CN103113473A - TAT-LBD-NEP1-40 fusion protein, construction method and application thereof - Google Patents
TAT-LBD-NEP1-40 fusion protein, construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a TAT-LBD-NEP1-40 fusion protein, a construction method and an application thereof for treating central lesion. The TAT-LBD-NEP1-40 fusion protein constructed by the invention remains selective antagonistic activity of NEP1-40 to a Nogo receptor and has the advantages of high transduction efficiency and easy transmission of blood and spine barriers and blood brain barrier, so that the limit of NEP1-40 is overcome so as to avoid the deficiency of extra damage to nervous tissues caused by micro-injection of NEP1-40 or micro pump implantation and difficulty for NEP1-40 to pass through blood and spine barriers and blood brain barrier to meet corresponding biological concentration. The infusion protein provided by the invention can be massively prepared, and is low in cost and high in activity. The infusion protein can be used to treat various CNS (Central Nervous System) damages such as cerebral ischemia and anoxia, cerebral hemorrhage, cerebral trauma and spinal cord injury, promote regeneration of nervous tissues and neural functional recovery, and treat CNS damages.
Description
Technical field
The invention belongs to the protein transduction field, be specifically related to that LBD is introduced TAT-NEP1-40 and merge preparation TAT-LBD-NEP1-40 fusion rotein.Simultaneously, the invention still further relates to the TAT-LBD-NEP1-40 fusion rotein and pass hemato encephalic barrier and be enriched in ischemic region, be used for the treatment of central nervous system injury.
Background technology
Wound, apoplexy, hematencephalon, Spinal injury etc. have the characteristics of high mortality and high disability rate.How to reduce the neural function extent of damage and the injured nerve meta function is improved better, improve patient's life quality, all significant to patient and family thereof, society, be a global difficult problem.At present, the neurological dysfunction that damage causes for CNS there is no effective medicine.Therefore, seeking novel effective treatment brain CNS damage medicine is study hotspot.
Generally believed in the past that the major cause that neurological dysfunction that Adult Mammals central nervous system (central nervous system, CNS) damage causes is difficult to recover was that neurone can not be regenerated.The existing breakthrough of the research that recent two decades comes thinks that at present CNS regenerates difficult reason except the nutritional factor deficiency that promotes regeneration, the inhibition regenerative environ-ment that another important reason is maincenter.In recent years, main gene-Nogo and acceptor (the Nogo receptor thereof that suppresses injured nerve regeneration, NgR) discovery has been raised new page for the research of CNS damage, and being described as is " the exploration central nervous system injury is repaired a milestone in the very long road ".Recently the research delivered of Neuron, applying gene knocks out (knockout) technology, discovery knocks out Nogo-A and NgR and improves regeneration and plastic reaction after the Spinal injury, uses NgR antagonist (NgRecto) treatment, the functional rehabilitation of promotion axon regeneration and Spinal injury.Same application Nogo-A antibody (IN-1,7B12) and NgR antagonist (NgRecto) treatment significantly improve the study of behaviour result that MACO causes, and promote aixs cylinder plasticity-.For this reason, Lee etc. write the paper of " Targeting the Nogo receptor to treat central nervous system injuries " at " Nature Review Drug Discovery ", propose " NgR as a drug discovery target ", thinking to regulate and control NgR is therapeutic goal, can greatly promote the rear neuronic functional rehabilitation of CNS damage, reverse its catastrophic consequence.NgR becomes study hotspot and the focus in CNS damage field at present, thinks that blocking it can affect development and the result of cerebral ischemia, promotes and strengthen neurological functional recovery.
Nogo-66 is the outer loop shape structures of Nogo-A molecule born of the same parents, thereby be combined the regeneration of triggering downstream signal path block nerves with NgR.NEP1-40 (Nogo extracellular peptide residues1-40) is the polypeptide that 40 residues of Nogo-66 aminoterminal form, and remains with the ability that Nogo-66 is combined with NgR, but does not trigger the signal transduction pathway in downstream.NEP1-40 has been applied to animal model with spinal cord damnification as the NgR antagonist, can promote the growth of cortex spinal cord aixs cylinder and sprouting of serotonin energy nerve fiber, and the formation of rise axon growth Protein S PRR1A and cynapse, can significantly promote the recovery of laboratory animal motor function.NEP1-40 has highly selective and validity and makes it be expected to become the new drug of the axon regeneration obstacle diseases such as treatment Spinal injury, cerebral trauma, apoplexy and chronic progressive external multiple sclerosis as the NgR antagonist.Yet, employed NEP1-40 is through chemosynthesis in the research at present, its cost is higher, and the existence owing to hemato encephalic barrier (BBB), blood ridge barrier, cytolemma etc., generally can not pass these barriers greater than 6 amino acid whose polypeptide, and do not reach effective pharmacological agent concentration, mainly be at present the form administration by operations such as micro-locating injection or implantable miniature pumps.There is potentially dangerouss such as bringing out intracerebral hemorrhage in microinjection, and can only keep short duration; The implantable miniature pump is better, but has the problem that is difficult to by blood ridge barrier, hemato encephalic barrier, and the further studies and clinical application of NEP1-40 is greatly limited.At present, the novel protein TAT-NEP1-40 fusion rotein of having developed by application TAT protein transduction technology can pass through hemato encephalic barrier smoothly, reduces cerebral ischemia.But the tropism is relatively poor for the TAT protein targets, enters rear extensively distribution in the brain, can not concentrate to act on the ischemic injuries position.Therefore, how to make the TAT-NEP1-40 targeting become the now emphasis of research in the CNS damage location.
