Method for establishing new coronavirus research model in nematode neurons and application of new coronavirus research model

Abstract

The invention relates to the technical field of biology, in particular to a method for establishing a novel coronavirus research model in nematode neurons and application of the novel coronavirus research model in nematode neurons, and the method comprises the following steps: optimizing an ORF3a protein amino acid residue sequence coded by a novel coronavirus as shown in SEQ ID No. 1 to obtain a DNA sequence of an orf3a gene as shown in SEQ ID No. 2; integrating the DNA sequence of the orf3a gene into a nematode genome to obtain a nematode model; the optimized ORF3a expression plasmid is randomly integrated into the genome of the nematode, the blank of the existing ORF3a protein nematode research model is filled, the neurotoxic effect and mechanism of the ORF3a protein in the nematode nervous system are researched, the action mechanism of the ORF3a protein on cells is better researched, and the nematode nematode expression model has a good application prospect. The research on the action mechanism of the compound in the human body and the related targeted drug screening work are promoted.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to a method for establishing a novel coronavirus research model in nematode neurons and its application. Background Art

[0002] The novel coronavirus (COVID-19) is a single-stranded RNA virus that has caused millions of deaths worldwide. To curb the spread of COVID-19 and control the pneumonia epidemic caused by COVID-19, scientific research has focused on understanding the mechanisms of viral infection and transmission. For example, ACE2 protein, the main receptor of COVID-19, and humanized ACE2 transgenic mice can be used to explore the mechanism of COVID-19 infection in vivo. In contrast, little is known about the pathological mechanisms induced by specific proteins encoded by COVID-19. The high mortality rate of pneumonia caused by COVID-19 indicates that several proteins encoded by COVID-19 are unusually toxic. After the immune system clears COVID-19, or the virus can no longer be detected in the body, infected people may still suffer from post-viral syndrome with obvious symptoms, such as neuropsychiatric symptoms and extreme fatigue that last for up to several months. With the use of vaccines, pneumonia caused by COVID-19 infection has begun to be effectively controlled. Therefore, in order to effectively mitigate a series of harmful effects after infection, it will become very important to study the pathogenesis of the body caused by COVID-19 proteins.

[0003] The SARS-CoV-2 genome contains 11 genes encoding 14 open reading frames (ORFs), which can produce a total of 29 proteins, including 16 non-structural proteins (NSP1-NSP16), 4 structural proteins (Spike protein [S], Membrane protein [M], Nucleocapsidprotein [N], Envelope protein [E]), and 9 auxiliary proteins (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c, and ORF10). The characteristics of the coronavirus-specific ORF3a protein were analyzed in the SARS coronavirus (SARS-CoV) that caused severe acute respiratory syndrome (SARS) in 2003. Studies have shown that the ORF3a protein of SARS-CoV can trigger pathogenic inflammatory responses by interacting with tumor necrosis factor receptor-interacting factor 3, ultimately leading to cell death and apoptosis, causing severe lung damage. In addition, the ORF3a protein of SARS-CoV can also induce vesicle formation and Golgi fragmentation. The ORF3a protein of the new coronavirus may have similar pathogenicity. In vitro studies have shown that the new coronavirus ORF3a can induce caspase-dependent apoptosis, and bioinformatics analysis shows that the mutation rate of ORF3a is positively correlated with the mortality rate of pneumonia caused by new coronavirus infection. In addition, the new coronavirus ORF3a protein accelerates viral replication by inhibiting the host cell autophagy mechanism. Although the ORF3a of the new coronavirus and SARS-CoV ORF3a are highly consistent, the latter does not have the effect of promoting viral replication. These studies have shown that the new coronavirus ORF3a has its own unique pathological mechanism in causing the high mortality rate of pneumonia caused by new coronavirus infection. Therefore, establishing an in vivo research model for explaining the pathogenic mechanism of the new coronavirus ORF3a is of great significance for the development of therapeutic drugs and means for pneumonia caused by new coronavirus infection.

