A method for constructing a mouse model simulating human Parkinson's disease and its application.

Abstract

This invention discloses a method for constructing an animal model simulating human Parkinson's disease. Using gene editing technology, the protein-coding region of a human SNCA gene carrying a mutation site is randomly or strategically inserted into the animal genome, or the animal SNCA gene protein-coding region is replaced. The constructed animal model expresses α-Syn protein carrying at least two mutations among A30P, E46K, and A53T. The animal model constructed using this method can effectively simulate the pathological characteristics and disease progression of clinical Parkinson's disease patients, and can also be used for efficacy evaluation of PD-related drugs.

Description

Technical Field

[0001] This invention belongs to the field of animal genetic engineering and gene modification, specifically relating to a method for constructing a mouse model that simulates human Parkinson's disease and its application. Background Technology

[0002] Parkinson's disease (PD) is a progressive neurodegenerative disease of the central nervous system, clinically characterized by motor system disorders such as resting tremor, muscle rigidity, and postural changes. Some patients also experience cognitive impairment, intellectual decline, and dementia. It is the second most common neurodegenerative disease worldwide. According to data from the World Health Organization (WHO), PD already affects more than 1% of the global population, totaling 6 million people, and this number may reach 12 million by 2030. Currently, the progressive loss of dopamine neurons (DA) in the substantia nigra pars compacta (SNpc) of the midbrain and the formation of Lewy bodies (LB) in the cytoplasm are generally considered the representative pathological features of PD. α-Synuclein (α-Syn) is the main filamentous component of Lewy bodies and Lewy neurites, characteristic pathological changes in PD, and is also a representative pathogenic factor in familial PD. Therefore, α-Syn is considered a molecular marker for some neurodegenerative diseases. In PD, these inclusion bodies are mainly distributed in the medulla oblongata, olfactory bulb, striatum, and substantia nigra, and to a lesser extent in various regions of the cortex. The gene encoding α-Syn, SNCA, has three major mutation sites: A53T (Ala-Thr), A30P (Ala-Pro), and E46K (Glu-Lys). These mutations can disrupt the original molecular spatial structure of the protein, preventing α-Syn from degrading normally and causing it to aggregate abnormally to form amyloid structures, thereby leading to neuronal degeneration.

[0003] Currently, the etiology and pathogenesis of Parkinson's disease (PD) are still unclear. Drugs targeting α-Syn clearance have entered clinical trials, and the development of effective PD treatments continues to receive widespread attention. According to data from the China New Drug Development Monitoring Center, among the 367 drugs under development for Parkinson's disease, the main drug targets are dopamine receptors, α-Syn, LRRK2, and monoamine neurotransmitter receptors, and most are small molecule drugs. Because human and mouse proteins have significant homology differences, directly using mice for drug efficacy evaluation cannot accurately simulate and reflect the drug's efficacy in humans. Therefore, using humanized mouse models or transgenic animals is a valuable tool for drug efficacy evaluation and mechanism research. Transgenic mouse models used in preclinical trials of new drugs involve randomly inserting human gene sequences carrying PD-causing mutations into mice, enabling the mice to express human proteins with different mutations, thereby mimicking the pathological and behavioral characteristics of PD.