Ln Laminin is the important extracellular matrix of CNS.When ischemic brain injury, laminin is high expression level (Liebkind RTE. in ischemic area, et al., 2007), therefore, it can become the treatment protein aggregation dives in conjunction with target area in one of the ischemic injuries zone, obviously strengthens drug level and therapeutic efficiency, makes the effect of the more effective performance targeted therapy for the treatment of albumen injured neuron.Agrin (Agrin) is a key molecule of neuromuscular junction postsynaptic differentiation, the aminoterminal of ln and agrin (N-terminal domain of agrin, NtA) very strong affinity is arranged, NtA can be used as the structural domain of being combined with ln.Have and studies show that: the fusion rotein that contains neurotrophic factor BDNF and ln calmodulin binding domain CaM NtA by structure, can be gathered in the cerebral ischemia zone by target, and the therapeutic action of efficient, lasting performance BDNF, and normal tissue and cell are without any side effect (Han, Q., et al., 2011).
Summary of the invention
The purpose of this invention is to provide a kind of TAT-LBD-NEP1-40 fusion rotein for the CNS injury in treating, it is merged by the TAT structural domain of human immunodeficiency virus and LBD structural domain and treatment function fragment NEP1-40 and makes.
Another object of the present invention provides a kind of method of TAT-LBD-NEP1-40 fusion rotein of the CNS of structure injury in treating.
A further object of the present invention is that the TAT-LBD-NEP1-40 fusion rotein is for the preparation of the application of the medicine of mammal brain central nervous system injury.This medicine can be used in the treatment that comprises cerebral ischemia/anoxia, hematencephalon, cerebral trauma, Spinal injury at the mammalian central nervous system damage disease.
In order to realize above-mentioned technical assignment, the present invention is achieved by following technical solution:
A kind of TAT-LBD-NEP1-40 fusion rotein is characterized in that: cDNA sequence and the protein sequence of this fusion rotein are as follows:
The cDNA sequence:
CTGCCGGGCGCATCGGGCACCTGTCCGGAACGCGCACTGGAACGTCGTGAAGAAGAAGCAAATGTTGTCCTGACGGGTACGGTTGAAGAAATTCTGAACGTTGATCCGGTCCAGCATACCTATAGCTGCAAAGTCCGTGTGTGGCGCTACCTGAAAGGCAAGGATCTGGTGGCACGTGAAAGTCTGCTGGACGGCGGTAATAAGGTGGTTATTTCCGGCTTTGGTGATCCGCTGATCTGTGACAACCAAGTCAGTACCGGTGATACGCGCATCTTTTTCGTTAATCCGGCACCGCCGTATCTGTGGCCGGCACATAAAAACGAACTGATGCTGAATAGCTCTCTGATGCGTATTACGCTGCGCAACCTGGAAGAAGTGGAATTTTGCGTTGAAGATAAACCGGGCGGTGGCGGTTCACGTATCTACAAAGGCGTTATTCAGGCGATCCAAAAGTCGGACGAAGGTCACCCGTTCCGCGCCTACCTGGAATCGGAAGTCGCAATCTCAGAAGAACTGGTGCAAAAATACAGCAACAGC。
Protein sequence:
-LPGASGTCPERALERREEEANVVLTGTVEEILNVDPVQHTYSCKVRVWRYLKGKDLVARESLLDGGNKVVISGFGDPLICDNQVSTGDTRIFFVNPAPPYLWPAHKNELMLNSSLMRITLRNLEEVEFCVEDKPGGGGSRIYKGVIQAIQKSDEGHPFRAYLESEVAISEELVQKYSNS。
Further, the TAT-LBD-NEP1-40 fusion rotein is that TAT, LBD and NEP1-40 fusion are made, and specifically makes up according to following step:
Step 1: obtain LBD gene and NEP1-40 sequence from Genebank, then will connect by the GGCGGTGGCGGTTCA sequence between LBD gene and the NEP1-40 sequence, synthetic LBD-NEP1-40 gene order, and so that Nco I and Xho I sequence are contained respectively in the gene order two ends of LBD-NEP1-40, the gene order of whole LBD-NEP1-40 is:
A
CCATGGCC-CT GCCGGGCGCAT CGGGCACCT G TCCGGAACGCGCAC TGGAACGTCGTGAAGAAGAAGCAAATGTTGTCCTGACGGGTACGGTTGAAGAAATTCTGAACGTTGATCCGGTCCAGCATACCTATAGCTGCAAAGTCCGTGTGTGGCGCTACCTGAAAGGCAAGGATCTGGTGGCACGTGAAAGTCTGCTGGACGGCGGTAATAAGGTGGTTATTTCCGGCTTTGGTGATCCGCTGATCTGTGACAACCAAGTCAGTACCGGTGATACGCGCATCTTTTTCGTTAATCCGGCACCGCCGTATCTGTGGCCGGCACATAAAAACGAACTGATGCTGAATAGCTCTCTGATGCGTATTACGCTGCGCAACCTGGAAGAAGTGGAATTTTGCGTTGAAGATAAACCGGGCGGTGGCGGTTCACGTATCTACAAAGGCGTTATTCAGGCGATCCAAAAGTCGGACGAAGGTCACCCGTTCCGCGCCTACCTGGAATCGGAAGTCGCAATCTCAGAAGAACTGGTGCAAAAATACAGCAACAGC-