[0004] As a small, simple worm, Caenorhabditis elegans (hereinafter referred to as "nematodes") has been widely recognized as an excellent model species in biomedical research. Compared with mammalian models, nematodes show unique advantages in studying the mechanisms of human diseases. First, based on bioinformatics analysis, it was found that 60-80% of human genes can find their corresponding homologous genes in nematodes. Many genetic factors associated with human diseases also have corresponding homologs in nematodes, which is conducive to mechanism-based scientific research. Second, the tiny size and rapid life cycle of nematodes greatly simplify the culture process in the laboratory. Adult nematodes are about 1 mm long and take only 3 days to develop from embryos to reproductive adults. On a plate with a diameter of 6 cm, nematodes can produce hundreds of offspring in a few days through self-pollination, which is particularly important for drug screening. Third, the transparent nature of nematodes allows a variety of fluorescent reporter proteins, such as green and red fluorescent proteins, to be used in real time in living nematodes to observe biological processes such as axon guidance, neurodegeneration, endocytosis and fat metabolism. Fourth, nematodes have a simple and clear neuronal lineage, which is convenient for conducting neurobiological functional research. Although nematodes have only 302 neurons, they have neurotransmitters similar to those of humans, including dopamine, acetylcholine, serotonin, γ-aminobutyric acid, and glutamate. Based on the functional division of neural circuits, nematodes contain four types of neurons: motor neurons, sensory neurons, interneurons, and multimodal neurons. In addition, many models of human neurodegenerative diseases have been developed using the nervous system of nematodes as a carrier, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Finally, the field of nematode research has a rich variety of genetic manipulation tools, such as RNAi libraries, CRISPR / Cas9 systems, and various mutants and transgenic strains preserved in the Nematode Genetic Center, which further expands its application scenarios as a model species. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for establishing a new coronavirus research model in nematode neurons and its application.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: A method for establishing a novel coronavirus research model in nematode neurons, comprising: The amino acid residue sequence of the ORF3a protein encoded by the novel coronavirus as shown in SEQ ID No. 1 was optimized to obtain the amino acid residue sequence as shown in SEQ ID No. 2 orf3a The DNA sequence of a gene; Will orf3a The DNA sequence of the gene is integrated into the genome of the nematode to obtain the nematode model.

[0007] Preferably, the optimization is performed to obtain the sequence shown in SEQ ID No. 2 orf3a The DNA sequence of the gene further includes: using the public nematode codon adaptation online tool orf3a Codon optimization was performed and orf3a Three synthetic introns were inserted into the cDNA to obtain the sequence shown in SEQ ID No. 2 orf3a The DNA sequence of a gene.

[0008] Preferably, orf3a The step of integrating the DNA sequence of the gene into the genome of the nematode further comprises: constructing an expression plasmid, wherein the expression plasmid comprises: The pan-neuron of nematodes was selected as the tissue for constructing the model, and the pan-neuron of nematodes shown in SEQ ID No. 3 was inserted into the rab-3 The promoter drives the expression of ORF3a protein; Construction of a plasmid for expression in a nematode model: using the trans-splicing sequence SL2 as shown in SEQ ID No. 4, and combining it with a green fluorescent protein coding sequence gfp Form an operon; the 3' non-coding region uses the UNC-54 The 3' non-coding region of the gene is finally expressed as shown in SEQ ID No. 5: rab-3 :: orf3a ::SL2:: gfp :: UNC-54 3′UTR; Construction of a plasmid expressed in control nematodes: GFP was expressed using the rab-3 promoter for pan-neuronal expression in nematodes, and the final expression was as shown in SEQ ID No. 6: rab-3 :: gfp :: UNC-54 3' UTR.

[0009] Preferably, the plasmid expressed in the nematode model and the plasmid expressed in the control nematode are injected into the gonads of wild-type nematodes, respectively.

[0010] Preferably, a pan-neuronal rab-3 Promoter expression gfp Fluorescence was used as a reporter gene to screen free-living transgenic nematodes in the model group and the control group.

[0011] Preferably, after irradiating the model group and control episomal transgenic nematodes with X-rays, the model group and control integrated transgenic nematodes with multiple copies of the plasmid integrated into the chromosome are obtained.

[0012] Preferably, the nematodes in the model group and the control group are obtained by backcrossing the model group and the control integrated transgenic nematodes with the wild-type background nematodes for multiple times, and the nematodes in the model group are selected as the nematode model.

[0013] Preferably, phenotypic and mechanistic analyses are performed on the nematode model.

[0014] Preferably, the dose of X-ray irradiation is 50 Gy.

