgaa gcatcagggc ctgaacaaga acatcaacct ggactctgcg 120

gatgggatgc cagtggcaag cactgatcag tggagtgagc tgaccgaggc agagcgactc 180

caagagaacc ttcaagctta tcgtaccttc catgttttgt tggccaggct cttagaagac 240

cagcaggtgc attttacccc aaccgaaggt gacttccatc aagctataca tacccttctt 300

ctccaagtcg ctgcctttgc ataccagata gaggagttaa tgatactcct ggaatacaag 360

atcccccgca atgaggctga tgggatgcct attaatgttg gagatggtgg tctctttgag 420

aaaaagctgt ggggcctaaa ggtgctgcag gagctttcac agtggacagt aaggtccatc 480

catgaccttc gtttcatttc ttctcatcag actgggtaa 519

<210>5

<211>11

<212>PRT

＜213〉artificial sequence

<400>5

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

1 5 10

<210>6

<211>16

<212>PRT

＜213〉artificial sequence

<400>6

Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys

1 5 10 15

<210>7

<211>36

<212>PRT

＜213〉artificial sequence

<400>7

Asp Ala Ala Thr Ala Thr Arg Gly Arg Ser Ala Ala Ser Arg Pro Thr

1 5 10 15

Glu Arg Pro Arg Ala Pro Ala Arg Ser Ala Ser Arg Pro Arg Pro Arg

20 25 30

Arg Pro Val Glu

35

Claims (16)

1. the protein transduction peptide merges truncated-type human ciliary nerve nutrition factor active segment, it is characterized in that this albumen comprises 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 comprises the aminoacid sequence shown in the SEQ ID NO.2.

3. the nucleotide sequence shown in the proteic SEQ ID NO.3 shown in the coding claim 1.

4. the nucleotide sequence shown in the proteic SEQ ID NO.4 shown in the coding claim 2.

5. the protein sequence of claim 1, wherein said protein transduction peptide sequence is made up of following several any aminoacid sequence or its functional fragment is formed.

(A) contain and be selected from the HIV-1 trans-activator (its aminoacid sequence comprises the sequence shown in the SEQ ID NO.5 for trans-activatortranscription, TAT) the protein transduction district in.

(B) fruit bat feeler albumen homology structural domain (ANTP), its aminoacid sequence comprises the sequence shown in the SBQ IDNO.6.

(C) the protein transduction sequence of hsv (HSV) VP22, its aminoacid sequence comprises the sequence shown in the SEQ ID NO.7.

6. a nucleic acid molecule and expression vector that contains coding claim 1 described proteic nucleotide sequence.

7. the nucleic acid molecule and the recombinant expression vector that contain the nucleotide sequence of the described truncation type active fragments of claim 2.

8. the preparation claim comprises: to the design of truncated-type human ciliary nerve nutrition factor active segment; Obtain the cDNA that the protein transduction peptide merges the truncated-type human ciliary nerve nutrition factor active segment gene; Construction recombination plasmid, transformed into escherichia coli carries out abduction delivering; Carry out renaturation with the dilution refolding method.

9. each described albumen of claim 1-4 or nucleic acid molecule are used for the treatment of the purposes of the medicine of nerve degenerative diseases in preparation, described disease comprises motor neurone diseases such as being not limited to 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.

Each described albumen of claim 1-4 or nucleic acid molecule preparation be used for the treatment of obesity or with the medicine of obesity relative disease in purposes.

11. each described albumen of claim 1-4 or nucleic acid molecule are used for the treatment of purposes in retina degenerative disease and the glaucomatous medicine in preparation.

12. a pharmaceutical composition, it contains each described albumen of claim 1-4 or nucleic acid molecule as activeconstituents and pharmaceutically acceptable carrier or excipient.

13. according to the pharmaceutical composition of claim 12, the administering mode of described pharmaceutical composition is selected from intravenously administrable, subcutaneous administration, muscle administration, intrathecal drug delivery or mucosa delivery.

14. according to the pharmaceutical composition of claim 13, described mucosa delivery is selected from nasal administration, mouth mucosa drug administration or mucous membrane of rectum administration.

15. according to the pharmaceutical composition of claim 12, the dosage form of described pharmaceutical composition is selected from the corresponding sustained-release preparation of oral preparations or parenteral formulation or its.

16. according to the pharmaceutical composition of claim 15, the parenteral formulation form of described pharmaceutical composition is 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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