[0004] Gene modification based on α-Syn has led to the development of mature SNCA TG models (Huα-Syn(A53T)transgenic line G2-3, A53Tα-Syn transgenic line M83) for studying the mechanisms and efficacy of Parkinson's disease (PD). In Huα-Syn(A53T)transgenic line G2-3 mice, human SNCAc DNA (carrying an A53T mutation) driven by the murine Prnp promoter was randomly integrated into C3H / HeJ x C57BL / 6J hybrid F1 mice. These mice only showed significant motor function decline at 13 months of age and died within 14-21 days of onset due to progressive motor dysfunction. Simultaneously, α-Syn inclusion bodies accumulated in the brain regions of mice aged 8-12 months, including progressive loss of dopamine alpha (DA) neurons and pathological accumulation of ubiquitin. The symptom progression in this model mice involved both behavioral phenotype and pathological changes, which does not conform to the clinical and pathological progression of PD patients. The A53T α-Syntransgenic line M83 model also randomly integrated a mouse Prnp promoter-driven human SNCA cDNA (carrying an A53T mutation) into C57BL / 6x C3H hybrid mice. At 8 months of age, some homozygous mice exhibited significant motor phenotypes, but by 14 months of age, all mice showed severe motor dysfunction. In the brains of mice aged 9-13 months, α-Syn inclusion bodies were widely distributed in the spinal cord, brainstem, and thalamus. The appearance of α-Syn inclusion bodies paralleled the development of the motor dysfunction phenotype, making it impossible to predict the behavioral phenotype of mice primarily from pathological results. This hinders preclinical drug efficacy evaluation and rapid advancement, limiting the model's application. Furthermore, since most existing mature SNCA TG models are donated to the Jackson Laboratory by research institutions, permission from the donors is required for sales to non-profit organizations (universities and research institutes), significantly restricting the use of these models by PD drug development companies and slowing down the development of PD treatments, especially for Chinese pharmaceutical companies. Therefore, developing a suitable PD model can not only solve the current shortage of PD models in China, but also advance the research on the pathogenesis of Parkinson's disease and the development of drugs in China. Summary of the Invention

[0005] This invention addresses the shortcomings of existing animal models carrying the A53T mutation, which exhibit delayed onset of DA neuron loss, α-Syn inclusion body aggregation, and motor function decline and impairment, and whose behavioral phenotype and pathology occur simultaneously, failing to reflect the clinical and pathological progression of PD patients. It establishes a method for creating an animal model that can realistically simulate human Parkinson's disease.

[0006] The specific technical solution of this invention is as follows:

[0007] A method for constructing an animal model simulating human Parkinson's disease involves using gene editing technology to randomly or precisely insert or replace the protein-coding region of a human SNCA gene carrying a mutation site into the animal genome, wherein the animal model expresses an α-Syn protein carrying at least two mutations among A30P, E46K, and A53T.

[0008] Preferably, the animal model expresses α-Syn protein carrying mutations of A30PE46K, E46KA53T, or A30PA53T.

[0009] In a specific example of the present invention, the animal model expresses an α-Syn protein carrying the E46KA53T mutation (α-Syn protein GenBank NM_000345.4, GI:1677495929).

[0010] The method described in this invention can utilize all feasible gene editing technologies under the prior art, including but not limited to one or more of homologous recombination, zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR / Cas9 system, and single-base editing technology.

[0011] In a specific example of this invention, homologous recombination technology is used to randomly insert the sequence of a foreign gene into the genome of a target animal through gene editing, so that the human SNCA protein carrying the mutation is specifically expressed in the brain of the target animal, thereby obtaining an animal model that can be stably inherited and has a spontaneous Parkinson's disease phenotype.

[0012] The construction method described in this invention involves obtaining a transgenic targeting vector using gene editing technology. The targeting vector can be injected into animal fertilized eggs via microinjection, or transfected into animal ES cells using ES cell targeting technology, or the target gene can be recombined into a retroviral vector using retroviral infection to infect pre- / post-implantation animal embryos, or the transgenic targeting vector can be introduced into sperm via electroporation using a sperm vector method and then combined with the egg.

[0013] A specific example of the construction method described in this invention includes the following steps:

[0014] (1) Design DNA homologous donors containing the protein-coding region of the human SNCA gene carrying the mutation site;

[0015] (2) The DNA homologous donor obtained in step (1) is injected into animal zygotes or transfected into ES cells by microinjection, and the protein coding region of the human SNCA gene carrying the mutation site is randomly inserted into the genome.