CTCGAGWherein the gene underscore is Nco I and Xho I restriction enzyme site;
Step 2: the gene order that will synthesize the LBD-NEP1-40 that obtains is loaded in the pUC57 carrier, obtains the pUC57-LBD-NEP1-40 carrier;
Step 3; Select the pTAT-HA of TAT sequence of the human immunodeficiency virus that contains restriction enzyme Nco I and Xho I as expression vector;
Step 4: cut pTAT-HA and pUC57-LBD-NEP1-40 carrier with restriction enzyme Nco I and Xho I enzyme, reclaim enzyme and cut product, wherein to cut product be linear pTAT carrier to the enzyme of pTAT, and the enzyme of the pUC57-LBD-NEP1-40 product of being sure to is the LBD-NEP1-40 gene fragment;
Step 5: it is that linear pTAT carrier is connected the T4 ligase enzyme to connect to obtain recombinant plasmid pTAT-LBD-NEP1-40 expression vector by colony screening with the TAT-LBD-NEP1-40 gene fragment that two kinds of enzymes that step 4 is obtained are cut product;
Step 6: after recombinant plasmid pTAT-LBD-NEP1-40 expression vector is correct through the dna sequencing analysis, be transformed in e. coli bl21 (DE3) bacterial strain, select the colony clone of 5～8 positives in the LB substratum of ammonia benzyl resistance, adopt the pre-abduction delivering TAT-LBD-NEP1-40 of IPTG fusion rotein;
Step 7: the whole bacterial protein behind the abduction delivering is adopted SDS-PAGE, Coomassie brilliant blue R250 dyeing successively, determine condition and the positive colony clone of abduction delivering, then at 16～22 ℃, great expression TAT-LBD-NEP1-40 fusion rotein under the 0.2mMIPTG condition;
Step 8: with the bacterium of the TAT-LBD-NEP1-40 fusion rotein of abduction delivering through the ultrasonication thalline, after discharging target protein, through the affinity chromatography column purification that the Ni ion is integrated, the albumen behind the purifying is 2mg/ml through ultrafiltration and concentration, and the refrigerator and cooled that leaves-80 ℃ in is hidden.
Characteristics of the present invention are: protein transduction domain (the protein transduction domain that will derive from human immunodeficiency virus HIV-1TAT, PTD), the LBD of specific combination ischemic region and NEP1-40 merge, make up the pTAT-LBD-NEP1-40 recombinant plasmid, obtain having bioactive TAT-LBD-NEP1-40 fusion rotein through expression, purifying.
TAT-LBD-NEP1-40 fusion rotein of the present invention can be used for comprising the treatment of the various CNS damages such as cerebral ischemia/anoxia, hematencephalon, cerebral trauma, Spinal injury.Described TAT-LBD-NEP1-40 fusion rotein has kept the selectivity antagonistic activity of NEP1-40 to NgR, have the transduction efficiency height, see through the advantage of BBB, overcome the limitation of NEP1-40, and LBD makes NEP1-40 have more targeting, be used for the brain injury treatment, can reduce medicine to the side effect of its hetero-organization, make the treatment targeting proteins be gathered in damage field.
TAT fusion rotein system is a kind of good albumen means of conveyance, break protein and usually can only entrained bioinformation be delivered to intracellular rule by cell surface receptor and signal transduction pathway, can effectively solve the problem that macro-molecular protein can not penetrate hemato encephalic barrier (BBB), in clinical application macromolecular drug treatment cerebrovascular disease, have broad application prospects.The present invention utilizes the method for TAT mediating protein, the encoding gene of TAT-PTD is connected with foreign protein LBD, NEP1-40 gene, expressed fusion protein, resulting TAT-LBD-NEP1-40 fusion rotein has not only kept the original penetration power of TAT, and does not affect the vigor of foreign protein.
