ACCS gene, protein and use thereof

Abstract

The present invention relates to an ACCS gene, a protein and the use thereof. An involved ACCS protein mutant is formed by means of mutating a proline residue at position 134 and a tryptophan residue at position 136 of an animal-derived wild-type ACCS protein into a glutamine residue and a tyrosine residue, respectively. The involved ACCS protein mutant can be used in the intervention and treatment of a variety of diseases comprising brain diseases, and provides a new regimen and approach for the intervention and treatment of brain diseases.

Description

ACCS gene, protein and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of protein mutation, in particular to an ACCS gene, protein and application thereof. BACKGROUND

[0002] ACCS is ACC synthase, which functions to convert S-adenosylmethionine into 1-Aminocyclopropane-1-carboxylic acid (abbreviated as ACC).

[0003] ACC can act on N-methyl-D-aspartate (NMDA) receptors, which are widely distributed in the tissues of animals. Abnormalities of NMDA receptors can cause various diseases, especially brain diseases. ACC can improve the symptoms of related diseases by regulating the functionally abnormal NMDA receptors. However, ACC as a drug for intervening and treating brain diseases has the disadvantages of short drug efficacy, the need for multiple drug administration and poor patient compliance, and the like. If ACC can be synthesized in the body of a patient through genetic manipulation, these disadvantages can be made up.

[0004] Genes encoding ACCS protein, i.e. ACCS genes, exist in plants and animals. However, the ACCS genes from animals cannot translate active ACCS protein. The human ACCS (HsACCS, AY026508) gene encodes a 501-amino-acid HsACCS protein (AAK11482). Recombinant HsACCS protein expressed in Pichia pastoris cannot catalyze the synthesis of ACC, i.e. does not have ACCS enzyme activity. In addition, many animals have orthologous genes of HsACCS, such as pigs, cows, mice, etc., but so far, there has been no report on the enzyme activity of animal ACCS. However, plants such as apple ACCS (MdACCS, XP_008342238.2) can catalyze the synthesis of ACC and have ACCS enzyme activity.

[0005] SUMMARY

[0006] The present application aims to provide a new ACCS protein mutant, a gene encoding the same and an application thereof.

[0007] The present application adopts the following technical solution:

[0008] In one aspect, the present application discloses an ACCS protein mutant, which is formed by mutating the proline residue at the 134th site and the tryptophan residue at the 136th site of a wild-type ACCS protein from an animal source into glutamine residue and tyrosine residue, respectively.

[0009] It is to be noted that the present application has found that the wild type ACCS protein from animal source can be used for intervention and treatment of various diseases including brain diseases by mutating the proline residue at position 134 into glutamine residue and the tryptophan residue at position 136 into tyrosine residue.

[0010] Preferably, the animal refers to mouse, i.e. the proline residue at position 134 and the tryptophan residue at position 136 of the wild type ACCS protein from mouse are mutated into glutamine residue and tyrosine residue respectively to obtain the ACCS protein mutant of the present application.

[0011] Preferably, the ACCS protein mutant is the sequence shown in Seq ID No. 1.

[0012] The present application further discloses a nucleic acid encoding the ACCS protein mutant of the present application.

[0013] Preferably, the nucleic acid is the sequence shown in Seq ID No. 2 or the RNA transcribed from the sequence shown in Seq ID No. 2.

[0014] It is to be noted that the nucleic acid of the present application, i.e. the nucleic acid encoding the ACCS protein mutant of the present application, can be DNA or RNA, and its main function is to encode the ACCS protein mutant of the present application in vivo or in vitro, thereby playing a role in intervention and treatment of diseases.

[0015] The present application further discloses a recombinant plasmid containing the nucleic acid of the present application.

[0016] The present application further discloses use of the ACCS protein mutant of the present application, or the nucleic acid of the present application, or the recombinant plasmid of the present application, or the ACCS protein from non-animal source, or the ACCS nucleic acid from non-animal source, or the recombinant plasmid containing the ACCS nucleic acid from non-animal source in preparation of a medicament for intervention and treatment of brain diseases.