[0016] Alternatively, based on CRISPR / Cas9 technology, Cas9 or its expression vector can be prepared, and an sgRNA or tracrRNA / crRNA binary complex or its respective expression vector can be designed at the 5' and 3' ends of the protein-coding region of the animal SNCA gene; or a co-expression vector of Cas9, sgRNA or crRNA and tracrRNA can be prepared, and the DNA homologous donor, Cas9, sgRNA or tracrRNA / crRNA binary complex, or their respective expression vectors or co-expression vectors can be injected into animal zygotes or transfected into animal ES cells, knocking out the protein-coding region of the animal SNCA gene while inserting the human SNCA gene protein-coding region carrying the mutation site into the genome;

[0017] (3) Fertilized eggs are transferred to surrogate mothers or positive ES cells are injected into animal blastocysts and the blastocysts are transferred to surrogate mothers for feeding. The offspring produced are F0 generation animals. After the F0 generation animals with correct genotype identification reach sexual maturity, they are bred with wild-type animals of the same background to obtain F1 generation, which is an animal model that simulates human Parkinson's disease.

[0018] The sequence of the human SNCA gene protein coding region carrying the mutation site (preferably E46KA53T) described in step (1) above is shown in SEQ ID No:1.

[0019] In the construction method described in this invention, preferably, the homologous donor has the Thy1.2 promoter.

[0020] In a preferred embodiment, the homologous donor is a Thy1 gene knockout plasmid, which retains the Thy1.2 promoter and replaces the coding regions of exons 2, 3, and 4 with cDNA of the human SNCA gene protein coding region carrying the E46KA53T mutation site.

[0021] In a preferred embodiment of the present invention, a kozak sequence may be introduced at the front end of the human SNCA gene cDNA carrying the mutation site (preferably E46KA53T) to help initiate the translation of the mRNA sequence.

[0022] DNA homologous donors can also be co-injected with Cas9 to improve targeting efficiency.

[0023] In a specific example of this invention, the Kozak sequence (GCCACCAUGG) and human SNCA cDNA carrying E46K and A53T mutations (excluding 5'UTR and 3'UTR) were cloned into the Thy1 gene knockout plasmid (Plasmid#20736, containing the mouse Thy1.2 promoter, 5'UTR and 3'UTR), replacing its original exons 2-4, to construct a Thy1-hSNCAE46KA53T vector (homologous donor) for targeted injection.

[0024] The DNA homologous donor sequence is shown in SEQ ID No:2.

[0025] In one specific embodiment of the present invention, the DNA homologous donor obtained in step (1) and Cas9 are injected into animal fertilized eggs or transfected into ES cells via microinjection, and the protein coding region of the human SNCA gene carrying the mutation site is randomly inserted into the genome.

[0026] The construction method of this invention uses mammals, preferably rodents, and more preferably rats or mice. In a preferred embodiment, the mouse is a C57BL / 6J, C57BL / 6N, BALB / c, NCG, FVB, C3H, or DBA mouse.

[0027] Another objective of this invention is to provide the application of the animal model of simulated human Parkinson's disease prepared by the above construction method in the screening and evaluation of the activity of drugs for the treatment of Parkinson's disease.

[0028] Advantages of this invention:

[0029] The mouse model constructed by the method described in this invention shows detectable loss of dopamine neurons in the brain as early as 3 months of age, with progressive loss occurring with increasing age. At 5 months of age, α-Syn inclusion bodies aggregate in various brain regions. The model mice exhibit significant motor function decline and impairment at 4 months of age, effectively simulating the pathological characteristics and disease progression of clinical Parkinson's disease patients. It can also be used for evaluating the efficacy of PD-related drugs. Compared to existing technologies, this significantly shortens the time to symptom onset and simulates pathological characteristics more closely resembling those of clinical patients. This model also fills the gap in domestic PD transgenic model products and breaks the monopoly of foreign PD models and surgically induced models in the Chinese market. Attached Figure Description

[0030] Figure 1 The image shows the plasmid pattern of GPT000415-04-01-plasmid-Thy1.2-hSNCA-A30P / A53T-TG.

[0031] Figure 2The image shows the plasmid pattern GPT000415-04-01-plasmid-Thy1.2-hSNCA-A30P-TG.

[0032] Figure 3 The image shows the plasmid pattern GPT000415-04-01-plasmid-Thy1.2-hSNCA-A30P / E46K-TG.

[0033] Figure 4 The image shows the plasmid pattern of GPT000415-04-01-plasmid-Thy1.2-hSNCA-A53T-TG.