In order to improve effective drug level, reduce simultaneously medicine to the side effect of its hetero-organization, therefore treating albumen how target is gathered in damage field is to need to solve a key issue.In cerebral ischemia brain injury process, ln (Laminin, LN) high expression level is in cerebral ischemia zone, the N terminal sequence of collectin agrin can with the LN specific binding, this sequence that can be combined with the LN characteristic is named as laminin-binding domain(LBD).By making up LBD and the fusion rotein that Brain Derived Neurotrophic Factor (Brain-derived neurotrophic factor, BDNF) combines, bring into play the effect of targeted therapy cerebral ischemia.
The present invention uses the C57 mouse, adopts the transduction of the method confirmation TAT-LBD-NEP1-40 fusion rotein of Fluorescent immunohistochemistry analysis and Westen Blot; the result shows that the TAT-LBD-NEP1-40 fusion rotein can pass hemato encephalic barrier and enter in the brain and reduce brain infarction area; the transduction that shows the TAT-LBD-NEP1-40 fusion rotein has cerebral protection to the C57 mouse.Simultaneously, the mode of the TAT-LBD-NEP1-40 of the present invention after with purifying by abdominal injection awards the C57 mouse behind the global brain ischemia model, detect the TAT-LBD-NEP1-40 fusion rotein to cerebral ischemia after variation and the cranial nerve function of Damaged axon whether be improved effect.The Fluorescent immunohistochemistry result shows; fusion rotein TAT-LBD-NEP1-40; TAT-β-Gal all is positive at Nerve Cells of Rat Brain; and the C57 mouse brain tissue of injection No-TAT-β-Gal fusion rotein is negative reaction; illustrate that but the TAT mediating protein transduces into cerebral tissue by hemato encephalic barrier; and TAT-LBD-NEP1-40 can significantly improve the neurological dysfunction that Focal Cerebral Ischemia causes; reduce infarct volume and promote injured neuron cynapse regeneration; show that TAT-LBD-NEP1-40 has the protein transduction effect and can enter cerebral tissue, cerebral ischemia is had provide protection.
TAT-LBD-NEP1-40 fusion rotein of the present invention has kept the selectivity antagonistic activity of NEP1-40 to NgR, have the transduction efficiency height, see through the advantage of BBB, overcome the limitation of NEP1-40, and LBD makes NEP1-40 have more targeting, be used for the brain injury treatment, can reduce medicine to the side effect of its hetero-organization, make the treatment targeting proteins be gathered in damage field, can be used for comprising the treatment of the various CNS damages such as cerebral ischemia/anoxia, hematencephalon, cerebral trauma, Spinal injury.The present invention can prepare in a large number, cost is low, activity is high, be used for comprising the treatment of the various CNS damages such as cerebral ischemia/anoxia, hematencephalon, cerebral trauma and Spinal injury, promote regeneration and the neurological functional recovery of nervous tissue, the medicine for the treatment of central nervous system injury.
Description of drawings
Fig. 1 .TAT-LBD-NEP1-40 fusion rotein passes hemato encephalic barrier and transduces into expressing synoptic diagram in the cerebral tissue.Figure is respectively 1h behind TAT-β-Gal, the TAT-LBD-NEP1-40 of abdominal injection 40nmol and the No-TAT-LBD-NEP1-40 of C57BL/6J mouse, 4h and 12h, and the brain of C57BL/6J mouse takes out row Westernblot and immuning fluorescent dyeing analysis.Figure 1A is Western blot analytical results; Figure 1B is the immuning fluorescent dyeing analysis result, and arrow represents the immunofluorescence positive cell, Scale bars=100 μ m.
Fig. 2 .TAT-LBD-NEP1-40 fusion rotein is on the impact of cerebral infarction volume.Fig. 2 A organizes representational TTC dyeing for each, and white is the cerebral infarction zone; Fig. 2 B is 4 groups of cerebral infarction volume (mm
3).
Fig. 3 .TAT-LBD-NEP1-40 fusion rotein promotes the axon growth synoptic diagram.Figure is 28d behind the TAT-LBD-NEP1-40 of C57BL/6J mouse peritoneal injection 40nmol and the PBS, and the brain of C57BL/6J mouse takes out the row immuning fluorescent dyeing analysis.Fig. 3 A is hippocampus immuning fluorescent dyeing analysis result; Fig. 3 B is cortical area immuning fluorescent dyeing analysis result, and arrow represents the aixs cylinder that grows.
Fig. 4 .TAT-LBD-NEP1-40 is to the improvement effect of C57BL/6J mouse neural function.Figure is the improvement effect data plot of using respectively water maze and overhead cross labyrinth assessment mouse neural function.Fig. 4 A is water maze; The overhead cross of Fig. 4 B labyrinth.
Below in conjunction with the drawings and specific embodiments particular content of the present invention is described in further detail.