[0015] In order to solve the above technical problems, another technical solution adopted by the present invention is: An application of the above-mentioned method for establishing a new coronavirus research model in nematode neurons, wherein the application is selected from at least one of the following applications: application in drug trials using the new coronavirus ORF3a protein nematode model as a carrier, application in low-throughput or high-throughput drug screening using the new coronavirus ORF3a protein nematode model as a carrier, and application using the new coronavirus ORF3a protein nematode model as a carrier to analyze its neurotoxicity mechanism.

[0016] The beneficial effect of the present invention is that a method for constructing a nematode model expressing the new coronavirus ORF3a protein in neurons provided by the present invention is to randomly integrate the optimized ORF3a expression plasmid into the nematode genome by microinjection and X-ray irradiation, which not only fills the gap in the existing ORF3a protein nematode research model, but also through studying the neurotoxic effects and mechanisms of ORF3a protein in the nematode nervous system, it can better study its mechanism of action on cells, which will greatly promote the study of its mechanism of action in the human body and related targeted drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A construction process of an ORF3a protein-related nematode model of a method for establishing a novel coronavirus research model in nematode neurons according to a specific embodiment of the present invention; Figure 2 An expression plasmid map of a model group constructed for a method of establishing a novel coronavirus research model in nematode neurons according to a specific embodiment of the present invention; Figure 3 An expression plasmid map of a control group constructed for a method of establishing a novel coronavirus research model in nematode neurons according to a specific embodiment of the present invention; Figure 4 Green fluorescence images of nematodes in the model group (left) and control group (right) of a method for establishing a novel coronavirus research model in nematode neurons according to a specific embodiment of the present invention; Figure 5 Comparison of the body length of nematodes in the model group and the control group of a method for establishing a novel coronavirus research model in nematode neurons according to a specific embodiment of the present invention; Figure 6 A comparison of the number of nematode body bends between a model group and a control group of a method for establishing a novel coronavirus research model in nematode neurons according to a specific embodiment of the present invention; Figure 7 A comparison of the number of nematode progeny between the model group and the control group of a method for establishing a novel coronavirus research model in nematode neurons according to a specific embodiment of the present invention; Figure 8 The paralysis mortality rate of nematodes in the model group and the control group of a method for establishing a novel coronavirus research model in nematode neurons according to a specific embodiment of the present invention; Fig. 9 This is a method for establishing a novel coronavirus research model in nematode neurons according to a specific embodiment of the present invention, in which differential gene phenotype enrichment analysis of nematodes in a model group and a control group is performed; Fig.10 This is a method for establishing a new coronavirus research model in nematode neurons according to a specific embodiment of the present invention, in which gene ontology analysis of nematodes in the model group and the control group is performed. DETAILED DESCRIPTION

[0018] In order to explain the technical content, achieved objectives and effects of the present invention in detail, the following is an explanation in combination with the implementation modes and the accompanying drawings.

[0019] The following is combined with specific embodiments and appendix Figure 1-10 , the present invention is further described in detail, and the protection content of the present invention is not limited to the following embodiments. Without departing from the spirit and scope of the inventive concept, the changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims are the scope of protection. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are common knowledge and common sense in this research field. The present invention has no special restrictions, such as according to MR Green, J. Sambrook's "Molecular Cloning Laboratory Guide (Fourth Edition)", WormBook, or according to the recommended conditions provided by the product supplier. Embodiment 1

[0020] A method for establishing a novel coronavirus research model in nematode neurons, comprising: Step 1: orf3a gene sequence optimization The amino acid residue sequence of the ORF3a protein in the present invention is from the virus strain isolated from Wuhan-Hu-1, and its accession number in NCBI-Gene is: 43740569. Through online bioinformatics tool analysis, it was found that the original orf3a The gene sequence (GenBank: NC_045512) has poor transcription and translation effects in nematodes. Therefore, the present invention firstly needs to orf3a The coding codon sequence was optimized, that is, the codons encoding the amino acid residues of ORF3a protein were subjected to synonymous mutations by utilizing the degeneracy of amino acid codons, and the orf3a The present invention utilizes the public online tool for nematode codon adaptation ( C. elegans Codon adapter, https: / / worm.mpi-cbg.de / codons / cgi-bin / optimize.py) Yes orf3a Codon optimization was performed and orf3a Insertion of three synthetic introns (indicated by lowercase letters) into the cDNA as shown in SEQ ID No. 2 can further enhance orf3a The optimized nucleic acid coding sequence was analyzed by bioinformatics tools and showed that it could significantly improve orf3a The gene sequence expression level is unchanged, and its amino acid residue sequence is as shown in SEQ ID No. 1.