[0034] Figure 5 The image shows the plasmid pattern GPT000415-04-01-plasmid-Thy1.2-hSNCA-E46K / A53T-TG.

[0035] Figure 6 The image shows the plasmid pattern GPT000415-04-01-plasmid-Thy1.2-hSNCA-E46K-TG.

[0036] Figure 7 The results show the fluorescence detection of Thy1-SNCA plasmid expression in PC12 cells. (G1: untreated group; G2: empty vector group; G3: GPT000415-04-01-plasmid-Thy1.2-hSNCA-E46K-TG;)

[0037] G4: GPT000415-04-01-plasmid-Thy1.2-hSNCA-A53T-TG;

[0038] G5: GPT000415-04-01-plasmid-Thy1.2-hSNCA-A30P-TG;

[0039] G6: GPT000415-04-01-plasmid-Thy1.2-hSNCA-E46K / A30P-TG;

[0040] G7: GPT000415-04-01-plasmid-Thy1.2-hSNCA-E46K / A53T-TG;

[0041] G8: GPT000415-04-01-plasmid-Thy1.2-hSNCA-A30P / A53T-TG).

[0042] Figure 8The results show the cell count and viability of PC12 cells transfected with the Thy1-SNCA plasmid.

[0043] Figure 9 This is a schematic diagram of the Thy1-SNCA firing carrier.

[0044] Figure 10 Gel image for PCR identification of the GPT000415-01-Thy1.2-hSNCA-A53TE46K TG plasmid.

[0045] Figure 11 Gel image showing enzyme digestion identification of GPT000415-01-Thy1.2-hSNCA-A53TE46K TG plasmid.

[0046] (DL2000 is the Marker, with strip sizes of 2000, 1000, 750, 500, 200, and 100; T14 is the EcoT14Idigest Marker, with strip sizes of 19329, 7743, 6223, 4254, 3472, 2690, 1882, and 1489).

[0047] Figure 12 The image shows the TG plasmid pattern GPT000415-01-Thy1.2-hSNCA-A53TE46K.

[0048] Figure 13 Gel images showing PCR identification of the Thy-hSNCA-E46KA53T Founder mouse gene. The image shows the identification of the Thy1-hSNCA gene, corresponding to PCR sequences ①, ②, and ③, respectively.

[0049] Figure 14 The results show the expression of TH neurons in the brain region of B6-hSNCA E46KA53T mice.

[0050] Figure 15 The results of grip strength testing are for B6-hSNCA E46KA53T mice.

[0051] Figure 16 Results of fatigue rotator bar test in B6-hSNCA E46KA53T mice. Detailed Implementation

[0052] Example 1: Effects of different SNCA mutation sites or combinations on PC12 cell viability

[0053] (1) Synthesize human SNCA CDS carrying A30P, E46K, A53T, A30PE46K, E46KA53T, or A30PA53T. The human SNCA CDS sequence is shown in SEQ ID No: 3. The sequence of hSNCA-A30P is the human SNCA CDS sequence in which nucleotides 88-90 are mutated from GCA to CCA; the sequence of hSNCA-A53T is the human SNCA CDS sequence in which nucleotides 157-159 are mutated from GCA to ACA; the sequence of hSNCA-E46K is the human SNCA CDS sequence in which nucleotides 136-138 are mutated from GAG to AAG; the sequences of hSNCA-A30P / E46K, hSNCA-E46K / A53T, and hSNCA-A30PA53T are combinations of the aforementioned single mutant sequences.

