ACCS gene, protein and its application

Abstract

The present application discloses an ACCS gene, a protein and its application. The ACCS protein mutant of the present application is formed by mutation of the proline residue at position 134 and the tryptophan residue at position 136 of the wild-type ACCS protein of animal origin into a glutamine residue and a tyrosine residue, respectively. The ACCS protein mutant of the present application can be used to intervene and treat a variety of diseases including brain diseases, providing a new scheme and approach for the intervention and treatment of brain diseases.

Description

Technical Field

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

[0002] ACCS is ACC synthase, which converts S-adenosylmethionine into 1-aminocyclopropane-1-carboxylic acid (ACC).

[0003] ACC can act on N-methyl-D-aspartate (NMDA) receptors, which are widely distributed in the body tissues of animals. Abnormalities of NMDA receptors can lead to a variety of diseases, especially brain diseases. ACC improves related disease symptoms by regulating dysfunctional NMDA receptors. However, ACC as a drug for intervention and treatment of brain diseases has shortcomings such as short efficacy, multiple doses and poor patient compliance. If ACC can be synthesized in patients through genetic manipulation, these shortcomings can be compensated.

[0004] Genes encoding ACCS proteins, namely ACCS genes, exist in both plants and animals; however, ACCS genes from animals cannot translate into active ACCS proteins. 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., it does not have ACCS enzyme activity. In addition, orthologous genes of the HsACCS gene exist in many animals, such as pigs, cattle, mice, etc., but to date, there are still no reports on the enzyme activity of animal ACCS. Plants such as apple ACCS (MdACCS, XP_008342238.2) can catalyze the synthesis of ACC and have ACCS enzyme activity. Summary of the invention

[0005] The purpose of the present application is to provide a new ACCS protein mutant and its encoding gene and application.

[0006] This application adopts the following technical solutions:

[0007] One aspect of the present application discloses an ACCS protein mutant, which is formed by mutating the proline residue at position 134 and the tryptophan residue at position 136 of the wild-type ACCS protein of animal origin into a glutamine residue and a tyrosine residue, respectively.

[0008] It should be noted that the present application found that the wild-type ACCS protein of animal origin, by mutating the proline residue at position 134 to a glutamine residue and the tryptophan residue at position 136 to a tyrosine residue, the mutant obtained in this way can be used to intervene in and treat a variety of diseases including brain diseases.

[0009] Preferably, the animal is especially a mouse, that is, the proline residue at position 134 and the tryptophan residue at position 136 of the wild-type ACCS protein from the mouse are mutated into a glutamine residue and a tyrosine residue, respectively, so as to obtain the ACCS protein mutant of the present application.

[0010] Preferably, the ACCS protein mutant is the sequence shown in Seq ID No.1.

[0011] Another aspect of the present application discloses a nucleic acid encoding the ACCS protein mutant of the present application.

[0012] Preferably, the nucleic acid is the sequence shown in Seq ID No.2, or RNA transcribed from the sequence shown in Seq ID No.2.

[0013] It should be noted that the nucleic acid of the present application, i.e., the nucleic acid encoding the ACCS protein mutant of the present application, may be DNA or RNA, and its main function is to play a role in intervening and treating diseases by encoding the ACCS protein mutant of the present application in vivo or in vitro.

[0014] Another aspect of the present application discloses a recombinant plasmid, wherein the recombinant plasmid contains the nucleic acid of the present application.

[0015] Another aspect of the present application discloses the 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 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 in the preparation of drugs for intervention and treatment of brain diseases.

[0016] It should be noted that the present application has found that animal-derived ACCS proteins need to undergo mutations to function, while non-animal-derived ACCS proteins and their encoding nucleic acids can be directly used to intervene and treat brain diseases. Therefore, the present application is the first to creatively use non-animal-derived ACCS proteins and their encoding nucleic acids to prepare drugs for intervention and treatment of brain diseases.

[0017] Another aspect of the present application discloses a drug delivery system, comprising a delivery vector 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 a recombinant plasmid containing the ACCS nucleic acid of non-animal origin.

[0018] 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 its encoding nucleic acid, or the corresponding recombinant plasmid; of course, it can also directly deliver the ACCS protein of non-animal origin or its encoding nucleic acid, or the corresponding recombinant plasmid containing non-animal origin ACCS nucleic acid.

[0019] In the present application, the non-animal-derived ACCS protein is the sequence shown in Seq ID No.3.