[0017] It is to be noted that the present application has found that the ACCS protein from animal source needs to be mutated to play a role, while the ACCS protein from non-animal source and its encoding nucleic acid can be directly used for intervention and treatment of brain diseases. Therefore, the present application creatively uses the ACCS protein from non-animal source and its encoding nucleic acid in preparation of a medicament for intervention and treatment of brain diseases.

[0018] Still another aspect of the present application discloses a drug delivery system, comprising a delivery carrier and a delivery drug, wherein the delivery drug is the ACCS protein mutant of the present application, or the nucleic acid of the present application, or the recombinant plasmid of the present application, or the ACCS protein of non-animal origin, or the ACCS nucleic acid of non-animal origin, or the recombinant plasmid containing the ACCS nucleic acid of non-animal origin.

[0019] It should be noted that the drug delivery system of the present application can directly deliver the ACCS protein mutant of the present application or the coding nucleic acid thereof, or the corresponding recombinant plasmid; of course, it can also directly deliver the ACCS protein of non-animal origin or the coding nucleic acid thereof, or the corresponding recombinant plasmid containing the ACCS nucleic acid of non-animal origin.

[0020] In the present application, the ACCS protein of non-animal origin is the sequence shown in Seq ID No. 3.

[0021] Preferably, the ACCS nucleic acid of non-animal origin is the sequence shown in Seq ID No. 4.

[0022] It should be noted that the sequence shown in Seq ID No. 4 is the ACCS nucleic acid of non-animal origin of the present application which is modified according to the codon preference of the host gene and can be used for intervention and treatment of brain diseases.

[0023] Preferably, the delivery carrier is at least one of a virus, a lipid nanoparticle, an extracellular vesicle and a probiotic.

[0024] It should be noted that the drug delivery system of the present application can in principle use the nucleic acid drug delivery carrier or the protein drug delivery carrier used in the prior art, and is not limited to viruses, lipid nanoparticles, extracellular vesicles and probiotics.

[0025] The beneficial effects of the present application are:

[0026] The present application mutates the proline residue at the 134th site of the wild-type ACCS protein of animal origin to a glutamine residue, and simultaneously mutates the tryptophan residue at the 136th site to a tyrosine residue, to obtain a new ACCS protein mutant, which can be used for intervention and treatment of various diseases including brain diseases, and provides a new scheme and approach for intervention and treatment of brain diseases. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a diagram of the enzyme activity of the site-directed mutation to restore the MmACCS1 protein in the embodiments of the present application; wherein A is a schematic diagram of the site-directed mutation of the MmACCS1 gene; B is a WB experiment diagram of the MmACCS1_W and MmACCS1_M proteins expressed by the E. coli cells; C is a column chart of the WB experiment results; D is a gas chromatogram of ethylene production of each group; E is a column chart of the enzyme activity of each group; WB is Western Blot; Vehicle is a carrier; MmACCS1 is mouse 1-aminocyclopropane-1-carboxylic acid synthetase 1; MmACCS1_W is the wild type MmACCS1 gene; MmACCS1_M is the mutant MmACCS1 gene; MmACCS1_W is the wild type MmACCS1 protein; MmACCS1_M is the mutant MmACCS1 protein; MdACCS is apple 1-aminocyclopropane-1-carboxylic acid synthetase; kD is kiloDalton; the experiment was repeated 3 times; *** indicates P<0.001; NS indicates no significant difference;

[0028] Figure 2 is a diagram of the enzyme activity of the MmACCS1_M protein expressed by the HEK293T cells in the embodiments of the present application; wherein A is a WB experiment diagram of the MmACCS1_W and MmACCS1_M proteins expressed by the HEK293T cells; B is a column chart of the WB experiment results; C is a gas chromatogram of ethylene production of each group; D is a column chart of the enzyme activity of each group; WB is Western Blot; Vehicle is a carrier; MmACCS1 is mouse 1-aminocyclopropane-1-carboxylic acid synthetase 1; MmACCS1_W is the wild type MmACCS1 protein; MmACCS1_M is the mutant MmACCS1 protein; kD is kiloDalton; the experiment was repeated 3 times; *** indicates P<0.001; NS indicates no significant difference;

