WO2005071071A1 - Ptd-human choline acetyltransferase fusion protein and its application - Google Patents

Abstract

The present invention relates to a PTD (Protein Transduction Domain)-human cholin acetyltransferase fusion protein and the nucleic acid molecule containing the nucleotide sequence encoding the fusion protein, also relates to the expression vector comprising the above said nucleic acid molecule. In addition, the invention involve the application of the said fusion protein for the preparation of the medicines to treat the neurodegeneration disease or prevent Alzheimer's disease, advance the memory of learning and improve the brains. Especially, this invention also provides a medicine composition comprising the fusion protein, which can be employed by multiple methods such as injection, sublingual medication, pulmonary absorption and be imbibed through the Scheiderian membranes, suppository or skin.

Description

 Transduction peptide-human choline acetyltransferase

 Fusion protein and application thereof

 The present invention relates to a transduction peptide-human choline acetyltransferase fusion protein and a nucleic acid molecule containing a nucleotide sequence encoding the fusion protein. The present invention also relates to an expression vector containing the aforementioned nucleic acid molecule. The invention also relates to the use of the fusion protein for preparing a medicine for treating neurodegenerative disease or preventing Alzheimer's disease, promoting learning and memory function, and promoting intelligence. The present invention particularly relates to a pharmaceutical composition containing the fusion protein, which can be administered by various methods such as injection, sublingual administration, lung aspiration, nasal mucosa, anal plug, or skin absorption. Background of the invention

 Acetylcholine (Ach) biosynthetic enzyme-choline acetyl transferase (ChAT, EC 2. 3. 1. 6), is an important choline neuron in the central and peripheral nervous system Enzymes. Cholinergic neurons are closely related to functions such as learning, memory, awakening, sleep, and exercise. Decreased ChAT content in the brain or changes in enzyme activity directly reflect changes in cholinergic neuron function. ChAT is therefore closely related to many cholinergic neurological diseases.

Alzheimer's disease D) is a progressive neurodegenerative disease, a type of dementia based on selective tissue damage and characteristic pathological changes. There is evidence that abnormalities in the cholinergic system are most closely related to the dementia characteristics of the disease. Specific loss of basal forebrain cholinergic neurons has been observed in AD patients. The biochemical manifestations are choline acetyltransferase and Acetylchol ines terase (AChE) in the cerebral cortex, especially the 10s G4 molecular type level of the enzyme is reduced, and the neurotransmitter acetylcholine synthesis level is low and high. Affinity choline intake decreased. The decrease in ChAT enzyme activity in AD patients, and insufficient Ach content in the quantum caused by neural impulses are the direct causes of AD. Cholinergic neurons are also affected in other neurodegenerative diseases such as amyotrophic lateral sclerosis and Huntington's disease. The midpontine cholinergic system has recently been found to be positively associated with schizophrenia and sudden infant death syndrome. The change of CMT is closely related to it.

 Ohno et al. (Tsuj ino, A. et al. Proc. Nat. Acad. Sci. 2001, 98: 2017-2022) found in patients with congenital myasthenic syndrome (CMS-EA) with frequent fatal asphyxia. ChAT was mutated. The CMT gene of 5 CMS-EA patients had 10 recessive mutations, one was a frameshift mutation and three nonsense mutations, which greatly reduced the expression and activity of ChAT. Five patients had symptoms of muscle weakness since birth or early infancy, but no anti-AchR antibody was detected. Therefore, mutations in the gene of CMT were the cause of muscle weakness in infants. .