[0021] Step 2: Construction of expression plasmid In order to construct the expression plasmid of the model group, the optimized intron-containing plasmid was synthesized from General Biotechnology (Anhui) Co., Ltd. orf3a The full gene is shown in SEQ ID No. 2, the product name is ORF3a(opt) plasmid, and the product order number is G0215286-1. From the synthesized ORF3a(opt) plasmid, orf3a The whole gene was assembled and combined with the pan-neuronal expression rab-3 The promoter sequence is shown in SEQ ID No. 3, and the SL2 sequence (i.e. gpd -2 / gpd -3 intergenic sequence, connecting ORF3a with a green fluorescent protein coding sequence gfp The operon was formed) as shown in SEQ ID No. 4 and cloned into the pPD95.77 backbone plasmid commonly used in nematodes. The 3' non-coding region used the UNC-54 The 3' non-coding region of the gene, the backbone plasmid already contains a universal gfp Gene sequences and UNC-54 3'UTR sequence. After the above fragments were connected and cloned, the sequence was verified to be completely correct by sequencing. The final complete expression plasmid sequence is shown in SEQ ID No. 5: rab-3 :: orf3a ::SL2:: gfp :: UNC-54 3'UTR, plasmid map as Figure 2 shown.

[0022] To construct the expression plasmid for the control group, the pan-neuronal expression plasmid was assembled by Gibson assembly. rab-3 The promoter sequence is shown in SEQ ID No. 3 and cloned into the same pPD95.77 backbone plasmid as above. The sequence is verified to be completely correct by sequencing. The final complete expression plasmid sequence is shown in SEQ ID No. 6: rab-3 :: gfp :: UNC-54 3'UTR, plasmid map as Figure 3 shown.

[0023] Step 3. Nematode microinjection The constructed plasmids were mixed with the pSL1190 cloning vector to prepare a mixed solution for nematode injection. The microinjection systems were as follows: the model group was P rab-3 :: orf3a ::SL2:: gfp :: UNC-54 3'UTR, 20 ng / μL; pSL1190, 25 ng / μL; prepared with deionized water. The control group was P rab-3 :: gfp :: UNC-54 3'UTR, 20 ng / μL; pSL1190, 25 ng / μL; prepared with deionized water. Injected into the gonads of young adults of wild-type nematodes via a microinjection platform. The pSL1190 cloning vector in the injection mixture of the model group was used as a supplementary plasmid to reduce orf3a The concentration of the expression plasmid was reduced, the expression level of ORF3a was reduced, and thus its toxicity was reduced, and the probability of obtaining free transgenic nematodes was increased. The concentrations of all components of the injection mixture in the model group were the optimal configuration concentrations for obtaining free transgenic nematodes. Accordingly, as the control group, except for the difference in the expression plasmid components in the injection mixture, other conditions were consistent with the model group. The wild-type nematode strain Bristol N2, which is widely used in laboratories, was selected as the background strain. This is also the wild background strain currently used in the construction of neurodegenerative disease models using nematodes as model organisms.

[0024] Step 4: Screening of free-type transgenic nematodes The free-type transgenic nematode strains were screened by using the GFP fluorescence expressed in pan-neurons of nematodes as a reporter marker. The criterion for selecting the free-type transgenic nematode strains was that two-thirds of the nematodes on the plate had green fluorescence (refer to Figure 4Model group (left) and control group (right) in the figure). Since the passage of plasmid array minichromosomes in free-type transgenic nematodes is non-Mendelian, the plasmid array minichromosomes will be lost as the nematodes continue to pass. Therefore, when culturing free-type nematodes, it is necessary to continuously enrich the transgenic nematodes expressing green fluorescence to maintain a certain initial number.

[0025] Step 5: X-ray irradiation and screening of integrated transgenic nematodes In order to obtain a stable genetic nematode that expresses ORF3a protein in neuronal cells, it is necessary to integrate the plasmid array minichromosome into the nematode genome. A certain dose of X-ray (e.g., 50 Gy, but not limited to 50 Gy, which can be adjusted according to actual needs) can randomly interrupt the chromosomes. In the subsequent gene repair process, the exogenous plasmid array minichromosome can be randomly integrated into the nematode genome. orf3a The nematodes expressing the plasmid array minichromosomes were irradiated with X-rays, and the integrated model group transgenic nematodes were screened in their offspring. Correspondingly, the integrated transgenic nematodes of the control group, that is, the nematode strain that only expressed GFP protein in neurons, were also obtained by the same way and method. The transgenic nematodes of the model group and the control group obtained by X-ray irradiation were multi-copy.