Embodiment
The TAT-LBD-NEP1-40 fusion rotein test of pesticide effectiveness
In order to prove beneficial effect of the present invention, the TAT-LBD-NEP1-40 fusion rotein that the contriver adopts the present invention to make up carries out following drug efficacy study, and test situation is as follows:
Need to prove the experimental technique of unreceipted actual conditions in the following test of pesticide effectiveness, usually according to people such as normal condition such as Sambrook, molecular cloning: the condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989); The people such as David and for example, the condition described in the cell experiment guide (New York:Cold Spring Harbor Laboratory Press, 1998).
(1) the transduction Function Identification of TAT-LBD-NEP1-40 fusion rotein
1, the TAT-LBD-NEP1-40 fusion rotein enters evaluation in the brain by hemato encephalic barrier
9 SD rats are divided into 3 groups at random: the TAT-LBD-NEP1-40 group, TAT-β-Gal group and No-TAT-β-Gal organize (n=3), the TAT-LBD-NEP1-40 of every group of rat difference abdominal injection 10nmol, TAT-β-Gal (positive control) and No-TAT-β-Gal (negative control) fusion rotein, respectively at 1h, 4h, behind the 12h, use 2% By Sodium Pentobarbital, open rapidly chest through left ventricle to the aorta ascendens intubate, first with physiological saline flush away blood, use subsequently cold (4 ° of C) to contain the 0.1mol/L phosphate buffered saline buffer (PBS of 4% Paraformaldehyde 96, pH7.4) then perfusion fixation 2h first quick and back slow gets that brain places 20% sucrose (4 ° of C) until tissue sinks to the bottom.The section of freezing microtome cutting continuous coronal, the thick 14 μ m of sheet place-20 ° of C to deposit.Section adds confining liquid room temperature sealing 1h, adds anti-6 * His mouse source monoclonal antibody (1: 2000), puts into 4 ° of C refrigerator overnight.The PBS washing is 3 times after taking out, each 5min, the anti-mouse IgG of donkey (1: 400) of adding FITC mark, lucifuge, room temperature reaction 2h.Again with PBS appropriateness washing, after the mounting at BX60 fluorescence microscopy Microscopic observation and adopt figure.
2, result
TAT-LBD-NEP1-40 passes hemato encephalic barrier and expresses in brain.
The Fluorescent immunohistochemistry result shows: fusion rotein TAT-LBD-NEP1-40, TAT-β-Gal all are positive at Nerve Cells of Rat Brain, and the rat cerebral tissue of injection No-TAT-β-Gal fusion rotein is negative reaction, illustrate that but the TAT mediating protein is by hemato encephalic barrier, referring to Fig. 1.
(2), the TAT-LBD-NEP1-40 fusion rotein is on the impact of cerebral infarction volume
1, animal grouping
32 male SD rats, weight (300-350) g, provided by The Fourth Military Medical University's Experimental Animal Center, be divided at random 4 groups: control group (Control), TAT-LBD-NEP1-40 group, No-TAT-LBD-NEP1-40 group and TAT-β-Gal organize (n=8).The rats after transient focal cerebral ischemia model method is as follows, each group abdominal injection PBS (pH7.3) 4ml, TAT-LBD-NEP1-40, No-TAT-LBD-NEP1-40 and TAT-β-Gal (be 40 μ mol and be dissolved in PBS4ml) when pouring into behind the embolism 120min again.
2, Focal Cerebral Ischemia Modelling
Fasting 12h before the rat art can't help water.Rat is kept depth of anesthesia with 4% isofluranum induced anesthesia, 2% isofluranum suction, keeps autonomous respiration.Body temperature connects the monitoring of multifunctional monitoring instrument by anus temperature probe in the art, and maintains 37～37.5 ° of C with roasting lamp.With reference to the report before us, brain MCAO model adopts right side internal carotid artery nylon wire line bolt method, be to get neck median incision after the Animal Anesthesia, expose right carotid, external carotid artery and internal carotid artery, ligation arteria carotis communis, external carotid artery are cut a kerf in arteria carotis communis bifurcated below, with a nylon wire (3-0 who burns till in advance round end with the ethanol lamp, Ethicon, Japan) insert internal carotid artery 17.5mm, until slight resistance sense is arranged.Extract nylon wire out behind the embolism 120min, recover again perfusion, skin suture.After reviving, puts back to rat anesthesia mouse cage, free diet.
3, cerebral infarction volumetric measurement
After pouring into 24h again, animal is put to death after with 2% By Sodium Pentobarbital, takes out rapidly the mouse brain, place icy salt solution 10min, get coronal-plane and evenly be cut into the thick brain sheet of 2mm, put into rapidly 2%TTC solution (37 ℃) dyeing 30min, then fix with 4% Paraformaldehyde 96 damping fluid.Use digital camera (Kodak behind the 24h, DC240, USA) take pictures, the input computer, with image processing software (Adobe, Photoshop7.0) calculate infarct size (the pink district is normal cerebral tissue, and white area is infarcted region), it is total infarct volume that each brain sheet infarct size sum multiply by thickness (2mm).