[0020] Preferably, the non-animal-derived ACCS nucleic acid is the sequence shown in Seq ID No.4.

[0021] It should be noted that the sequence shown in Seq ID No. 4 is a non-animal-derived ACCS nucleic acid that can be used for brain disease intervention and treatment after host gene codon preference modification in the present application.

[0022] Preferably, the delivery vehicle is at least one of a virus, a lipid nanoparticle, an extracellular vesicle and a probiotic.

[0023] It should be noted that the drug delivery system of the present application, in principle, the nucleic acid drug delivery vectors or protein drug delivery vectors used in the prior art can be applicable to the present application, and are not limited to viruses, lipid nanoparticles, extracellular vesicles and probiotics.

[0024] The beneficial effects of this application are:

[0025] The present application obtains a new ACCS protein mutant by mutating the proline residue at position 134 of the wild-type ACCS protein of animal origin into a glutamine residue, and simultaneously mutating the tryptophan residue at position 136 into a tyrosine residue. The ACCS protein mutant of the present application can be used to intervene in and treat a variety of diseases including brain diseases, providing a new solution and approach for the intervention and treatment of brain diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1: is a diagram of restoring the enzyme activity of MmACCS1 protein by site-directed mutagenesis in the embodiment of the present application; wherein A is a schematic diagram of site-directed mutagenesis of MmACCS1 gene; B is a WB experimental diagram of MmACCS1_W and MmACCS1_M proteins expressed by Escherichia coli cells; C is a bar graph of WB experimental results; D is a gas chromatogram of ethylene production in each group; E is a bar graph of enzyme activity in each group; WB is immunoblotting; Vehicle is a vector; MmACCS1 is mouse 1-aminocyclopropane 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-carboxylate synthase; kD is kilodaltons; the experiment was repeated 3 times; *** indicates P < 0.001; NS indicates no significant difference;

[0027] Figure 2 It is the enzyme activity diagram of MmACCS1_M protein expressed by HEK293T cells in the embodiment of the present application; wherein, A is the WB experimental diagram of MmACCS1_W and MmACCS1_M proteins expressed by HEK293T cells; B is a bar graph of WB experimental results; C is a gas chromatogram of ethylene production in each group; D is a bar graph of enzyme activity in each group; WB is immunoblotting; Vehicle is a vector; MmACCS1 is mouse 1-aminocyclopropane-1-carboxylate synthase 1; MmACCS1_W is a wild-type MmACCS1 protein; MmACCS1_M is a mutant MmACCS1 protein; kD is kilodaltons; the experiment was repeated 3 times; *** indicates P<0.001; NS indicates no significant difference;

[0028] Figure 3 It is a WB experimental diagram of mouse hippocampal tissue carrying MmACCS1_M protein in the embodiment of the present application; wherein A is a WB experimental diagram of MmACCS1_M protein expressed in hippocampal tissue; B is a bar graph of WB experimental results; WB is immunoblotting; Vehicle is a vector; MmACCS1 is mouse 1-aminocyclopropane-1-carboxylate synthetase 1; MmACCS1_M is a mutant MmACCS1 protein; kD is kilodaltons; the experiment was repeated 3 times; *** indicates P<0.001;

[0029] Figure 4: This is an open field experiment diagram of mice carrying MmACCS1_M protein in the hippocampus in the embodiment of the present application; wherein A is an open field movement trajectory diagram of each group of mice; B is a diagram showing the proportion of exploration distance of each group of mice in the central area of ​​the open field; C is a diagram showing the proportion of exploration time of each group of mice in the central area of ​​the open field; D is a diagram showing the number of explorations of each group of mice in the central area of ​​the open field; Saline is normal saline; MK801 is dizocilpine; Vehicle is a carrier; MmACCS1 is mouse 1-aminocyclopropane-1-carboxylate synthetase 1; MmACCS1_M is a 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; the green dot is the starting position of the movement, and the red dot is the ending position of the movement;

[0030] Figure 5 : is a Y-maze experiment diagram of mice carrying MmACCS1_M protein in the hippocampus in the embodiment of the present application; wherein A is a Y-maze motion trajectory diagram of mice in each group; B is a bar graph of the spontaneous alternation rate of mice in each group in the Y-maze; Saline is normal saline; MK801 is dizocilpine; Vehicle is a carrier; MmACCS1 is mouse 1-aminocyclopropane-1-carboxylate synthase 1; MmACCS1_M is a mutant MmACCS1 protein; the number of mice in each group is ≧8; * indicates P<0.05; NS indicates no significant difference;