[0029] Figure 3 is a WB experiment diagram of the hippocampus tissue carrying the MmACCS1_M protein of the mouse in the embodiments of the present application; wherein A is a WB experiment diagram of the MmACCS1_M protein expressed by the hippocampus tissue; B is a column chart of the WB experiment results; WB is Western Blot; Vehicle is a carrier; MmACCS1 is mouse 1-aminocyclopropane-1-carboxylic acid synthetase 1; MmACCS1_M is the mutant MmACCS1 protein; kD is kiloDalton; the experiment was repeated 3 times; *** indicates P<0.001;

[0030] Figure 4 is a diagram of the open field test of mice carrying MmACCS1_M protein in the hippocampus in the embodiments of the present application; wherein A is the open field movement trajectory diagram of mice in each group; B is the proportion of exploration distance of mice in the central area of the open field; C is the proportion of exploration time of mice in the central area of the open field; D is the exploration times of mice in the central area of the open field; Saline is physiological saline; MK801 is dizocilpine; Vehicle is a carrier; MmACCS1 is mouse 1-aminocyclopropane-1-carboxylic acid synthetase 1; MmACCS1_M is mutant MmACCS1 protein; the number of mice in each group is ≧8; ** indicates P<0.01; *** indicates P<0.001; NS indicates no significant difference; green dots are the starting position of movement, and red dots are the end position of movement;

[0031] Figure 5 is a diagram of the Y maze test of mice carrying MmACCS1_M protein in the hippocampus in the embodiments of the present application; wherein A is the Y maze movement trajectory diagram of mice in each group; B is the spontaneous alternation rate column diagram of mice in the Y maze; Saline is physiological saline; MK801 is dizocilpine; Vehicle is a carrier; MmACCS1 is mouse 1-aminocyclopropane-1-carboxylic acid synthetase 1; MmACCS1_M is mutant MmACCS1 protein; the number of mice in each group is ≧8; * indicates P<0.05; NS indicates no significant difference;

[0032] Figure 6 is a diagram of the water maze test of mice carrying MmACCS1_M protein in the hippocampus in the embodiments of the present application; wherein A is the water maze movement trajectory diagram of mice in each group; B is the number of times of crossing the platform in the water maze of mice in each group; Saline is physiological saline; MK801 is dizocilpine; Vehicle is a carrier; MmACCS1 is mouse 1-aminocyclopropane-1-carboxylic acid synthetase 1; MmACCS1_M is mutant MmACCS1 protein; the number of mice in each group is ≧8; * indicates P<0.05; NS indicates no significant difference; green dots are the starting position of swimming, red dots are the end position of swimming, and the platform is a yellow circle in the third quadrant;

[0033] Figure 7 is a diagram of the novel object recognition test of mice carrying MmACCS1_M protein in the hippocampus in the embodiments of the present application; wherein A is a schematic diagram of the novel object recognition test; B is a column diagram of the exploration rate of mice to the new object; Saline is physiological saline; MK801 is dizocilpine; Vehicle is a carrier; MmACCS1 is mouse 1-aminocyclopropane-1-carboxylic acid synthetase 1; MmACCS1_M is mutant MmACCS1 protein; the number of mice in each group is ≧8; * indicates P<0.05; NS indicates no significant difference; the green square object is the old object, and the yellow cylindrical object is the new object;

[0034] Figure 8 is a behavior experiment diagram of a depression model mouse carrying MdACCS gene probiotics by gavage in the embodiments of the present application; wherein A is a graph of the proportion of exploration distance of each group of depression model mice in the central area of the open field; B is a graph of the proportion of exploration time of each group of depression model mice in the central area of the open field; C is a column chart of the proportion of immobility time of each group of depression model mice in the tail suspension test; D is a column chart of the proportion of immobility time of depression model mice in the forced swimming test; Vehicle is a carrier; MdACCS is apple 1-aminocyclopropane-1-carboxylic acid synthetase; the number of mice in each group is ≧8; * indicates P<0.05.