[0054] (2) The hSNCA-A30P, hSNCA-A53T, hSNCA-E46K, hSNCA-A30P / E46K, hSNCA-E46K / A53T, and hSNCA-A30PA53T obtained in step 1 were sliced ​​with GPT000367-Thy1-first (a vector already available from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., Chinese patent document CN113957095A), Flag, IRES, and EGFP, respectively. Competent cells were transformed, plated, and cultured overnight at 37°C. Single clones were picked from the plates and cultured using primers (hSNCA-TF 5'-TCTCTGAGTGGCAAAGGACC-3', hSNCA-TR 5'-GCCAAAAGACGGCAA). PCR was performed on the bacterial culture using TATGG-3'. Positive clones obtained from PCR were then confirmed by enzyme digestion. The correctly identified clones were named GPT000415-04-01-plasmid-Thy1.2-hSNCA-A30P / A53T-TG, GPT000415-04-01-plasmid-Thy1.2-hSNCA-A30P-TG, and GPT000415-04-01-plasmid-Thy1.2-hSNCA-A30P-TG, respectively. The plasmid spectra are as follows: mid-Thy1.2-hSNCA-A30P / E46K-TG, GPT000415-04-01-plasmid-Thy1.2-hSNCA-A53T-TG, GPT000415-04-01-plasmid-Thy1.2-hSNCA-E46K / A53T-TG, GPT000415-04-01-plasmid-Thy1.2-hSNCA-E46K-TG. Figures 1-6 As shown.

[0055] (3) After PC12 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) reached 70-80% confluence, transfection experiments were performed. Take 2 μg of each mutant plasmid prepared in step (2) and the transfection empty vector (GPT000367-Thy1-first carrying the flag tag and EGFP) and dissolve them in 125 μL of Opti-MEM medium. Take 5 μg of P3000 Reagent and dissolve it in Opti-MEM medium. After pipetting 3-5 times, let it stand at room temperature for 5 min. Take 5 μg of Lipofectamine 3000 Reagent and dissolve it in 125 μL of Opti-MEM medium. After pipetting 3-5 times, let it stand at room temperature for 5 min. Mix the two solutions and let them stand at room temperature for 15 min. PC12 cells were washed with PBS, 2 mL of 1640 complete medium was added, followed by 250 μL of transfection mixture. The mixture was shaken well and incubated at 37°C for 4-6 hours. The culture medium was then replaced with fresh 1640 medium, and the cells were cultured for a total transfection time of 24 hours. The transfection effect was observed under a fluorescence microscope. Figure 7 As shown in the figure. The results showed that green fluorescence was observed in PC12 cell lines (G3-G8) transfected with plasmids carrying different mutations under a fluorescence microscope, indicating that SNCA with different mutations was expressed in PC12 cells. Green fluorescence was also observed in PC12 cells transfected with empty vector (G2), while no green fluorescence was detected in PC12 cells (G1) that were not transfected.

[0056] After fluorescence imaging, the culture medium for PC12 cells was aspirated, and 1 mL of trypsin was added for digestion for 1 min. Digestion was then stopped by adding 1 mL of 1640 medium. The cells were gently pipetted off, centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in 1 mL of culture medium. 20 μL of the cell suspension was mixed with an equal volume of trypan blue solution, and cell counting and viability testing were performed. Results are as follows: Figure 8 As shown.

[0057] The results showed that the viability of PC12 cell lines transfected with plasmids carrying different mutation sites was significantly reduced (G3-G8). The viability of cells transfected with plasmids carrying a single mutation site (G3-G5) was higher than that of cells transfected with plasmids carrying three different mutation combinations (G6-G8). Among the three different mutation site combinations, the cell viability of the E46KA53T mutation combination was the lowest (G7), indicating that the E46KA53T mutation combination had the most significant impact on cell survival and was the most toxic. Therefore, E46KA53T was selected for further animal model construction.

[0058] Example 2: Construction of a mouse model simulating human Parkinson's disease

[0059] 1. Construction of Thy1-SNCA targeting vector

[0060] A schematic diagram of the Thy1-SNCA firing carrier is shown below. Figure 9 As shown.

[0061] (1) Using human SNCA CDS carrying the A53TE46K mutation as a template, the target fragment was amplified using the primers in Table 1 and then recovered for later use.

[0062] Table 1. List of primers for amplifying human SNCA-A53TE46K CDS fragment

[0063]

[0064] (2) GPT000415-hSNCA1, GPT000415-hSNCA2, and GPT000367-Thy1-first (a vector available from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were sliced ​​together, competent cells were transformed, plated, and incubated overnight at 37°C. Single colonies were picked from the plates and cultured. PCR identification of the bacterial culture was performed using the primers in Table 2. The results are as follows: Figure 10 As shown in Table 3. Positive clones obtained by PCR identification were then confirmed by enzyme digestion, and the results are shown in Table 3. Figure 11 As shown. The correctly identified clone was named GPT000415-01-Thy1.2-hSNCA-A53TE46K TG. The plasmid map is shown below. Figure 12 As shown.