 Ach is the first neurotransmitter to be discovered (Loewi 0, Pf lugers Arch Gesamte Phyiol, 1921, 189: 239-242), and plays important roles in the brain such as learning, memory, sleep, etc. In addition, the insufficient release of Ach from the gastrointestinal sphincter, osteoblastic neuromuscular junction, and pre-autonomic ganglion fibers and post-sympathetic ganglion fibers in the elderly is caused by weakness and slowness of action in many elderly people, as well as reflux induced by gastric and intestinal fluids. Causes of inflammation and canceration. It is also one of the causes of poor sexual function. Choline acetyltransferase is an enzyme that catalyzes the biosynthesis of the neurotransmitter ACh, which catalyzes the transfer of the acetyl group of acetyl-CoA to bilirubin. ChAT catalyzes the synthesis of Ach in only one step, which is the rate-limiting enzyme for Ach synthesis. Therefore, increasing the expression of the CHAT gene can promote the synthesis of the neurotransmitter Ach, thereby effectively improving learning and memory and various aging symptoms caused by insufficient Ach secretion in the elderly.

 There are many ChAT raRNAs in human choline acetyltransferase, but there is no difference in the sequence of the translated ChAT mature protein. Gene expression of CMT has been performed in various expression systems, but ChAT expressed in prokaryotic cells has no enzymatic activity.

ChAT is a large molecular protein with a molecular weight of 69kD, so it cannot cross the blood-brain barrier. Central nervous system diseases must be administered centrally. At present, central administration methods mainly include The implantation method, transplantation method, lateral ventricle injection method, and neural progenitor cell brain transplantation method all have problems such as complicated operation, high technical requirements, and high risk (infection, accident, etc.).

 The advent of transduction peptide technology has provided an effective new therapeutic approach for ChAT protein molecules to efficiently treat neurological diseases through the blood-brain barrier.

 Transduction peptide is a protein transduction domain (PTD) that can efficiently pass through biological membranes. It can transduce molecules such as peptides, proteins, and DNA that are covalently linked into almost all tissues. And cells, including osteoclasts, peripheral blood mononuclear cells, and primary cells, which are difficult to transfect by conventional methods, can even cross the blood-brain barrier (Schwarze SR, Dowdy SF, Trends Pharmacol Sci. 2000, 21 (2 ): 45-8). In 1988, Green (Cell. 1988, 55: 1179-1188) discovered that the Tat protein of HIV can efficiently enter and exit cells freely. 1994 Fawel KProc Nat l Acad Sci US A. 1994 Jan 18; 91 (2): 664-8.) After chemically linking the 36 Aa region of TAT to a heterologous protein, the cross-linked protein can also be transferred into cells Inside.

Schwarze SR, et al. (Science, 1999, 285: 1569-1572.) After fusion and expression of a short peptide of the PTD region of TAT and a large molecule galactosidase in prokaryotic cells, the fusion protein was injected in mice and found that Active enzyme molecules are expressed in various tissues in the body, and it is particularly important that short peptides can carry large molecules across the blood-brain barrier. Specifically, the transduction peptide is an arginine-rich polypeptide. The ability of TAT to cross the cell membrane automatically is due to the role of a peptide YGRKKRRQRRRGG (SEQ ID NO: 1) located on the TAT protein at positions 47-57. In addition to the TAT peptide, the polypeptide sequence from Drosophila antennal foot homeotype transcription factor Antp at positions 43-58 (Deross i D, et al. The Journal of Biological chemistry. 1996, 27 (30): 18188- 18193) ; The 267-300 sequence (El lott C, Ohare P. Cel l. 1997, 88: 223-233) from the sores single virus DNA binding protein VP22 has a transduction function and transduction mechanism completely similar to TAT peptide They are collectively referred to as PTD (Ford KG, et al. Gene Therapy. 2001, 8: 1-4).