[0035] Figure 9 is a new object recognition experiment diagram of an Alzheimer's disease model mouse carrying MdACCS_O gene probiotics by gavage in the embodiments of the present application; wherein Vehicle is a carrier; MdACCS is apple 1-aminocyclopropane-1-carboxylic acid synthetase; the number of mice in each group is ≧8; * indicates P<0.05. DETAILED DESCRIPTION

[0036] The present application found that the ACCS protein encoded by the ACCS gene of animal origin has no enzyme activity, mainly because the amino acid residues of the key site of enzyme activity have been mutated, resulting in the loss of enzyme activity. The present application modifies the wild type ACCS gene sequence of animal origin by artificial gene site-directed mutagenesis, changes the amino acid residues, and finds the effect of the synthesized ACC in brain disease intervention and treatment. At the same time, the present application also found that the non-animal source ACCS gene can also be used in brain disease intervention and treatment after host gene codon preference modification.

[0037] The ACCS gene of the present application can be of animal origin or non-animal origin. The ACCS protein of animal origin needs to be mutated to be suitable for the present application, i.e. the ACCS protein mutant of the present application. Although the ACCS protein of non-animal origin has activity, it cannot be directly used in the present application and needs to be modified according to the host gene codon preference before it can be applied. The host of the present application can be animals and humans, and can also be microorganisms such as probiotics, etc.

[0038] The ACCS gene of the present application can be constructed into a corresponding biological expression vector and delivered into animals or humans by virus, lipid nanoparticles, extracellular vesicles, probiotics, etc. to play a role; the ACCS gene can also be packaged in artificial delivery carriers (such as lipid nanoparticles and extracellular vesicles) in the form of artificial synthetic mRNA and delivered into the human body to play a role.

[0039] The ACCS gene of the present application can function through the ACC synthesized by the protein encoded thereby, through other substances synthesized by the protein, through other functions of the protein, or through the mRNA of the ACCS gene. The mRNA of the ACCS gene can be linear mRNA or circular mRNA.

[0040] The ACCS protein mutant of the present application can be used for intervention and treatment of various diseases, not limited to brain diseases.

[0041] In order to facilitate the relevant research work, the mouse ACCS1 (MmACCS1) gene is cloned by taking the mouse as a model animal. First, the mouse orthologous gene MmACCS1 of the gene is searched in the GenBank database of NCBI by using BLAST with HsACCS as a reference gene. The sequence alignment result shows that the nucleotide sequence consistency of MmACCS1 (NM_001290782.1) and HsACCS is 84%. The amino acid sequence consistency of MmACCS1 (NP_001277711.1) and HsACCS is 83%, and the amino acid similarity is 89%. The recombinant MmACCS1 protein is expressed by prokaryotic and eukaryotic cells, and the result shows that it does not have enzyme activity. MmACCS1 and MdACCS are modeled together by using VMD software, although MmACCS1 and MdACCS have certain similarity in the three-dimensional structure, but they differ in multiple amino acid residue sites. All sites that may cause MmACCS1 to lose enzyme activity are first predicted, then these sites are arranged and combined, each arrangement and combination will form a unique amino acid residue sequence, a unique amino acid residue sequence corresponds to a unique protein, and finally each protein is detected and evaluated for enzyme activity. The construction and use of the mutant are introduced by taking one of the combinations as an example, which is that the proline residue at the 134th site and the tryptophan residue at the 136th site of the MmACCS1 protein are respectively mutated into glutamine residue and tyrosine residue.

[0042] It can be understood that the MmACCS1 protein mutant obtained by mutating the proline residue at the 134th site and the tryptophan residue at the 136th site of the MmACCS1 protein into glutamine residue and tyrosine residue is only an ACCS protein mutant that has been proved to be able to be used for intervention and treatment of brain diseases in one implementation manner of the present application, and under the same inventive concept, it does not exclude that other ACCS protein mutants with similar functions can also be screened out.