[0065] Table 2. List of TG identification primers for GPT000415-01-Thy1.2-hSNCA-A53TE46K

[0066]

[0067] Table 3. List of GPT000415-01-Thy1.2-hSNCA-A53TE46K TG plasmids identified by enzyme digestion.

[0068]

[0069] (3) The correctly identified clone #2 was sequenced for confirmation. GPT000415-01-Thy1.2-hSNCA-A53TE46K TG was digested with AgeI enzyme, and the target fragment (7249bp) was recovered for injection and named GPT000415-Thy1-hSNCA-A53TE46K-inj-a (SEQ ID No:2).

[0070] (4) GPT000415-Thy1-hSNCA-A53TE46K-inj-a and Cas9 were combined to form an injection system, which was injected into the fertilized eggs of C57BL / 6J (B6J) mice at 0.5 days of age via microinjection. The embryos were then transferred into pseudopregnant female mice at 0.5 days of age. After birth, the tails of the mice were clipped to extract the genome, and Thy1-hSNCA-A53TE46K was identified by PCR. The results are as follows: Figure 13 As shown. Positive mice were selected through gene identification and named Thy-hSNCA-E46KA53T. A total of 27 Thy-hSNCA-E46KA53T positive mice (Founder mice) were obtained, with mouse numbers 5#, 7#, 12#, 14#, 15#, 18#, 24#, 28#, 30#, 34#, 36#, 37#, 40#, 41#, 50#, 55#, 65#, 67#, 71#, 75#, 81#, 91#, 94#, 96#, 99#, 103#, and 109#. These Founder mice were backcrossed with background mice (B6J) to obtain F1. After selection, the offspring of mouse 99#, F1 mouse #2, was selected for propagation, line establishment, and subsequent experimental verification (i.e., line 2, strain number T054322, abbreviated as B6-hSNCA E46KA53T).

[0071] Table 4. List of primers for PCR identification of Thy1-hSNCA gene

[0072]

[0073] Example 3: Pathological Detection of B6-hSNCA E46KA53T Mouse Model

[0074] 1. Detection of TH neurons in the brain region of B6-hSNCA E46KA53T mice

[0075] Brains were collected from B6-hSNCA E46KA53T and WT mice of different ages. After dehydration, embedding, and sectioning, immunohistochemical staining was performed to detect the loss of TH neurons in the mouse brain regions. The results are as follows: Figure 14 As shown in the figure. The results showed that the loss of TH-positive neurons could be detected in the substantia nigra region of the brain of 3-month-old B6-hSNCA E46KA53T mice, and the number of TH-positive neurons gradually decreased with increasing age.

[0076] 2. Behavioral testing of mice

[0077] To assess the limb strength and motor function of B6-hSNCA E46KA53T mice, the mice underwent a grip strength test and a fatigue rotating rod test to evaluate their motor function. The results are as follows: Figure 15 , 16As shown in the figure. The experimental results showed that, compared with littermate negative mice, 3-month-old B6-hSNCAE46KA53T mice had significantly reduced limb strength and drop time in the grip strength and fatigue rotating rod tests, indicating that B6-hSNCA E46KA53T mice had impaired motor function.

[0078] The method described in this invention constructs a vector with Thy1-hSNCA E46K and A53T mutations. Randomly inserting this vector into the mouse genome yields a stably inherited and spontaneously generated animal model of classic Parkinson's disease phenotype. B6-hSNCA E46KA53T mice constructed using this method show a reduction in TH-positive neurons at 3 months of age, which decreases with increasing age, exhibiting an age-dependent pattern, accompanied by an increase in α-Syn inclusion bodies pathologically. Furthermore, B6-hSNCAE46KA53T mice, despite the loss of TH-positive neurons, show decreased limb strength and motor function at 4 months of age. However, the phenotypes appear earlier than those in commonly used PD models, providing a broader experimental window for PD drug treatment and shortening the mouse waiting period. Therefore, the B6-hSNCA E46KA53T model can be used for PD-related gene function analysis, disease pathogenesis research, discovery of new drug targets, and preclinical efficacy evaluation of drugs.