[0031] Figure 6 : It is a diagram of a water maze experiment of mice carrying MmACCS1_M protein in the hippocampus in the embodiment of the present application; wherein A is a diagram of the water maze movement trajectory of mice in each group; B is a bar graph of the number of times each group of mice crosses the platform in the water maze; Saline is normal saline; MK801 is dizocilpine; Vehicle is a carrier; MmACCS1 is mouse 1-aminocyclopropane-1-carboxylate synthetase 1; MmACCS1_M is a mutant MmACCS1 protein; the number of mice in each group is ≧8; * indicates P<0.05; NS indicates no significant difference; the green dot is the starting position of swimming, the red dot is the ending position of swimming, and the platform is the yellow circle in the third quadrant;

[0032] Figure 7: This is a diagram of a new object recognition experiment of mice carrying MmACCS1_M protein in the hippocampus in the embodiment of the present application; wherein A is a schematic diagram of the new object recognition experiment; B is a bar graph of the exploration rate of new objects by mice in each group; Saline is normal saline; MK801 is dizocilpine; Vehicle is a carrier; MmACCS1 is mouse 1-aminocyclopropane-1-carboxylate synthetase 1; MmACCS1_M is a mutant MmACCS1 protein; the number of mice in each group is ≧8; * indicates P<0.05; NS indicates no significant difference; green square objects are old objects, and yellow cylindrical objects are new objects;

[0033] Figure 8 It is a behavioral experiment diagram of depression model mice gavaged with probiotics carrying the MdACCS gene in the embodiment of the present application; wherein A is a graph showing the proportion of exploration distance of each group of depression model mice in the central area of ​​the open field; B is a graph showing the proportion of exploration time of each group of depression model mice in the central area of ​​the open field; C is a bar graph showing the proportion of immobility time of each group of depression model mice in the tail suspension test; D is a bar graph showing 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;

[0034] Fig. 9 This is a diagram of a novel object recognition experiment in Alzheimer's disease model mice gavaged with probiotics carrying the MdACCS_O gene in an embodiment of the present application; wherein Vehicle is a vector; MdACCS is apple 1-aminocyclopropane-1-carboxylic acid synthetase; the number of mice in each group is ≧8; * indicates P<0.05. DETAILED DESCRIPTION

[0035] The present application has found that the ACCS protein encoded by the ACCS gene of animal origin has no enzymatic activity, mainly because the amino acid residues at the key sites of its enzymatic activity have mutated, causing it to lose its enzymatic 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 role of its synthesized ACC in brain disease intervention and treatment. At the same time, the present application also finds that the ACCS gene of non-animal origin can also be used in brain disease intervention and treatment after the host gene codon preference is modified.

[0036] 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 applicable to the present application, i.e., the ACCS protein mutant of the present application. Although the ACCS protein of non-animal origin is active, it cannot be directly used in the present application and needs to be modified according to the host gene codon preference before application. The host of the present application can be animals and humans or microorganisms, such as probiotics.

[0037] The ACCS gene of the present application can be constructed into a corresponding biological expression vector and delivered to animals or humans through viruses, lipid nanoparticles, extracellular vesicles, probiotics, etc. to exert its effects; the ACCS gene can also be packaged in an artificial delivery vector (such as lipid nanoparticles and extracellular vesicles) in the form of artificially synthesized mRNA and delivered to the human body to exert its effects.

[0038] The ACCS gene of the present application can play a role through the ACC synthesized by the protein it encodes, can also play a role through other substances synthesized by the protein, can also play a role through other functions of the protein, and can also play a role through the mRNA of the ACCS gene. The mRNA of the ACCS gene can be a linear mRNA or a circular mRNA.

[0039] The ACCS protein mutant of the present application can be used for the intervention and treatment of a variety of diseases, not limited to brain diseases.