[0043] The present application is further described in detail through specific embodiments. The following embodiments only further illustrate the present application, and should not be understood as limiting the present application.

[0044] Example One

[0045] I. AAV brain drug carrying mouse ACCS1 gene mutant

[0046] The protein encoded by mouse ACCS1 gene (named MmACCS1, NCBI number NM_001290782.1) is MmACCS1 (NCBI number NP_001277711.1).

[0047] In this experiment, we first predict the sites that may cause MmACCS1 to lose enzyme activity, then arrange and combine these sites, each arrangement and combination will form a unique sequence of amino acid residues, a unique sequence of amino acid residues corresponds to a unique protein, and finally detect and evaluate the enzyme activity of each protein. Here we take one of the combinations as an example to introduce the construction and drug process of the mutant. This combination is to mutate the proline residue at site 134 and the tryptophan residue at site 136 of the wild-type MmACCS1 protein (named MmACCS1_W) into glutamine residue and tyrosine residue respectively, that is, MmACCS1-P134Q-W136Y, as shown in Figure 1A, so that we get the mutant MmACCS1 protein (named MmACCS1_M), that is, the MmACCS1 protein mutant in this example.

[0048] The MmACCS1 protein mutant is the sequence shown in Seq ID No. 1, and the corresponding coding gene is the sequence shown in Seq ID No. 2.

[0049] Seq ID No. 1:

[0050] Seq ID No. 2:

[0051] To detect the enzyme activity of MmACCS1_M protein, the gene sequence corresponding to the amino acid residue sequence of MmACCS1_M (MmACCS1_M) was synthesized, and then the synthesized MmACCS1_M gene was constructed into a prokaryotic cell overexpression vector (also known as a recombinant plasmid). The gene synthesis and vector construction were completed by GenScript (Shanghai) Co., Ltd. To simultaneously verify the enzyme activity of MmACCS1_W protein, the MmACCS1_W gene corresponding to the protein was also synthesized, and the gene was also constructed into a prokaryotic cell overexpression vector. Then, the vectors were transformed into E. coli competent cells BL21 using an electroporation instrument, and positive colonies were screened on LB solid medium containing kanamycin. A single colony with good growth was inoculated into LB liquid medium, and after culture, E. coli cells were collected, crude protein was extracted, and Western blot (WB) detection was performed. The results are shown in FIG. 1, panels B and C. The sizes of the MmACCS1_W and MmACCS1_M proteins with expression tags were both about 60 kilodaltons (kD), which was consistent with their theoretical molecular weights. This indicated that the MmACCS1_W and MmACCS1_M proteins were successfully expressed in E. coli cells.

[0052] In the enzyme activity detection experiment, the MmACCS1_W and MmACCS1_M proteins were mixed with a solution containing S-adenosylmethionine (substrate) and pyridoxal phosphate (cofactor), respectively. After the reaction was completed, copper chloride solution and a mixture of sodium hypochlorite and sodium hydroxide were added to the solution in sequence, with the purpose of converting the ACC generated in the previous step into ethylene. Then, the amount of ethylene was detected using a gas chromatograph, and the enzyme activity of MmACCS1_W and MmACCS1_M proteins was calculated through the standard curve of ACC generating ethylene. The enzyme activity detection results showed that the MmACCS1_W protein expressed in E. coli cells had no enzyme activity, while the MmACCS1_M protein had enzyme activity, as shown in FIG. 1, panels D and E.