4, experimental result
The cerebral infarction volume
The brain section (2mm * 6) of Fig. 2 A after for Ischemia and Reperfusion in vivo in Rats 24h of each group, the pink district is normal cerebral tissue, white area is infarcted region.Ischemia-reperfusion 24h cerebral infarction volume, control group are (309.5 ± 36.4) mm
3, TAT-LBD-NEP1-40 group is (296.2 ± 72.5) mm for (107.6 ± 23.1) mm3, No-TAT-LBD-NEP1-40 group
3, TAT-β-Gal group is (269.3 ± 68.0) mm
3TAT-LBD-NEP1-40 group infarct volume is significantly less than control group, No-TAT-LBD-NEP1-40 group and TAT-β-Gal and organizes (P=0.002 and 0.004), and Control group, No-TAT-LBD-NEP1-40 group and TAT-β-Gal group compares indifference (P=0.903), sees Fig. 2 B.
(3), the TAT-LBD-NEP1-40 fusion rotein promotes axon growth
1, animal grouping
12 male C57/BL6 mouse, body weight 20 ~ 25g is provided by The Fourth Military Medical University's Experimental Animal Center, is divided at random 2 groups: control group (Control), TAT-LBD-NEP1-40 organize (n=6).The global brain ischemia model is as follows, and each group abdominal injection PBS (pH7.3) 0.5ml, TAT-LBD-NEP1-40(when pouring into behind the embolism 20min are dissolved among the 0.5mlPBS with the dosage of 250mg/kg again)
2, global brain ischemia Modelling
Fasting 6h before the art prohibits water 2h.2% isofluranum sucks keeps depth of anesthesia, keeps autonomous respiration.Detect cerebral blood flow in the art, model success standard descends 70% for the cerebral blood flow that folder closes 5min.Get neck median incision, carefully separate bilateral common carotid arteries (CCA), folder recovers again perfusion, skin suture after closing 20min.After reviving, puts back to mouse anesthesia mouse cage, free diet.
3, tissue slice preparation
Continuously 28d administration behind the Cerebral Ischemia-reperfusion in Mice, under dark anesthesia in the 28th day, open chest and expose heart, cut off left auricle of heart through the aorta ascendens intubate, with getting express developed about physiological saline 20mL, use subsequently the about 30mL of 4% Paraformaldehyde 96 phosphate buffered saline buffer (4 ° of C, pH7.4) perfusion to fix, the careful brain that takes out behind the rear fixedly 2h, immerse 20% sucrose solution, be positioned in 4 ° of C refrigerators to brain and be sunken to the bottom.With the section of freezing-microtome continuous coronal, the thick 10 μ m of sheet preserve section for-20 ℃.
4, identified by immunofluorescence axon growth
Section adds confining liquid room temperature sealing 1h, adds anti-MAP mouse source monoclonal antibody (1: 400), puts into 4 ° of C refrigerator overnight.The PBS washing is 3 times after taking out, and each 5min adds rabbit anti-mouse igg (1: 1000) and Neurotrace (1:2000), lucifuge, room temperature reaction 2h.Again with PBS appropriateness washing, after the mounting at BX60 fluorescence microscopy Microscopic observation and adopt figure.
5, experimental result
The Fluorescent immunohistochemistry result shows: fusion rotein TAT-LBD-NEP1-40 group aixs cylinder obviously increases than the PBS group.Fig. 3 A is the hippocampus, and Fig. 3 B is the cortex zone.
(4), TAT-LBD-NEP1-40 is to the improvement effect of mouse neural function.
1, animal grouping
16 male C57/BL6 mouse, body weight 20 ~ 25g is provided by The Fourth Military Medical University's Experimental Animal Center, is divided at random 2 groups: control group (Control), TAT-LBD-NEP1-40 organize (n=8).The global brain ischemia model is as follows, and each group abdominal injection PBS (pH7.3) 0.5ml, TAT-LBD-NEP1-40(when pouring into behind the embolism 20min are dissolved among the 0.5mlPBS with the dosage of 250mg/kg again) the global brain ischemia model production method is the same.
2, study of behaviour experimental technique
(1) water maze
Acquired training (Acquisition phase): the pond is divided into 4 quadrants, the be placed in one central authorities in a quadrant district of platform.The mouse head is put into water towards pool wall, put into the position and get at random one of four zero positions of East, West, South, North.The record animal is found the time (s) of underwater platform.In front several times training, if this time surpasses 60s, then guide animal to arrive platform, allow animal stop 15s at platform.