[0040] In order to facilitate the relevant research work, the present application uses the model animal mouse as the research object to clone the mouse ACCS1 (MmACCS1) gene. First, using HsACCS as the reference gene, the mouse orthologous gene MmACCS1 of the gene was searched in the GenBank database of NCBI using BLAST. The sequence alignment results show that the nucleotide sequence identity of MmACCS1 (NM_001290782.1) and HsACCS is 84%. The amino acid sequence identity of MmACCS1 (NP_001277711.1) and HsACCS is 83%, and the amino acid similarity is 89%. The present application expresses the recombinant MmACCS1 protein through prokaryotic and eukaryotic cells, and it is found that it has no enzymatic activity. The present application uses VMD software to perform molecular modeling of MmACCS1 and MdACCS together. Although MmACCS1 and MdACCS have certain similarities in three-dimensional structure, they have differences in multiple amino acid residue sites. This application first predicts all sites that may cause MmACCS1 to lose its enzymatic activity, and then arranges and combines these sites. Each arrangement and combination will form a unique amino acid residue sequence, and a unique amino acid residue sequence corresponds to a unique protein. Finally, the enzyme activity of each protein is detected and evaluated. This application takes one of the combinations as an example to introduce the construction and medication process of the mutant. This combination is to mutate the proline residue at position 134 and the tryptophan residue at position 136 of the MmACCS1 protein into a glutamine residue and a tyrosine residue, respectively.

[0041] It can be understood that the MmACCS1 protein mutant obtained by mutating the proline residue at position 134 and the tryptophan residue at position 136 of the MmACCS1 protein into a glutamine residue and a tyrosine residue, respectively, is only an ACCS protein mutant that has been confirmed to be able to intervene in and treat brain diseases in one implementation of the present application. Under the same inventive concept, it is not ruled out that other ACCS protein mutants with similar functions can also be screened out.

[0042] The present application is further described in detail below through specific examples. The following examples are only used to further illustrate the present application and should not be construed as limiting the present application.

[0043] Embodiment 1

[0044] 1. Brain administration of AAV carrying mouse ACCS1 gene mutant

[0045] The protein encoded by the mouse ACCS1 gene (named MmACCS1, NCBI number NM_001290782.1) is MmACCS1 (NCBI number NP_001277711.1).

[0046] This experiment first predicts the sites that may cause MmACCS1 to lose its enzyme activity, and then arranges and combines these sites. Each arrangement and combination will form a unique amino acid residue sequence. A unique amino acid residue sequence corresponds to a unique protein. Finally, the enzyme activity of each protein is detected and evaluated. Here we take one of the combinations as an example to introduce the construction and medication process of the mutant. This combination is to mutate the proline residue at position 134 and the tryptophan residue at position 136 of the wild-type MmACCS1 protein (named MmACCS1_W) into a glutamine residue and a tyrosine residue, respectively, that is, MmACCS1-P134Q-W136Y, such as Figure 1 As shown in A, the mutant MmACCS1 protein (named MmACCS1_M) was obtained, that is, the MmACCS1 protein mutant in this example.

[0047] 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.

[0048] Seq ID No.1:

[0049] MFCLPQQESTAPTTCTGSASTQDMDSGYGDGLQGECLRKPDQTQPKLYGVGDPTATFSSDSSCLSSRGRVIKWFWDSAEEGYRTYHMDEYDEDKNPSGIINLGTSENKLCFDLLSWRLTQGDMLHVEPSLLQYQDYRGHLFLREEVAKFLSFY CKSPAPLKPENVVVLNGCASLFSALATVLCEAGEALLIPTPYYGAITQHIYLYGNVRLAYVYLDSKVTGLNTRPFQLTVEKLEMVLQGVSSEGVKVKGLILINPQNPLGDVYSPEELQDFLRFAMRHKLHVIMDEVYMLSVFEESLGYRSVLSLERLPDPQRTHVMWATSKDFGM SGLRFGVLYTENQHVATAVASLCRYHGLSGLVQHQMAQLLRDHDWISQVYLPENHARLKAAHTYVSEELRALGIPFVSRGAGFFIWVDLRKYLCKGTFEEEALLWRQFLDNKVLLSSGKTFECKEPGWFRVVFSDKENRLRLGMQRMRQVLEGQSQVVEDASPCHAQEPQSQPR*

[0050] Seq ID No.2:

[0051]