[0053] In order to study the enzyme activity of MmACCS1_W and MmACCS1_M proteins expressed by animal cells, the present experiment commissioned and Yuan Biotechnology (Shanghai) Co., Ltd. to construct a lentivirus overexpression vector (i.e. recombinant plasmid) of MmACCS1_W and MmACCS1_M genes and packaged into a lentivirus, and then transfected the lentivirus into human embryonic kidney cells HEK293T, added puromycin (1-5 μg / mL) to kill HEK293T cells not transfected with the lentivirus, thereby screening HEK293T cells transfected with MmACCS1_W and MmACCS1_M genes. Finally, the HEK293T cells transfected with MmACCS1_W and MmACCS1_M genes were identified by PCR technology. The PCR primers were 5'-GCTGCAGTACCAGGACTACAG-3' and 5'-TCTTCCGGGGAGTAGACGTCA-3', the PCR reaction annealing temperature was 56°C, and the PCR reaction was set for 30 cycles. The HEK293T cells carrying MmACCS1_W and MmACCS1_M genes were cultured, collected, and the crude protein was extracted after ultrasonic disruption, followed by WB detection. The results are shown in Figures 2A and 2B, and the HEK293T cells successfully expressed 60kD proteins, which is consistent with the theoretical molecular weight of MmACCS1_W and MmACCS1_M proteins, suggesting that MmACCS1_W and MmACCS1_M proteins are successfully expressed by HEK293T cells. Then, we detected the enzyme activity of MmACCS1_W and MmACCS1_M proteins expressed by HEK293T cells (the same as the method for detecting the enzyme activity of MmACCS1_W and MmACCS1_M proteins expressed by E. coli cells). The enzyme activity detection results showed that MmACCS1_W had no enzyme activity, while MmACCS1_M protein had significantly higher enzyme activity than MmACCS1_W group and empty vector group, suggesting that MmACCS1_M protein expressed by HEK293T cells has enzyme activity, as shown in Figures 2C and 2D.

[0054] In order to further study the effect of MmACCS1_M protein on animal behavior, the present experiment commissioned and Yuan Biotechnology (Shanghai) Co., Ltd. to construct an adeno-associated virus overexpression vector (i.e. recombinant plasmid) of MmACCS1_M gene and package it into an adeno-associated virus (AAV), and then injected the AAV carrying the empty vector and the AAV carrying the MmACCS1_M gene into the CA1 region of the mouse hippocampus through a brain stereotaxic apparatus. The specific parameters are as follows: bilateral hippocampal stereotactic injection of AAV carrying MmACCS1_M gene, 1×10 8 ~4×10 10vg (vg represents viral genome), 1 micro liter AAV was injected slowly on each side, and the needle was kept for 10 minutes after injection. After the behavior experiment, the mice were euthanized, and the hippocampus was extracted for WB detection. The results showed that the hippocampus expressed a protein of about 60 kD, which was consistent with the theoretical molecular weight of MmACCS1_M protein, indicating that the MmACCS1_M protein was successfully expressed in the hippocampus of mice, as shown in FIG. 3A and FIG. 3B.

[0055] In order to evaluate the effect of MmACCS1_M protein on the behavior of schizophrenia model mice, the present experiment carried out open field experiment, Y maze experiment, water maze experiment and novel object recognition experiment.

[0056] The results of mouse open field experiment are shown in FIG. 4. Under physiological conditions, MmACCS1_M protein does not affect the normal behavior of mice. MK801 can induce schizophrenia-like behavior in mice. The results of the present experiment show that administration of MK801 can significantly increase the movement behavior of empty mice. However, MmACCS1_M protein can significantly reduce the activity distance, crossing area and activity time of MK801-administered mice in the central area, indicating that MmACCS1_M protein can significantly reduce the movement behavior of MK801-administered mice.

[0057] The results of mouse Y maze experiment are shown in FIG. 5. MmACCS1_M protein does not affect the physiological behavior of normal mice. After administration of MK801, the spontaneous alternation rate of mice in each group increased significantly, but MmACCS1_M protein significantly reduced the high alternation rate of MK801-administered mice.

[0058] The results of mouse water maze experiment are shown in FIG. 6. MmACCS1_M protein has no significant effect on the activity of normal mice in the target area and the spatial learning and memory ability. MK801 reduces the spatial learning and memory ability of mice, while MmACCS1_M protein significantly improves the spatial learning and memory ability of MK801-administered mice.