 The present inventor uses a transduction peptide to construct a transduction peptide-human choline acetylase fusion protein, directly introduces ChAT into the body, solves the problem of low levels of Ach released by synapses from the source, and improves Ach in cholinergic synapses. The content in the bubble effectively solves the problem of insufficient ACh in the bubble. Summary of the invention

 One aspect of the invention relates to a transduction peptide-human choline acetyltransferase fusion protein. In the present invention, the transduction peptide used for transducing the human choline acetyltransferase can be selected from the PTD sequence of the transactivator protein TAT, the Drosophila homeologous transcription factor ANTP, and the herpes simplex virus type I VP22 PTD sequence of a transcription factor or a functional analog or fragment thereof. Particularly preferably, the transduction peptide consists of the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof. In fact, as long as a transduction peptide capable of transducing the human choline acetyltransferase according to the present invention is applicable to the present invention. The nucleotide sequence encoding the transduction peptide in the present invention is shown in SEQ ID NO: 2.

 In the present invention, the human-derived choline acetyltransferase used to construct the fusion protein contains the amino group of SEQ ID NO: 3 or a functional fragment thereof. Preferably, the human-derived choline acetyl acyltransferase consists of SEQ ID NO: 3. The nucleotide sequence encoding the human choline acetyltransferase is shown in SEQ ID NO: 4.

 In a preferred embodiment, the transduction peptide-human choline acetyltransferase fusion protein constructed according to the present invention has the amino acid sequence of SEQ ID NO: 5 or a functionally equivalent variant thereof. In the present invention, the amino group of the functionally equivalent variant of the fusion protein is derived from a conservative modification of the amino acid sequence described in SEQ ID NO: 5 without substantially altering the function of the fusion protein, including the modification of the fusion protein. Substitution, addition or deletion of one or more conservative amino acid residues in an amino acid sequence.

Another aspect of the present invention relates to a nucleic acid molecule containing a nucleoside sequence encoding a transduction peptide-human choline acetyltransferase fusion protein of the present invention. More specifically, all The nucleic acid molecule contains the nucleotide sequence of SEQ ID NO: 6, or a degenerate sequence thereof. Another aspect of the present invention relates to an expression vector containing a nucleic acid molecule encoding the transduction peptide-human choline acetyltransferase fusion protein of the present invention. Preferably, the expression vector of the present invention is a prokaryotic or eukaryotic expression vector, including but not limited to pGEX, pET, pBV220 and pcD.

 In one embodiment of the present invention, a human ChAT fusion-expressed with a transduced peptide is obtained by prokaryotic cell culture by introducing the nucleic acid molecule into a prokaryotic expression vector pGEX-4T-1.

 The expression of the fusion protein of the present invention is easy to operate, low in cost, and high in transduction efficiency. On the one hand, the purification of the obtained fusion protein can be performed under denaturing conditions, which greatly simplifies the purification process of the fusion protein in the form of inclusion bodies. The natural conformation of the denatured protein becomes a relatively flexible chain structure, which is free from the constraints of spatial conformation, has higher energy, and greatly improves the transduction efficiency.

 When the prokaryotic expression fusion protein constructed by the present invention is transferred into a cell, it is refolded under the action of a chaperone molecule to restore the natural conformation, so that ChAT can exert its biological activity, and the protein transduction domain linked to it does not affect the cell. Normal structure or function.

 Another aspect of the present invention relates to the use of the transduction peptide-human choline acetyltransferase fusion protein for the preparation of a medicament for treating progressive neurodegenerative disease or preventing Alzheimer's disease, promoting learning and memory functions, and promoting intelligence. The neurodegenerative diseases described in the present invention include, but are not limited to, Alzheimer's disease, Parkinson's disease, myasthenia gravis, and muscular atrophy.

The invention also relates to a pharmaceutical composition containing the transduction peptide-human choline acetyltransferase fusion protein, which is characterized in that the pharmaceutical composition contains a commonly used pharmaceutically acceptable carrier or excipient. The dosage forms described in the present invention include, but are not limited to, dosage forms administered by (intravenous) injection, sublingual administration, lung aspiration, nasal mucosa and anal plug or skin absorption. Those of ordinary skill in the art know that they can penetrate skin, mucous membrane, cornea, conjunctiva, serosa, muscle membrane, blood vessels and lymphatic membrane, choroid, nerve membrane, blood-brain barrier, etc. All cell membrane and biofilm dosage forms can be used in the pharmaceutical composition of the present invention. By using the above-mentioned transduction peptide technology, the present inventors successfully achieved the purpose of inputting human-derived choline acetyltransferase (ChAT) into the body and efficiently crossing the blood-brain barrier, thereby making clinical medication safer and more convenient and making it a nerve Long-term self-administration, preventive and nootropic care for degenerative diseases has become a reality.