[0052] In order to detect the enzymatic activity of the MmACCS1_M protein, this experiment synthesized the gene sequence (MmACCS1_M) corresponding to the amino acid residue sequence of MmACCS1_M, and then constructed the synthesized MmACCS1_M gene into a prokaryotic cell overexpression vector (also known as a recombinant plasmid). Gene synthesis and vector construction were commissioned to Heyuan Biotechnology (Shanghai) Co., Ltd. In order to simultaneously verify the enzymatic activity of the MmACCS1_W protein, this experiment also synthesized the MmACCS1_W gene corresponding to the protein, and also constructed the gene into a prokaryotic cell overexpression vector. These vectors were then transferred into Escherichia coli competent cells BL21 using an electroporation transformer, and positive colonies were screened on LB solid culture medium plates with kanamycin. Single colonies with good growth were picked and inoculated into LB liquid culture medium. After culture, the E. coli cells were collected, and the crude protein was extracted and then subjected to immunoblotting (WB) detection. The results are as follows. Figure 1 As shown in Figures B and C, the sizes of MmACCS1_W and MmACCS1_M proteins with expression tags are both about 60 kilodaltons (kD), which is consistent with their theoretical molecular weights. This suggests that MmACCS1_W and MmACCS1_M proteins were successfully expressed in E. coli cells.

[0053] In the enzyme activity detection experiment, this experiment mixed the MmACCS1_W and MmACCS1_M proteins with solutions containing S-adenosylmethionine (substrate) and pyridoxal phosphate (cofactor), respectively. After the reaction was completed, copper chloride solution and a mixed solution of sodium hypochlorite and sodium hydroxide were added to the solution in order to convert the ACC generated in the previous step into ethylene. The amount of ethylene was then detected by gas chromatograph, and the enzyme activity of MmACCS1_W and MmACCS1_M proteins was calculated using a standard curve of ACC generating ethylene. The results of the enzyme activity detection showed that the MmACCS1_W protein expressed by Escherichia coli cells had no enzyme activity, while the MmACCS1_M protein had enzyme activity, such as Figure 1 As shown in Figures D and E.

[0054] In order to study the enzymatic activity of MmACCS1_W and MmACCS1_M proteins expressed in animal cells, this experiment commissioned Heyuan Biotechnology (Shanghai) Co., Ltd. to construct the lentiviral overexpression vector (i.e., recombinant plasmid) of MmACCS1_W and MmACCS1_M genes and package them into lentiviruses. Then, the lentiviruses were transfected into human embryonic kidney cells HEK293T, and puromycin (1-5 μg / mL) was added to kill the HEK293T cells that were not transfected with lentiviruses, thereby screening out HEK293T cells transfected with MmACCS1_W and MmACCS1_M genes. Finally, these 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. HEK293T cells carrying MmACCS1_W and MmACCS1_M genes were cultured, collected, and their crude proteins were extracted after ultrasonic disruption, followed by WB detection. Figure 2 As shown in Figures A and B, HEK293T cells successfully expressed a 60kD protein, which is consistent with the theoretical molecular weight of the MmACCS1_W and MmACCS1_M proteins, indicating that the MmACCS1_W and MmACCS1_M proteins were successfully expressed by HEK293T cells. Next, we detected the enzymatic activity of the MmACCS1_W and MmACCS1_M proteins expressed by HEK293T cells (the same method as the enzyme activity detection method of the MmACCS1_W and MmACCS1_M proteins expressed by Escherichia coli cells). The results of the enzyme activity detection showed that MmACCS1_W had no enzyme activity, while the MmACCS1_M protein had significantly higher enzyme activity than the MmACCS1_W group and the empty load group, indicating that the MmACCS1_M protein expressed by HEK293T cells had enzyme activity, as shown in Figure 1. Figure 2 As shown in Figures C and D.

[0055] In order to further study the effect of MmACCS1_M protein on animal behavior. This experiment commissioned Heyuan Biotechnology (Shanghai) Co., Ltd. to construct an adeno-associated virus overexpression vector (i.e., recombinant plasmid) of the MmACCS1_M gene and package it into an adeno-associated virus (AAV), and injected the AAV carrying the empty vector and the MmACCS1_M gene into the CA1 region of the mouse hippocampus through a brain stereotaxic instrument. The specific parameters are: bilateral hippocampal stereotaxic injection of AAV carrying the MmACCS1_M gene, each mouse injected with 1×10 8 ~4×10 10vg (vg stands for viral genome), 1 microliter of AAV was slowly injected on each side, and the needle was retained for 10 minutes after the injection. After the behavioral experiment, the mice were euthanized, and the protein of the hippocampus was extracted for WB detection. The results showed that the hippocampus expressed a protein of about 60kD, which was consistent with the theoretical molecular weight of the MmACCS1_M protein, indicating that the MmACCS1_M protein was successfully expressed in the mouse hippocampus. Figure 3 As shown in Figures A and B.