[0059] The results of mouse novel object recognition experiment are shown in FIG. 7A. In the novel object recognition experiment, the mice were first trained with object 1 and object 2 for 5 days, and object 1 was replaced with a new object during the test. The cognitive function of mice was evaluated according to the exploration time of the new object. The experimental results are shown in FIG. 7B. MmACCS1_M protein has no significant effect on the cognitive function of normal mice. MK801 reduces the exploration behavior of mice to new objects, damages the cognitive function of mice, and causes cognitive impairment in mice. However, MmACCS1_M protein can improve the cognitive impairment caused by MK801.

[0060] II. Intraperitoneal administration of AAV carrying MmACCS1_M gene

[0061] Repeat "I, AAV brain medication carrying mouse ACCS1 gene mutant", but the difference is that the brain medication is replaced by intraperitoneal injection, the specific parameters are: intraperitoneal injection of AAV carrying MmACCS1_M gene, the number of AAV injected into each mouse is 1 x 10 9 ~ 1 x 10 11 vg, the injection volume is 50 microliters.

[0062] By intraperitoneal injection of AAV carrying MmACCS1_M gene, the symptoms of mouse mental illness caused by MK801 are significantly improved, and the drug effect lasts for 2-3 months. Moreover, the drug effect of intraperitoneal injection is equivalent to that of brain medication.

[0063] Three, intravenous medication of AAV carrying MmACCS1_M gene

[0064] Repeat "I, AAV brain medication carrying mouse ACCS1 gene mutant", but the difference is that the brain medication is replaced by tail vein injection, the specific parameters are: tail vein injection of AAV carrying MmACCS1_M gene, the number of AAV injected into each mouse is 1 x 10 9 ~ 1 x 10 11 vg, the injection volume is 50 microliters.

[0065] By intravenous medication of AAV carrying MmACCS1_M gene, the symptoms of mouse mental illness caused by MK801 are significantly improved, and the drug effect lasts for 2-3 months. Moreover, the drug effect of tail vein injection is better than that of brain medication.

[0066] Example two

[0067] In this experiment, probiotics carrying plant ACCS gene were used for gavage medication, as follows.

[0068] The plant ACCS gene, such as apple ACCS (MdACCS, NCBI No. XP_008342238.2) gene, is named as MdACCS_O gene after being optimized according to the prokaryotic cell codon bias. Although the MdACCS gene and the MdACCS_O gene are different in nucleotide sequence, the amino acid sequences of the proteins they encode are completely identical, and the protein they encode is the MdACCS protein. The gene synthesis and vector construction are completed by GenScript (Shanghai) Co., Ltd. The prokaryotic cell expression vector (i.e., recombinant plasmid) carrying the MdACCS_O gene is transformed into probiotics, such as Bifidobacterium, Escherichia coli Nissle1917, mucinophilic Akkermansia, lactic acid bacteria, etc., and the transformation and positive strain identification method is the same as the Escherichia coli BL21 transformation and identification method in Example 1. In this example, Escherichia coli Nissle 1917 is selected, which is purchased from Ningbo Tastuk Bio-technology Co., Ltd. After identification of the positive Escherichia coli Nissle 1917 strain, the Nissle 1917 cells carrying the MdACCS_O gene are cultured and collected, and then used for gavage of the model mice of neurological or mental diseases, 2 times a week, 1×10 7 ~1×10 9 live bacteria per mouse each time, for 6 times of continuous gavage.

[0069] The MdACCS_O gene is the sequence shown in Seq ID No. 4, and the MdACCS protein is the sequence shown in Seq ID No. 3.

[0070] Seq ID No. 3:

[0071] Seq ID No. 4:

[0072] In order to evaluate the effect of the probiotics carrying the MdACCS_O gene on the behavior of the model mice of depression, the open field experiment, the tail suspension experiment and the forced swimming experiment were performed.

[0073] The results of the open field experiment are shown in FIG. 8A and FIG. 8B. Compared with the model mice of depression gavaged with probiotics carrying the empty vector, the model mice of depression gavaged with probiotics carrying the MdACCS_O gene showed significantly increased movement behavior, which indicates that the MdACCS protein has the function of improving the movement willingness of the model mice of depression.