 Compared with the traditional methods of introducing proteins into cells, such as microinjection, perforated protein, and lipid shield method, the transduction peptide-mediated protein transduction has significant advantages. BRIEF DESCRIPTION OF THE DRAWINGS

 figure 1. SDS-PAGE and Western blot results of the fusion protein GST-TAT-CHAT and TAT-CHAT.

 figure 2. C T levels in the brains of mice after intravenous injection of different amounts of human recombinant TAT-ChAT or ChAT.

 image 3. The concentration of human recombinant TAT-ChAT or ChAT was intravenously injected into the brains of mice. Figure 4. Kinetics of intravenous TAT-CHAT or CHAT. (A) Time-dependent ACh levels of exogenous recombinant TAT-CHAT or CHAT; (B) Concentration-dependent ACh levels of exogenous recombinant TAT-CHAT or CHAT.

 Figure 5 Schematic diagram of the construction of the recombinant vector pGEM-ChAT of the ChAT gene clone.

 Figure 6 shows the construction of the recombinant vector pcDNA-ChAT. Example 1 Construction of transduction peptide-CHAT fusion protein carrier

The s-type of the human ChAT gene donated by Professor Engel from the Mayo Institute of Clinical Neurology in the United States was subjected to PCR technology (primer design: 5 upstream primers, GAATTCACCC TACTCCACAC CAGAGG (SEQ ID NO: 11); 5 downstream primers, ct taagctaaccctccac (SEQ ID NO: 12) ₀ PCR reaction conditions, 94. (: Denaturation 60 sec, 55 ° (refolding 60 860, 72 ° C extension 90 sec, 30 cycles,) to convert S-ChAT to M-type ChAT. According to the construction route shown in FIG. 5 and FIG. 6, on the basis of obtaining the cloning vector GEM-ChAT, a eukaryotic expression vector pcDNA-ChAT was further constructed. pcDNA is an eukaryotic highly efficient long-term and transiently expressed mammalian cell expression vector. Its promoter CMV allows the vector to be expressed at a high level of stability and non-replicative transients in most mammalian cells. Both pGEM-ChAT and pcDNA3.1 (Invitrogen) were digested with EcoRI, and then the ChAT gene was inserted into the pcDNA3.1 vector to obtain the recombinant plasmid pcDNA3.1 / CHAT. Primers were designed according to the CHAT nuclear sequence (SEQ ID NO: 4), and BamHI, Kpnl and EcoRI digestion sites were introduced. The upstream primer is 5 and GCAGGATCCGCAGGTACCATGGCAGC AAAAACTCCC3 '(SEQ ID NO: 7); the downstream primer is 5 and GCT GAATTC TCAAGGTTGGTGTCCCTG3' (SEQ ID NO: 8). Using this pair of primers, pfuTaq high-fidelity enzyme (product of Beijing Tianwei Times Co., Ltd.) was used for PCR amplification (PCR reaction conditions, denaturation at 94 ° C for 60 seconds, renaturation at 55 ° C for 60 seconds, extension at 72 ° C for 90 seconds, 30 Cycles) Amplify the gene encoding CHAT from the pcDNA / CHAT plasmid template.