[0056] In order to evaluate the effect of MmACCS1_M protein on the behavior of schizophrenia model mice, this experiment conducted open field test, Y maze test, water maze test and novel object recognition test.

[0057] The results of the open field test in mice are as follows Figure 4 As shown in the figure, 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 this experiment show that administration of MK801 can significantly increase the motor behavior of empty-load mice. However, MmACCS1_M protein can significantly reduce the activity distance, number of cross-zones and activity time of mice administered MK801 in the central area, indicating that MmACCS1_M protein can significantly reduce the motor behavior of mice administered MK801.

[0058] The results of the mouse Y maze experiment are as follows Figure 5 As shown, MmACCS1_M protein did 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 mice administered MK801.

[0059] The results of the mouse water maze experiment are as follows Figure 6 As shown in the figure, MmACCS1_M protein had no significant effect on the activity of normal mice in the target area and their spatial learning and memory ability. MK801 reduced the spatial learning and memory ability of mice, while MmACCS1_M protein significantly improved the spatial learning and memory ability of mice treated with MK801.

[0060] Mouse novel object recognition experiment Figure 7 As shown in Figure A, in the novel object recognition experiment, the mice were first trained with Object 1 and Object 2 for 5 days. During the test, Object 1 was replaced with a new object, and the cognitive function of the mice was evaluated based on the time the mice spent exploring the new object. Figure 7 As shown in Figure B, MmACCS1_M protein has no significant effect on the cognitive function of normal mice. MK801 reduces the mice's exploration behavior of new objects, damages the mice's cognitive function, and causes cognitive impairment in mice. However, MmACCS1_M protein can improve the cognitive impairment caused by MK801.

[0061] 2. Intraperitoneal administration of AAV carrying the MmACCS1_M gene

[0062] Repeat “I. Brain administration of AAV carrying mouse ACCS1 gene mutant”, but the difference is that brain administration is replaced by intraperitoneal administration. Specific parameters: intraperitoneal injection of AAV carrying MmACCS1_M gene, the number of AAV injected per mouse is 1×10 9 ~1×10 11 vg, injection volume was 50 μl.

[0063] Intraperitoneal administration of AAV carrying the MmACCS1_M gene significantly improved the symptoms of MK801-induced psychiatric disorders in mice, with a drug effect lasting 2-3 months. In addition, the efficacy of intraperitoneal injection is comparable to that of brain administration.

[0064] 3. Intravenous administration of AAV carrying the MmACCS1_M gene

[0065] Repeat "I. Brain administration of AAV carrying mouse ACCS1 gene mutant", but the difference is that brain administration is replaced by tail vein administration. Specific parameters: tail vein injection of AAV carrying MmACCS1_M gene, the number of AAV injected per mouse is 1×10 9 ~1×10 11 vg, injection volume was 50 μl.

[0066] The intravenous administration of AAV carrying the MmACCS1_M gene significantly improved the symptoms of MK801-induced psychiatric disorders in mice, with a drug effect lasting 2-3 months. Moreover, the efficacy of the drug administered through the tail vein was better than that of the drug administered through the brain.

[0067] Embodiment 2

[0068] In this experiment, probiotics carrying the plant ACCS gene were administered by intragastric administration, as follows.

[0069] Plant ACCS gene, such as apple ACCS (MdACCS, NCBI numbering XP_008342238.2) gene, is named MdACCS_O gene after being optimized according to prokaryotic codon preference. Although MdACCS gene is different from MdACCS_O gene in nucleotide sequence, the amino acid sequence of their encoded protein is exactly the same, and the protein they encode is MdACCS protein. Gene synthesis and vector construction are entrusted to Heyuan Biotechnology (Shanghai) Co., Ltd. to complete. Probiotics, such as bifidobacterium, Escherichia coli Nissle1917, Akkermansia muciphila, lactic acid bacteria, etc., are transformed with positive bacteria identification method and Escherichia coli BL21 transformation and identification method in Example 1. Escherichia coli Nissle 1917 is selected in this embodiment, and the bacteria is purchased from Ningbo Testo Biotechnology Co., Ltd. After positive E. coli Nissle 1917 strains were identified, Nissle 1917 cells carrying the MdACCS_O gene were cultured and collected, and then used to gavage mice with neurological or psychiatric diseases twice a week, with 1×10 7 ~1×10 9 Live bacteria were administered intragastrically for 6 times.