[0074] The tail suspension test is a classical and quick method for evaluating the efficacy of antidepressants. The results of the tail suspension test in this experiment showed that the immobility time of the depression model mice administered with the probiotic carrying the MdACCS_O gene was significantly reduced compared to the depression model mice administered with the probiotic carrying the empty vector, as shown in Figure 8C, which suggests that the MdACCS protein has an antidepressant effect.

[0075] The forced swimming test is another method for evaluating the antidepressant effect of drugs. The results of the forced swimming test in this experiment showed that the immobility time of the depression model mice administered with the probiotic carrying the MdACCS_O gene returned to the level of normal mice compared to the depression model mice administered with the probiotic carrying the empty vector, as shown in Figure 8D, which again suggests that the MdACCS protein has an antidepressant effect.

[0076] In order to evaluate the effect of the probiotic carrying the MdACCS_O gene on the behavior of Alzheimer's disease model mice, this application conducted a novel object recognition test.

[0077] The results of the experiment are shown in Figure 9, and the results of the novel object recognition test in this application showed that the cognitive function of the Alzheimer's disease model mice administered with the probiotic carrying the MdACCS_O gene was significantly improved compared to the Alzheimer's disease model mice administered with the probiotic carrying the empty vector, which suggests that the MdACCS protein has an anti-Alzheimer's disease effect.

[0078] On the 60th day after the end of gavage, the expression of the MdACCS_O gene was detected in the feces of the depression model mice and the Alzheimer's disease model mice administered with the probiotic carrying the MdACCS_O gene by PCR and WB techniques. These results suggest that the probiotic carrying the MdACCS_O gene successfully colonized the intestinal tract of the depression model mice and the Alzheimer's disease model mice and survived in the intestinal tract of the disease model mice for more than 60 days.

[0079] The above examples demonstrate that the implantation of animal or plant ACCS genes with enzyme activity implemented in this experiment can improve the symptoms of neurological or mental diseases for up to 2 months or even longer.

[0080] The above is a further detailed description of the present application in conjunction with specific embodiments, and cannot be considered as limiting the specific implementation of the present application to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. An ACCS protein mutant, characterized in that: The ACCS protein from animal source is mutated from proline residue at position 134 and tryptophan residue at position 136 to glutamine residue and tyrosine residue respectively.

2. The mutant ACCS protein according to claim 1, characterized in that: The animal is a mouse.

3. The mutant ACCS protein according to claim 1 or 2, characterized in that: The ACCS protein mutant is the sequence shown in Seq ID No.

1.

4. A nucleic acid encoding the ACCS protein mutant of any one of claims 1-3.

5. The nucleic acid of claim 4, wherein: The nucleic acid is the sequence shown in Seq ID No. 2, or the RNA transcribed from the sequence shown in Seq ID No.

2.

6. A recombinant plasmid, characterized by: The recombinant plasmid contains the nucleic acid of claim 4 or 5.

7. Use of the ACCS protein mutant of any one of claims 1-3, or the nucleic acid of claim 4 or 5, or the recombinant plasmid of claim 6, or the ACCS protein from non-animal source, or the ACCS nucleic acid from non-animal source, or the recombinant plasmid containing the ACCS nucleic acid from non-animal source in the preparation of a medicament for the intervention and treatment of brain diseases.

8. A drug delivery system characterized by: The delivery carrier and the delivery drug, wherein the delivery drug is the ACCS protein mutant of any one of claims 1-3, or the nucleic acid of claim 4 or 5, or the recombinant plasmid of claim 6, or the ACCS protein from non-animal source, or the ACCS nucleic acid from non-animal source, or the recombinant plasmid containing the ACCS nucleic acid from non-animal source.

9. The use of claim 7 or the drug delivery system of claim 8, wherein: The ACCS protein from non-animal source is the sequence shown in Seq ID No.

3. Preferably, the ACCS nucleic acid from non-animal source is the sequence shown in Seq ID No.

4.

10. The drug delivery system of claim 8, wherein: The delivery carrier is at least one of a virus, a lipid nanoparticle, an extracellular vesicle, and a probiotic.

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