(2) overhead cross labyrinth
The labyrinth comprises two open ends (16 centimetres * 5 centimetres), two closed ends (12 centimetres of 16 cm x, 5 cm x), intermediate platform (5 centimetres of 5 cm x), 25 centimetres above ground level.Every experimentation on animals once, every mouse is put into separately the end of opening end, in the face of the center, labyrinth, mouse says that with four limbs the time of usefulness is mobile delay time (TLT) from the opening end to the closed end, in case mouse does not enter closed end in 180s, soft it being pushed closed end and count TLT is 180s, and animal allows freely to explore 10s after having surveyed TLT.
3, experimental result
(1) water maze: shown in Fig. 4 A, at the training second day: fusion rotein TAT-LBD-NEP1-40 group has notable difference (P＜0.01) with the PBS group.Fusion rotein TAT-LBD-NEP1-40 group can obviously reduce the swimming latent period of mouse.
(2) overhead cross labyrinth: shown in Fig. 4 B, have notable difference (P＜0.05) between fusion rotein TAT-LBD-NEP1-40 group and PBS group group.Using the memory function of the rear mouse of fusion rotein TAT-LBD-NEP1-40 treatment obviously improves.
Claims (4)
1. TAT-LBD-NEP1-40 fusion rotein, it is characterized in that: cDNA sequence and the protein sequence of this fusion rotein are as follows:
The cDNA sequence:
ATG?CGG?GGT?TCT?CAT?CAT?CAT?CAT?CAT?CAT?GGT?ATG?GCT?AGCATG?ACT?GGT?GGA?CAG?CAA?ATG?GGT?CGG?GAT?CTG?TAC?GAC?GATGAC?GAT?AAG?GAT?CGA?TGG?GGA?TCC?AAG?CTT?GGC?TAC?GGC?CGCAAG?AAA?CGC?CGC?CAG?CGC?CGC?CGC?GGT?GGA?TCC?ACC?ATG?GCCCTGCCGGGCGCATCGGGCACCTGTCCGGAACGCGCACTGGAACGTCGTGAAGAAGAAGCAAATGTTGTCCTGACGGGTACGGTTGAAGAAATTCTGAACGTTGATCCGGTCCAGCATACCTATAGCTGCAAAGTCCGTGTGTGGCGCTACCTGAAAGGCAAGGATCTGGTGGCACGTGAAAGTCTGCTGGACGGCGGTAATAAGGTGGTTATTTCCGGCTTTGGTGATCCGCTGATCTGTGACAACCAAGTCAGTACCGGTGATACGCGCATCTTTTTCGTTAATCCGGCACCGCCGTATCTGTGGCCGGCACATAAAAACGAACTGATGCTGAATAGCTCTCTGATGCGTATTACGCTGCGCAACCTGGAAGAAGTGGAATTTTGCGTTGAAGATAAACCGGGCGGTGGCGGTTCACGTATCTACAAAGGCGTTATTCAGGCGATCCAAAAGTCGGACGAAGGTCACCCGTTCCGCGCCTACCTGGAATCGGAAGTCGCAATCTCAGAAGAACTGGTGCAAAAATACAGCAACAGC；
Protein sequence:
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSKLGYGRKKRRQRRRGGSTMALPGASGTCPERALERREEEANVVLTGTVEEILNVDPVQHTYSCKVRVWRYLKGKDLVARESLLDGGNKVVISGFGDPLICDNQVSTGDTRIFF?VNPAPPYLWPAHKNELMLNSSLMRITLRNLEEVEFCVEDKPGGGGSRIYKGVIQAIQKSDEGHPFRAYLESEVAISEELVQKYSNS。
2. make up TAT-LBD-NEP1-40 fusion rotein claimed in claim 1, it is characterized in that: the TAT-LBD-NEP1-40 fusion rotein is that TAT, LBD and NEP1-40 fusion are made, and specifically makes up according to following step:
Step 1: obtain LBD gene and NEP1-40 sequence from Genebank, then will connect by the GGCGGTGGCGGTTCA sequence between LBD gene and the NEP1-40 sequence, synthetic LBD-NEP1-40 gene order, and so that Nco I and Xho I sequence are contained respectively in the gene order two ends of LBD-NEP1-40, the gene order of whole LBD-NEP1-40 is:
A
CCATGGCC-CTGCCGGGCGCATCGGGCACCTGTCCGGAACGCGCACTGGAACGTCGTGAAGAAGAAGCAAATGTTGTCCTGACGGGTACGGTTGAAGAAATTCTGAACGTTGATCCGGTCCAGCATACCTATAGCTGCAAAGTCCGTGTGTGGCGCTACCTGAAAGGCAAGGATCTGGTGGCACGTGAAAGTCTGCTGGACGGCGGTAATAAGGTGGTTATTTCCGGCTTTGGTGATCCGCTGATCTGTGACAACCAAGTCAGTACCGGTGATACGCGCATCTTTTTCGTTAATCCGGCACCGCCGTATCTGTGGCCGGCACATAAAAACGAACTGATGCTGAATAGCTCTCTGATGCGTATTACGCTGCGCAACCTGGAAGAAGTGGAATTTTGCGTTGAAGATAAACCGGGCGGTGGCGGTTCACGTATCTACAAAGGCGTTATTCAGGCGATCCAAAAGTCGGACGAAGGTCACCCGTTCCGCGCCTACCTGGAATCGGAAGTCGCAATCTCAGAAGAACTGGTGCAAAAATACAGCAACAGC-
CTCGAGWherein the gene underscore is Nco I and Xho I restriction enzyme site;
Step 2: the gene order that will synthesize the LBD-NEP1-40 that obtains is loaded in the pUC57 carrier, obtains the pUC57-LBD-NEP1-40 carrier;