[0070] Among them, 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.

[0071] Seq ID No.3:

[0072] MRMLSRNATFNSHGQDSSYFLGWQEYEKNPYHEVHNTNGIIQMGLAENQLCFDLLESWLAKNPEAAAFKKNGESIFAELALFQDYHGLPAFKKAMVDFMAEIRGNKVTFDPNHLVLTAGATSANETFIFCLADPGEAVLIPTPYYPGFDRDLKWRTGVEIVPIHCTSSNGFQITETALEEAYQEAEKRNLRVKGVLVTNPSNPLGTTMTRNELYLLLSFVEDKGIHLISDEIYSGTAFSSPSFISVMEVLKDRNCDENSEVWQRVHVVYSLSKDLGLPGFRVGAIYSNDDMVVAAATKMSSFGLVSSQTQHLLSAMLSDKKLTKNYIAENHKRLKQRQKKLVSGLQKSGISCLNGNAGLFCWVDMRHLLRSNTFEAEMELWKKIVYEVHLNISPGSSCHCTEPGWFRVCFANLPERTLDLAMQRLKAFVGEYYNVPEVNGGSQSSHLSHSRRQSLTKWVSRLSFDDRGPIPGR*

[0073] Seq ID No.4:

[0074] ATGCGCATGCTGTCTCGTAACGCTACTTTCAACTCTCACGGCCAGGATAGCTCTTACTTCCTGGGCTGGCAGGAATACGAAAAAAACCCGTATCATGAGGTTCACAATACGAACGGTATCATTCAGATGGGTCTGGCGGAAAATCAGCTGTGCTTCGATCTGCTGGAGTCCTGGCTGGCCAAAAATCCGGAAGCCGCAGCGTTCAAAAAAAACGGTGAAAGCATCTTCGCCGAACTGGCGCTGTTCCAGGATTACCATGGTCTGCCGGCCTTCAAAAAAGCCATGGTTGATTTTATGGCGGAAATTCGCGGTAACAAAGTGACTTTTGACCCGAACCACCTGGTACTGACTGCAGGTGCGACTTCCGCGAACGAAACCTTTATTTTCTGCCTGGCTGACCCAGGTGAAGCGGTGCTGATTCCGACCCCTTACTACCCGGGCTTTGACCGTGATCTGAAATGGCGCACTGGCGTTGAAATTGTACCGATTCATTGCACCAGCTCTAACGGCTTCCAGATCACTGAGACTGCACTGGAAGAAGCGTACCAGGAAGCTGAAAAACGTAACCTGCGCGTAAAAGGTGTTCTGGTTACCAACCCGTCTAACCCACTGGGCACCACTATGACCCGTAACGAACTGTACCTGCTGCTGTCTTTCGTGGAAGACAAAGGCATCCACCTGATTAGCGACGAAATTTATAGCGGCACCGCATTTTCCAGCCCGTCTTTCATCTCTGTAATGGAAGTTCTGAAAGACCGCAACTGCGATGAAAACTCTGAAGTTTGGCAACGTGTCCACGTTGTTTACTCTCTGTCTAAAGACCTGGGTCTGCCGGGTTTCCGTGTTGGCGCAATCTACAGCAATGATGATATGGTCGTCGCGGCCGCTACCAAGATGTCCTCTTTTGGCCTGGTTTCCAGCCAGACTCAACATCTGCTGTCCGCCATGCTGTCTGACAAAAAACTGACCAAAAACTACATCGCCGAAAACCACAAGCG TCTGAAACAGCGTCAGAAAAAACTGGTGTCTGGCCTGCAGAAATCTGGCATCTCTTGCCTGAACGGTAACGCTGGTCTGTTCTGTTGGGTGGACATGCGCCACCTGCTGCGCTCCAACACTTTCGAGGCGGAAATGGAACT GTGGAAAAAAATCGTCTACGAGGTTCACCTGAACATTTCTCCGGGTTCTTCCTGCCATTGTACCGAACCGGGTTGGTTCCGTGTATGCTTCGCCAACCTGCCTGAACGTACCCTGGACCTGGCTATGCAGCGTCTGAAAGC GTTCGTGGGTGAGTATTACAACGTCCCGGAAGTCAACGGTGGTAGCCAGAGTCCCCATCTGTCTCACTCTCGTCGCCAATCTCTGACCAAATGGGTAAGCCGCCTGAGCTTCGATGACCGTGGCCCGATCCCGGGTCGTTGA

[0075] In order to evaluate the effect of probiotics carrying the MdACCS_O gene on the behavior of depression model mice, this experiment conducted open field test, tail suspension test and forced swimming test.