After double digestion with BamHI and EcoRI, it was directly introduced into a glutathione transferase (GST) fusion protein expression vector-pGEX-4T vector (product of Amershara biosciences). After verifying the correctness of PCR and digestion, perform double digestion with BamHI and Kpnl, insert the transduction peptide-PTD gene fragment (SEQ ID NO: 2): the sense strand is 5, GATCCTATGGTCGTAAAAAGCGACGCCAACGTAGACGTGG TGGTGGTAC3 '(SEQ ID NO: 9); antisense The chain is 5, CACCACCACGTCTACGTTGGCGTCGCTTTTTACGACCAT AG3 '(SEQ ID NO: 10). Recombinant transduction peptide-CHAT fusion protein gene sequencing analysis (Beijing Boya) was correct. Example 2 Expression and purification of transduction peptide-CHAT fusion protein The recombinant plasmid pGEX-ChAT prepared by the method described in Example 1 was transferred into E. coli BL21 (E. coli Ul (DE3) pLysS was purchased from Beijing Tianwei Times Company), and the transduction peptide-CHAT fusion protein was induced to be expressed.

 After culturing the cells overnight, re-inoculate 10% into the new LB medium the next day, shake the bacteria to about OD600-0. 6 and add IPTG to the medium at a final concentration of l, 0mmol / L, 30 ° C was induced for 4 hours. The cells were lysed and GST-transduced peptide-CHAT was extracted. The transduced peptide-CHAT was digested and purified with thrombin, and detected by Western blotting.

 Wes tern blot t ing was performed by conventional methods for electrophoresis and transfection. Sheep anti-human CHAT polyclonal antibody (Chemicon International Co.) was added. Rabbit anti-goat IgG labeled with horseradish peroxidase was used as the secondary antibody. MB (two Aminobenzidine) staining.

 The results showed that the fusion protein GST-transduction peptide-CHAT and transduction peptide-CHAT all combined with polyclonal antibodies to develop color. Among them, GST-transduction peptide-CHAT has a molecular weight of about 96KD, GST 26KD, and transduction peptide-CHAT 70KD. The measured protein bands are consistent with the theoretical values (see Figure 1). Example 3 Kinetic characteristics of CHAT content in the brain of mice after intravenous injection of transduction peptide-CHAT or CHAT

 1 experimental grouping

Healthy male mice (Shanghai species, class II animals), weighing 25 ± lg. Provided by Animal Experiment Center of Academy of Military Medical Sciences. Mice were randomly divided into 2 groups of 3 mice each. Group 1 mice were injected intravenously 10 () transduced or peptide -CHAT CHAT μ ₈ respectively at different time points (0, 0. 5, 1, 2, 5, 12h) The brain after injection, CHAT activity Determination. The mice in group 2 were injected with transconducting peptide-CHAT or CHAT 0.2 ml at different concentrations (0-50 (^ g / mL)) in the tail vein. The brains were collected 2 hours later and the CHAT activity was measured. Each experiment was repeated 3 times. .

 2 Determination of CHAT activity

The animals of each group were sacrificed by decapitation, and the brain was removed as soon as possible. After removing the cerebellum and brain, they were immediately placed at -20. C Refrigerator, freeze for future use. Made with ice-cold saline in an ice bath during the experiment 10 ° /. Homogenate (10,000 rpm, 10s each time, 30s interval, 2 times in total). CHAT activity was determined by colorimetry (ChaoLP. Spectrophotometric determination of choline acetyltransferase if the presence of dithiothritol. Anal Biochem. 1972, 85, 20-22). The reaction system uses 0.5mol / L sodium phosphate buffer (pH7.0), 6.2xlO " ³ raol / L acetyl-CoA, 1. Oraol / L choline chloride, 7.6xlO" ⁴ mol / L methylsulfate ^{neostigmine, 3mol / L NaCl, 1.1x10- 3} mol / L EDTA, 0.5mol / L hydrochloric acid and creatinine 6.5 face ol / LDTT each 20μ1, plus leavened distilled water to 0.4mL. After incubating at 37 ° C for 5 minutes, add 200 μl of tissue homogenate, 37. C water bath continued to warm for 20min. Add an equal amount of boiled slurry to a blank tube. Then add 2niin of each reaction tube to boiling water, add 2.5 to make ol / L sodium arsenate 0.8mL. Centrifuge at room temperature. Take 1.0 mL of the supernatant, add 3nmol / L 4-PDS 20 L, and incubate at 25 ° C for 15 minutes, then read the extinction value (A) at 3 ² 4 nm in an ultraviolet-visible spectrophotometer. The extinction value of each tube is corrected by the blank tube. Enzyme activity is expressed as nmo / h · mgPr. The standard tube replaced the tissue homogenate with 0.1 mmol / L CoA. The protein content was measured using the Lowry method.