[0026] Step 6: Backcross Four rounds of backcrossing are required to eliminate the unknown genetic background; in addition to integrating exogenous DNA, gene repair after X-ray irradiation may also produce other unknown mutations elsewhere in the genome. In order to eliminate the effects of these unknown mutations on the phenotype of nematodes and allow the nematodes to directly express the true transgenic phenotype, the usual practice is to backcross with the background nematodes before irradiation. In the model group, wild-type Bristol N2 males were backcrossed with the model group hermaphroditic nematodes, and homozygous nematodes were screened in the F2 generation. This was repeated for four rounds of backcrossing. In the control group, the control group hermaphroditic nematodes were backcrossed with wild-type Bristol N2 males, and homozygous nematodes were screened in the F2 generation. This was repeated for four rounds of backcrossing. Confocal imaging was performed on the final model group nematodes (the nematodes in the model group were selected as the nematode model) and the control group nematodes as shown in the figure. Figure 4 As shown, there are significant differences in the axon structure of neurons in the model group nematodes and those in the control group nematodes.

[0027] Step 7: Phenotypic analysis The function of the nervous system of nematodes has a wide range of effects on their growth and development. In order to verify the neurotoxicity of ORF3a protein expressed in the nervous system of nematodes, a series of phenotypic evaluation tests were conducted on the nematodes obtained by backcrossing the model group and the control group, including developmental ability, motor ability, reproductive ability and neural function. Figure 5 As shown in the figure, the impact on athletic ability is as follows: Figure 6 As shown in the figure, the effect on reproductive capacity is shown in the figure below. Figure 7 As shown in Figure 2, the paralysis-related mortality rate caused by ORF3a neurotoxicity is Figure 8 As shown, the results showed that there were significant differences between the two groups, indicating that the expression of ORF3a protein in nematode pan-neurons can cause significant neurotoxicity. It also proves that the present invention successfully constructed a nematode neurotoxicity research model of SARS-CoV-2 ORF3a.

[0028] Potential mechanism description In order to further verify the potential mechanism of ORF3a protein's effect on nematode neurotoxicity, transcriptome sequencing analysis was performed on the model group and control group nematodes, and the enrichment analysis tool in the nematode database (https: / / wormbase.org / tools / enrichment / tea / tea.cgi) was used to perform phenotypic enrichment analysis on differentially expressed genes in neurons. Figure 8 The genes affected by the expression of ORF3a viral protein are shown to be involved in the following phenotypes: movement variant, paralyzed, body morphology variant, and muscle system morphology variant. In addition, the online gene enrichment analysis tool g:Profiler (https: / / biit.cs.ut.ee / gprofiler / gost) was used to conduct Gene Ontology (GO) analysis based on molecular function (MF), specific biological process (BP), and cellular component (CC), so as to better understand how changes in gene expression patterns caused by the expression of ORF3a in nematode neurons affect the function and structure of nematodes, and how these changes are associated with disease states. The GO analysis results show that Fig.10As shown in the figure, the differentially expressed genes in MF mainly involve functions such as the structural constituent of cuticle; in BP, they mainly involve processes such as transmembrane transport; and in CC, they mainly cover striated muscle thin filaments. This indicates that the expression of ORF3a protein in neurons causes neurotoxicity and movement disorders in nematodes by affecting the structure of neuronal cells, the transport of neurotransmitters and other substances. Embodiment 2

[0029] An application in a drug trial using a nematode model of the novel coronavirus ORF3a protein as a carrier, comprising: First, a potential candidate drug is prepared, which may have an effect on the nematode model of the model group nematodes obtained in Example 1, that is, the ORF3a protein nematode model according to different mechanisms of action. The drug test is divided into a test group and a control group, and the ORF3a protein nematode model is exposed to a test group containing a drug, and a control group that is not treated or exposed to an inactive solvent. After a period of time, by evaluating the differences in relevant indicators such as the motor ability, fertility, and body shape of the ORF3a protein nematode model in the test group and the control group, it is finally confirmed whether the potential drug has an effect on the neurotoxicity of the ORF3a protein to nematodes. Embodiment 3