[0076] The results of the open field test are as follows Figure 8 As shown in Figures A and B, compared with the depression model mice gavaged with empty vector probiotics, the movement behavior of the depression model mice gavaged with MdACCS_O gene probiotics was significantly increased, suggesting that MdACCS protein has the function of enhancing the willingness of depression model mice to exercise.

[0077] The tail suspension test is a classic and rapid method for evaluating the efficacy of antidepressant drugs. The results of the tail suspension test in this experiment showed that compared with depression model mice gavaged with empty vector probiotics, the immobility time of depression model mice gavaged with MdACCS_O gene probiotics was significantly reduced. Figure 8 As shown in Figure C, this suggests that MdACCS protein has antidepressant effects.

[0078] The forced swimming test is another method to evaluate the antidepressant effect of drugs. The results of the forced swimming test in this experiment showed that compared with the depression model mice gavaged with empty vector probiotics, the immobility time of depression model mice gavaged with MdACCS_O gene probiotics was restored to the level of normal mice, such as Figure 8 As shown in Figure D, this again suggests that MdACCS protein has antidepressant effects.

[0079] In order to evaluate the effect of probiotics carrying the MdACCS_O gene on the behavior of Alzheimer's disease model mice, the present application conducted a novel object recognition experiment.

[0080] The experimental results are as follows Fig. 9 As shown, the results of the new object recognition experiment of the present application showed that compared with the Alzheimer's disease model mice gavaged with probiotics carrying empty vectors, the cognitive function of the Alzheimer's disease model mice gavaged with probiotics carrying the MdACCS_O gene was significantly improved, which suggests that the MdACCS protein has anti-Alzheimer's disease efficacy.

[0081] On the 60th day after intragastric administration, the expression of MdACCS_O gene was detected in the feces of depression model mice and Alzheimer's disease model mice gavaged with probiotics carrying MdACCS_O gene by PCR and WB technology. These results suggest that probiotics carrying MdACCS_O gene successfully colonized the intestines of depression model mice and Alzheimer's disease model mice, and survived in the intestines of disease model mice for more than 60 days.

[0082] The above examples illustrate that the experiment implemented by implanting animal or plant ACCS genes with enzyme activity can achieve the efficacy of improving the symptoms of neurological or psychiatric diseases for up to 2 months or even longer.

[0083] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians 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 proline residue at position 134 and the tryptophan residue at position 136 of the wild-type ACCS protein from animal sources are mutated into a glutamine residue and a tyrosine residue, respectively. The sequence of the ACCS protein mutant is shown in Seq ID No.

1.

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

3. A nucleic acid encoding the ACCS protein mutant according to claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that: The nucleic acid sequence is shown in Seq ID No.2, or is RNA transcribed from the sequence shown in Seq ID No.

2.

5. A recombinant plasmid, characterized in that: The recombinant plasmid contains the nucleic acid according to claim 3 or 4.

6. Use of the ACCS protein mutant according to claim 1 or 2, or the nucleic acid according to claim 3 or 4, or the recombinant plasmid according to claim 5 in the preparation of a drug for intervention and treatment of brain diseases, wherein the brain disease is schizophrenia.

7. An ACCS protein of non-animal origin, or ACCS Nucleic acids, or containing non-animal sources ACCS The use of a recombinant plasmid of nucleic acid in the preparation of brain disease intervention and treatment drugs, wherein the non-animal source ACCS protein sequence is shown in Seq ID No.3; the non-animal source ACCS The nucleic acid sequence is shown in Seq ID No.4 or RNA transcribed from the sequence shown in Seq ID No.4, and the brain disease is Alzheimer's disease or depression.

8. A drug delivery system, characterized in that: The method comprises a delivery vector and a delivery drug, wherein the delivery drug is the ACCS protein mutant according to claim 1 or 2, or the nucleic acid according to claim 3 or 4, or the recombinant plasmid according to claim 5.

9. The drug delivery system according to claim 8, characterized in that: The delivery vehicle is at least one of a virus, a lipid nanoparticle, an extracellular vesicle and a probiotic.

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