 3 Experimental results

 The experimental results showed that with the increase of protein concentration, the CHAT activity in the brain of the transduction peptide-CHAT injection group increased, while the CHAT activity in the CHAT intravenous injection group showed no sign of increase, indicating that the transduction peptide-CHAT could pass through the blood-brain barrier When the cell membrane enters the cell and refolds into the active form within the cell, CHAT is difficult to pass through the blood-brain barrier and the cell membrane, which clearly shows the transduction peptide's guiding effect on the protein across the membrane. The aging curve after 2 hours of transduction peptide-CHAT intravenous injection showed that the CHAT activity reached a peak, and with time, CHAT activity decreased, and the half-life was about 12 hours (see Figures 2 and 3). Example 4 Kinetic characteristics of ACh content in mouse brain after intravenous injection of transduction peptide-CHAT or CHAT

 1 experimental grouping

Healthy male mice (Shanghai species, class II animals), weighing 25 ± lg. Military Medical Department Provided by the College Animal Experiment Center. Mice were randomly divided into 2 groups of 3 mice each. In the first group of mice, 100 μ _§ of the transduction peptide-CHAT or CHAT was injected into the tail vein, and the brains were taken at different time points (0, 0.5, 1, 2, 5, 12 h) after injection for ACh measurement. The mice in the second group were injected with transconducting peptide-CHAT or CHAT 0.2 ml at different concentrations (0-500 g / mL) in the tail vein, and brains were collected 2 h later for ACh measurement. Each experiment was repeated 3 times. .

 Determination of 2ACh content

 Using basic hydroxylamine colorimetric method. Take 0.8mL of brain homogenate, mix with 1.4mL of distilled water, add 1.5mmol / L physostigmine 0.2mL, slowly add 1.84mol / L trichloroacetic acid

0.8mL, mix thoroughly, centrifuge 1mL of the supernatant, add 1.0mL of basic hydroxylamine, and leave at room temperature for 15min, then add 4mol / L HC1, 0. 37mol / L FeCl ₃ each 0.5ml, the above steps All are well mixed. With double distilled water as a blank control, the colorimeter was measured at 54 Onm with a spectrophotometer. ACh content is expressed as nmol / mgProtein. Standard tube with 0.2 μΰΐο1 / ιΛ ACh 0.8 mL instead of homogenate

 3 Experimental results

 The experimental results show that as the protein concentration increases, the level of ACh in the brain is increased by the transduction peptide-CHAT, indicating that the transduction peptide-CHAT enters the cell to fold into an active form, and the synthesis of ACh increases. CHAT did not affect the ACh content after intravenous injection. The aging curve showed that the ACh content reached a peak 1 to 2 hours after intravenous injection of the transduction peptide-CHAT, and the ACh level decreased with time. It is basically consistent with the change of CHAT activity. See Figure 4. Example 5 Production of Alzheimer's (AD) disease animal model

 Αβ (22-35, Sigma) was dissolved in sterile normal saline at a concentration of

1. OmmoU refers to the method of Maur ice (Maurice T, Lockhart BP, Privat A. Amnes ia induced in mice by central ly adminis tered β-amyloid pept ides involves chol inergic dysfunct ion. Brain Res. 1996, 706, 181-193) , After being sealed in a 37 ° C incubator for 96 hours to make it condensed, mice were injected with 4 L intraventricularly, and the learning and memory index test was performed 11 days later. Example 6 Detect experiment (s tep-through tes t) to detect the effect of transduction peptide-chat on learning and memory of AD animal model