[0030] An application of a nematode model of novel coronavirus ORF3a protein as a carrier in low-throughput or high-throughput drug screening, comprising: First, the purpose and scope of drug screening are studied, and the corresponding drug library is prepared. These drugs may have an effect on the nematode model of the model group nematodes obtained in Example 1, that is, the ORF3a protein nematode model, according to different mechanisms of action. For low-throughput screening: the ORF3a protein nematode model is selected to be exposed to a small amount of representative drugs for testing one by one; for high-throughput screening: using automated equipment and software, using microplate technology, different drugs are placed in each well, and the ORF3a protein nematode model is exposed to different drug microwells using automated sorting equipment, and a large number of drugs or compounds are quickly screened. All low-throughput and high-throughput screenings are set up with a control group that is not treated or exposed to an inactive solvent. After a period of time, by evaluating the difference in the motor ability indicators of the ORF3a protein nematode model in the test group and the control group, it is finally confirmed whether the potential drugs in the flux screening have an effect on the neurotoxicity of the ORF3a protein to nematodes. Embodiment 4

[0031] An application of using the nematode model of the novel coronavirus ORF3a protein as a carrier to analyze its neurotoxicity mechanism, including: First, the nematode model of the model group nematodes obtained in Example 1, that is, the ORF3a protein nematode model, is subjected to forward genetic screening (e.g., EMS mutagenesis) or reverse genetic screening (e.g., RNAi, CRISPR / Cas9 gene editing). Then, the nematode strains with significant phenotype differences (compared with the ORF3a protein nematode model) are screened. Subsequent analysis methods, such as transcriptome sequencing, proteomics and other technologies are used to analyze the changes in gene expression profiles and protein expression profiles in nematodes, and then combined with bioinformatics analysis to predict and verify targets and signaling pathways related to neurotoxicity.

[0032] SEQ ID No .1 MDLFMRIFTIGTVTLKQGEIKDATPSDFVRATATIPIQASLPFGWLIVGVALLAVFQSASKIITLKKRWQLALSKGVHFVCNLLLLFVTVYSHLLLVAAGLEAPFLYLYALVYFLQSINFVRIIMRLWLCWKCRSKNP LLYDANYFLCWHTNCYDYCIPYNSVTSSIVITSGDGTTTSPISEHDYQIGGYTEKWESGVKDCVVLHSYFTSDYYQLYSTQLSTDTGVEHVTFFIYNKIVDEPEEHVQIHTIDGSSGVVNPVMEPIYDEPTTTTSVPL- SEQ ID No. 2 ATGGATCTTTTCATGCGCATCTTCACTATCGGAACCGTTACTCTTAAGCAAGGAGAAATCAAGGATGCTACCCCATCCGATTTCGTTCGTGCTACTGCTACCATCCCAATCCAAGCTTCTCTTCCATTCGGATGGCTTATCGTTGGAGTCGCCCTTCTTGCTGTTTTCCAATCTGCTTCTAAGATCATCACTCTTAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagAAGCGTTGGCAACTTGCTCTTTCCAAGGGAGTTCACTTCGTTTGCAACCTTCTTCTTCTTTTCGTTACCGTTTACTCCCACCTTCTTCTTGTCGCTGCTGGACTTGAGGCCCCATTCCTTTACCTTTACGCCCTTGTTTACTTCCTTCAATCTATCAACTTCGTTCGCATCATCATGCGTCTTTGGCTTTGCTGGAAGTGCCGCTCTAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagAACCCACTTCTTTACGACGCTAACTACTTCCTTTGCTGGCATACTAACTGCTACGATTACTGCATTCCATACAACTCTGTTACCTCTTCTATCGTTATCACCTCTGGAGATGGAACCACCTCTCCAATCTCTGAACATGATTACCAGATCGGAGGATACACTGAAAAGTGGGAGTCTGGAGTTAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagGATTGCGTTGTTCTTCACTCTTACTTCACCTCTGATTACTACCAACTTTACTCTACCCAACTTTCCACCGATACCGGAGTTGAGCACGTTACTTTCTTCATTTACAACAAGATTGTTGATGAACCAGAAGAGCACGTTCAAATCCACACTATCGATGGATCCTCCGGAGTTGTTAACCCAGTTATGGAACCAATCTACGATGAGCCAACTACCACCACCTCTGTCCCACTTTAA SEQ ID No .3 SEQ ID No .4 ggatcccgctgtctcatcctactttcacctagttaactgcttgtcttaaaatctatgcttctctttagtatctaaaattttcctagaagcttacaagtatataaatggtctcttctcaataaaggttgtatatttattcatcttattgaatctgccatttcctcgtttttgcgagtttatataccttccaattttctttctattgtattttcaacttctaattttaattcagggaaactgcttcaacgcatc SEQ ID No .5 SEQ ID No .6 The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention's specification and drawings, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for establishing a novel coronavirus research model in nematode neurons, characterized in that: include: The amino acid residue sequence of the ORF3a protein encoded by the novel coronavirus as shown in SEQ ID No. 1 was optimized to obtain the amino acid residue sequence as shown in SEQ ID No. 2 orf3a The DNA sequence of a gene; Will orf3a The DNA sequence of the gene is integrated into the genome of the nematode to obtain the nematode model.