Step 3; Select the pTAT-HA of TAT sequence of the human immunodeficiency virus that contains restriction enzyme Nco I and Xho I as expression vector; Wherein the nucleotides sequence of TAT is classified as: ATA GGC AGG AAG AAG CGT AGA CAG AGA CGT AGA;
Step 4: cut pTAT-HA and pUC57-LBD-NEP1-40 carrier with restriction enzyme Nco I and Xho I enzyme, reclaim respectively enzyme and cut product, wherein to cut product be linear pTAT carrier to the enzyme of pTAT-HA, and it is the TAT-LBD-NEP1-40 gene fragment that the enzyme of pUC57-LBD-NEP1-40 is cut product;
Step 5: the enzyme that step 4 obtains is cut product, and namely linear pTAT carrier is connected the T4 ligase enzyme to connect and obtains recombinant plasmid pTAT-LBD-NEP1-40 expression vector by colony screening with the TAT-LBD-NEP1-40 gene fragment;
Step 6: after recombinant plasmid pTAT-LBD-NEP1-40 expression vector is correct through the dna sequencing analysis, be transformed in e. coli bl21 (DE3) bacterial strain, select the colony clone of 5～8 positives in the LB substratum of ammonia benzyl resistance, adopt the pre-abduction delivering TAT-LBD-NEP1-40 of IPTG fusion rotein;
Step 7: the TAT-LBD-NEP1-40 fusion rotein behind the abduction delivering is adopted SDS-PAGE, Coomassie brilliant blue R250 dyeing successively, determine condition and the positive colony clone of abduction delivering, then at 16～22 ℃, great expression TAT-LBD-NEP1-40 fusion rotein under the 0.2mM IPTG condition;
Step 8: with the bacterium of the TAT-LBD-NEP1-40 fusion rotein of abduction delivering through the ultrasonication thalline, after discharging target protein, through the affinity chromatography column purification that the Ni ion is integrated, the albumen behind the purifying is 2mg/ml through ultrafiltration and concentration, leaves-80 ℃ refrigeration in.
3. TAT-LBD-NEP1-40 fusion rotein claimed in claim 1 is for the preparation of the application of the medicine of mammal brain central nervous system injury.
4. application as claimed in claim 3 is characterized in that: described medicine is used for comprising at the mammalian central nervous system damage disease treatment of cerebral ischemia/anoxia, hematencephalon, cerebral trauma, Spinal injury.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724203A (en) * | 2019-11-08 | 2020-01-24 | 中国人民解放军第四军医大学 | Short peptide for promoting TFEB (T-Epstein-Barr) nuclear translocation, linear short peptide based on short peptide and application of short peptide in relieving cerebral ischemic injury |
| CN116036239A (en) * | 2023-03-28 | 2023-05-02 | 中国人民解放军军事科学院军事医学研究院 | Application of NEP1-40 in preparation of medicine for specifically inhibiting illusion effect |
-
2013
- 2013-01-28 CN CN201310032694.2A patent/CN103113473B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| GOU X.等: "TAT-NEP1-40 as a novel therapeutic candidate for axonal regeneration and functional recovery after stroke", 《J DRUG TARGET》 * |
| HAN Q.等: "The promotion of cerebral ischemia recovery in rats by laminin-binding BDNF", 《BIOMATERIALS》 * |
| SUN W.等: "Improvement of sciatic nerve regeneration using laminin-binding human NGF-beta", 《PLOS ONE》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724203A (en) * | 2019-11-08 | 2020-01-24 | 中国人民解放军第四军医大学 | Short peptide for promoting TFEB (T-Epstein-Barr) nuclear translocation, linear short peptide based on short peptide and application of short peptide in relieving cerebral ischemic injury |
| CN110724203B (en) * | 2019-11-08 | 2021-04-30 | 中国人民解放军第四军医大学 | Short peptide for promoting TFEB (T-Epstein-Barr) nuclear translocation, linear short peptide based on short peptide and application of short peptide in relieving cerebral ischemic injury |
| CN116036239A (en) * | 2023-03-28 | 2023-05-02 | 中国人民解放军军事科学院军事医学研究院 | Application of NEP1-40 in preparation of medicine for specifically inhibiting illusion effect |
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