 Animals were randomly divided into 5 groups of 6 animals each: (1) control group: intraventricular injection of the same amount of normal saline; (2) Αβ group; (3) transduction peptide-CHAT + Αβ group: 2h intravenous injection before test 2. Omg / kg transduction peptide-CHAT; (4) CHAT + Αβ group: same method as (3); (4) Huperzine A (Hup) + Αβ group: Αβ model mice before testing 40min intraperitoneally injected Hup 0. lrag / kg.

 Use JZZ94 multi-functional avoidance conditional reflectometer. Experiments were performed using the darkening nature of mice and the ability to remember electric shocks. The mice were first allowed to move freely in the box for 5 minutes to adapt to the environment, and then the animals were placed in the bright room with their backs facing toward the hole, at the same time the automatic recording device was started and a 30V voltage was applied in the dark room. After 3 shocks, the mice usually learned their lessons and formed memories, and they no longer entered the dark room for formal practice. Those who failed to obtain memory after 3 shocks were eliminated.

 Memory test was performed 24 hours after obsessive training. The mice were placed in the bright room with their backs facing away from the hole, and the automatic recording device was started to record the "errors" and "memory retention time" (error interval time) of the test mice entering the dark room within 2 minutes.

The experimental results show that compared with the normal control group, the number of errors in the Αβ model group is increased, and the interval between entering the dark room, that is, the memory retention time is shortened (P <0.01), indicating that Αβ can significantly reduce memory acquisition in mice. Administration of transduced peptides-CHAT or Hup can significantly reduce the number of errors in AD model mice caused by Αβ entering the dark room and prolong their memory retention time. There is no significant difference compared with the normal control group (P> 0.05). However, the CHAT administration did not improve the learning and memory ability of mice (Table 1). It shows that the transduction peptide-CHAT is similar to Hup, which can improve the memory impairment caused by Αβ and enhance the memory. Table 1 TAT -CHAT, CHAT and Hup remission avoidance test in memory loss caused by the Αβ ^{(Δ Ρ> 0 05, **} Ρ <0 01: compared with the control ^{group; ## Ρ <0 01..} .: Compared with Αβ group). Group error number memory retention control 0. 10 ± 0. 32 119. 40 ± 1. 90

 Αβ 2. 28 ± 0. 49 "17. 17 ± 15. 61"

TAT-CHAT + Αβ 0. 56 ± 0. 38 ^{## Δ} 115. 62 ± 12. 37 ^Δ

 CHAT + Αβ 2. 87 ± 0. 99 "28. 38 ± 17. 55"

Hup + Αβ 0. 37 ± 0. 26 ^{## Δ} 107. 25125. 30 ^{## Δ} Example 7 Water maze test was used to determine the effect of transduction peptide-CHAT on the learning and memory of AD animal models

The animals were randomly divided into 5 groups of 6 animals each: (1) control group: intraventricular injection of the same amount of normal saline; (2) Αβ group; (3) transduction peptide-CHAT + Αβ group: 2h intravenous injection before test 2. Omg / kg of transduced peptide-CHAT; (4) CHAT + Αβ group: 2 hours before test 2. Omg / kg of transduced peptide-CHAT; (4) Huperzine A (Hup) + Αβ group: Αβ model mice were intraperitoneally injected with Hup 0.1 mg / kg ₀ 40 minutes before the test

 The water maze automatic control device is developed by the Institute of Materia Medica, Chinese Academy of Medical Sciences. The pool is 130cm long, 85cra wide, 24cm deep, and the water temperature is controlled at (20 ± 0.5). C. (1) Discrimination and cognitive training: Put the mouse on the end step for 30s to make it recognize and memorize the position. Then place the mouse in water at the starting point, and if no ladder is found within 3 minutes, guide them to the end point. (2) Swimming experiment: The mice after the above training were tested for swimming. Record the swimming time required for the mice to swim from the start point to the end point (those with a length of more than 3 minutes will be counted as 3 minutes). Each mouse was tested once a day for two consecutive days.