2. The method for establishing a novel coronavirus research model in nematode neurons according to claim 1, characterized in that: Optimize to obtain the SEQ ID No. 2 orf3a The DNA sequence of the gene further includes: using the public nematode codon adaptation online tool orf3a Codon optimization was performed and orf3a Three synthetic introns were inserted into the cDNA to obtain the sequence shown in SEQ ID No. 2 orf3a The DNA sequence of a gene.

3. The method for establishing a novel coronavirus research model in nematode neurons according to claim 2, characterized in that: Will orf3a The step of integrating the DNA sequence of the gene into the genome of the nematode further comprises: constructing an expression plasmid, wherein the expression plasmid comprises: The pan-neuron of nematodes was selected as the tissue organ for constructing the model, and the pan-neuron of nematodes shown in SEQ ID No. 3 was inserted into the rab-3 The promoter initiates the expression of ORF3a protein; Construction of a plasmid for expression in a nematode model: using the trans-splicing sequence SL2 as shown in SEQ ID No. 4, and combining it with a green fluorescent protein coding sequence gfp Form an operon; the 3' non-coding region uses the UNC-54 The 3' non-coding region of the gene is finally expressed as shown in SEQ ID No. 5: rab-3 :: orf3a ::SL2:: gfp :: UNC-54 3′UTR; Construction of a plasmid expressed in control nematodes: GFP was expressed using the rab-3 promoter for pan-neuronal expression in nematodes, and the final expression was as shown in SEQ ID No. 6: rab-3 :: gfp :: UNC-54 3' UTR.

4. The method for establishing a novel coronavirus research model in nematode neurons according to claim 3, characterized in that: The plasmid expressed in the nematode model and the plasmid expressed in the control nematode were injected into the gonads of wild-type nematodes, respectively.

5. The method for establishing a novel coronavirus research model in nematode neurons according to claim 4, characterized in that: Using Pan Neuron rab-3 Promoter expression gfp The reporter gene was used to screen the free-type transgenic nematodes in the model group and the control group.

6. The method for establishing a novel coronavirus research model in nematode neurons according to claim 5, characterized in that: After irradiating the model group and the control episomal transgenic nematodes with X-rays, the model group and the control integrated transgenic nematodes with multiple copies of the plasmid integrated into the chromosome were obtained.

7. The method for establishing a novel coronavirus research model in nematode neurons according to claim 6, characterized in that: The model group and control group nematodes were obtained by multiple backcrossing of the model group and control integrated transgenic nematodes with the wild-type background nematodes, and the nematodes in the model group were selected as the nematode model.

8. The method for establishing a novel coronavirus research model in nematode neurons according to claim 7, characterized in that: Perform phenotypic and mechanistic analyses on the nematode model.

9. The method for establishing a novel coronavirus research model in nematode neurons according to claim 6, characterized in that: The dose of X-ray irradiation was 50 Gy.

10. An application of the method for establishing a novel coronavirus research model in nematode neurons as described in any one of claims 1 to 9, characterized in that: The application is selected from at least one of the following applications: application in drug trials using the nematode model of the new coronavirus ORF3a protein as a carrier, application in low-throughput or high-throughput drug screening using the nematode model of the new coronavirus ORF3a protein as a carrier, and application in analyzing the neurotoxicity mechanism of the nematode model of the new coronavirus ORF3a protein as a carrier.

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