Claims

1. A method for constructing an animal model simulating Parkinson's disease in humans, characterized by The animal model is obtained by using gene editing technology to randomly or positionally insert the human SNCA gene protein coding region carrying the mutation site into the animal genome or replace the protein coding region of the animal SNCA gene, so that the animal model expresses the alpha-Syn protein carrying the E46KA53T mutation.

2. The construction method of claim 1, wherein The gene editing technology includes one or more of homologous recombination, zinc finger nuclease technology, transcription activator-like effector nuclease technology, CRISPR / Cas9 technology, and single-base editing technology.

3. The construction method of claim 1, wherein The transgenic targeting vector is obtained by using gene editing technology, the targeting vector is injected into the animal zygote by microinjection, or the targeting vector is transfected into the animal ES cell by ES cell targeting technology, or the target gene is recombined into the retrovirus vector by retrovirus infection method, and the pre-implantation / post-implantation animal embryo is infected, or the transgenic targeting vector is introduced into the sperm by electroporation and combined with the egg.

4. The construction method of claim 3, wherein The method comprises the following steps: (1) designing a DNA homologous donor containing the human SNCA gene protein coding region carrying the mutation site; (2) injecting the DNA homologous donor prepared in step (1) into the animal zygote or transfecting it into the ES cell by microinjection, and randomly inserting the human SNCA gene protein coding region carrying the mutation site into the genome; or based on the CRISPR / Cas9 technology, preparing Cas9 or its expression vector, designing one sgRNA or a tracrRNA / crRNA binary complex or their respective expression vectors at the 5' end and 3' end of the protein coding region of the animal SNCA gene; or preparing a co-expression vector of Cas9, sgRNA or crRNA and tracrRNA; injecting the DNA homologous donor, Cas9, sgRNA or tracrRNA / crRNA binary complex, or their respective expression vectors or co-expression vectors into the animal zygote or transfecting them into the animal ES cell, knocking out the protein coding region of the animal SNCA gene while inserting the human SNCA gene protein coding region carrying the mutation site into the genome; (3) transplanting the zygote into a surrogate mother or injecting the targeting positive ES cell into the animal blastocyst and transplanting the blastocyst into a surrogate mother, breeding, and the offspring produced is the F0 generation animal, which is mated with the same background wild type animal after sexual maturation to obtain the F1 generation, which is an animal model simulating human Parkinson's disease.

5. The construction method of claim 4, wherein The human SNCA gene protein coding region sequence carrying the E46KA53T mutation site is shown as SEQ ID No:

1.

6. The method of construction of claim 5, wherein The homologous donor has a Thy1.2 promoter.

7. The construction method of claim 6, wherein The homologous donor is a Thy1 gene knockout plasmid, which retains the Thy1.2 promoter, and the coding regions of the original exons 2, 3 and 4 are replaced by the human SNCA gene protein coding region cDNA carrying the E46KA53T mutation site.

8. The construction method of claim 7, wherein The homologous donor carries a kozak sequence upstream of the human SNCA gene protein coding region cDNA with an E46K A53T mutation site.

9. The method of construction of claim 7, wherein The homologous donor sequence is shown as SEQ ID No:

2.

10. The construction method according to any one of claims 1-9, characterized in that The animal is a mammal.

11. The construction method of claim 10, wherein The animal is a rodent.

12. The method of construction of claim 11, wherein The animal is a rat or a mouse.

13. The method of construction of claim 12, wherein The animal is a C57BL / 6J, C57BL / 6N, BALB / c, NCG, FVB, C3H, DBA mouse.

14. The animal model simulating Parkinson's disease of human prepared by the construction method according to any one of claims 1-13 is used in drug screening and activity evaluation for treating Parkinson's disease.

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