The spatial discrimination and memory ability of mice were measured by water maze method. The results showed that compared with normal control mice, Αβ significantly prolonged The swimming time (P <0.01) indicates that the mice in the model group have significant spatial discrimination and memory dysfunction. After transduction of peptides-CHAT and Hup, swimming time was significantly shortened (Table 2). It shows that the transduction peptide-CHAT is similar to Hup and has the effect of enhancing memory and cognition.

Table 2 TAT -CHAT, CHAT, and spatial discrimination in mice Hup recovery and memory disorders induced by the Αβ ^{(Δ Ρ> 0 05, **} Ρ <0 01 In the water maze test: compared with the control group; Ρ.. <0. 01: Compared with Αβ group)

 Swimming and swimming time

 1st 2nd 3rd Control 45. 9 ± 24. 35 36. 4 ± 28. 46 27. 70 ± 20. 82

Αβ 151. 12 ± 31. 21 "122. 12 ± 27. 84 ** 85. 87 ± 15. 30"

TAT-CHAT + Αβ 51. 67 + 32. 45 ^{## Δ} 20. 89 ± 10. 53 ^{# Δ} 31. 33 ± 23. 38 ^{# Δ}

CHAT + Αβ 148. 88 + 34. 28 "151. 25 ± 41. 53" 104. 62 ± 39. 12 "

Hup + Αβ 41. 12 + 37. 02 ^{## Δ} 18. 00 ± 16. 62 ^m 19. 12 ± 15. 01 ^{## Δ}

Claims

1. A transduction peptide-human choline acetyltransferase fusion protein, wherein the transduction peptide is selected from the PTD sequence of the transactivator protein TAT, Drosophila homeotype transcription factor ANTP, and herpes simplex virus type I VP22 The PTD sequence of a transcription factor or a functional analog or fragment thereof, and the human choline acetyltransferase contains the amino acid sequence of SEQ ID NO: 3 or a functional fragment thereof.  The fusion protein according to claim 1, wherein the transduction peptide consists of the amino acid sequence of SEQ ID NO: 1 or a functional fragment thereof, and the human choline acetyltransferase comprises the amino acid sequence of SEQ ID NO: 3 composition.  3. The fusion protein according to claim 1, which consists of the sequence shown in SEQ ID NO: 5.  A nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein according to claim 1.  The nucleic acid molecule according to claim 4, which is encoded by the nucleotide sequence of SEQ ID NO: 6 or a degenerative sequence thereof.  An expression vector comprising the nucleic acid molecule according to claim 4.  7. The fusion protein according to claim 1 for use in the preparation of a medicament for treating progressive neurodegenerative disease or preventing Alzheimer's disease, promoting learning and memory function, and promoting intelligence.  8. The use according to claim 7, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, myasthenia gravis, muscle atrophy. 9. A pharmaceutical composition comprising the fusion protein according to claim 1 and a pharmaceutically acceptable carrier or excipient, and it is selected from the group consisting of skin, mucosa, cornea, conjunctiva, serosa, sarcolemma, blood vessels And all forms of cell membranes and biofilms such as lymphatic membrane, choroid, nerve membrane, blood-brain barrier, etc., preferably, it is selected from the group consisting of suitable for injection, sublingual administration, lung aspiration, nasal mucosa and anal plug or skin absorption Dosage form. 10. A method for treating a patient with progressive neurodegenerative disease or preventing presenile dementia, comprising administering to the patient a therapeutically effective amount of the fusion protein of claim 1 or the pharmaceutical composition of claim 9.