Method for establishing NSP5 protein research model in caenorhabditis elegans neurons and application of NSP5 protein 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 NSP5 protein amino acid residue sequence coded by a novel coronavirus as shown in SEQ ID No. 1 to obtain a DNA sequence of an nsp5 gene as shown in SEQ ID No. 2; integrating the DNA sequence of the nsp5 gene into a nematode genome to obtain a nematode model; the optimized NSP5 expression sequence is integrated into the genome of the nematode at a fixed point, so that the blank of an existing NSP5 protein nematode research model is filled, the toxic effect and mechanism of the NSP5 protein in a nematode nervous system are researched, the action mechanism of the NSP5 protein on cells can be better researched, and the nematode nematode expression model has a good application prospect. Therefore, the research on the action mechanism of the compound in the human body and the related targeted drug screening work can be greatly promoted.

Description

Technical Field

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

[0002] In the scientific community, extensive research has been conducted on the replication and pathogenesis of SARS-CoV-2, and relevant vaccines and treatment methods have been developed. Due to the importance of the viral spike protein in virus infection, transmission, and vaccine development, the academic community has mainly focused on studying the structure, function, and evolution of the spike protein so far; however, other viral proteins also play a crucial role in virus replication, transmission, and the pathogenic process. For example: the accessory protein ORF3a of novel coronavirus can promote lysosome exocytosis by recruiting the BORC complex and SNARE proteins related to lysosome exocytosis, and then mediate virus release; the accessory protein ORF7a of novel coronavirus promotes autophagy by degrading SNAP29 and restricts the fusion of autophagosomes and lysosomes, thereby promoting virus replication. Therefore, constructing a protein model related to novel coronavirus lays a foundation for in-depth study of its potential mechanism of action in organisms and provides new targets for the treatment of related diseases caused by coronaviruses.

[0003] The SARS-CoV-2 genome contains 11 genes that can encode 14 open reading frames (ORFs), which can produce a total of 29 proteins, including 16 non-structural proteins (Non-Structure Protein, NSP1-NSP16), 4 structural proteins (Spike protein [S], Membrane protein [M], Nucleocapsidprotein [N], Envelope protein [E]) and 9 accessory proteins (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c and ORF10). Coronavirus protease NSP5 (also known as Mpro or 3CLpro) is a protease extremely important in the reproduction process of coronaviruses. It is responsible for the hydrolysis and processing of viral replicase polyproteins, that is, cleaving the protein precursors translated from the viral genome to obtain multiple non-structural proteins. NSP5 plays an essential and conserved role in coronaviruses and is a research hotspot in the field of human anti-coronavirus. Since the outbreak of SARS-CoV in 2003, scientists have been working hard to develop inhibitors against NSP5, and several compounds targeting the active site of NSP5 inhibition have been designed and tested, including esters, covalent or non-covalent peptide mimetics. NSP5 interacts with other viral proteins or host proteins to regulate viral replication and innate immune evasion, and these interactions may also affect the function of NSP5. Therefore, blocking these interactions can inhibit viral replication. For example, it has been reported that human MAGED2 can be cleaved by NSP5 protease, and abolishing the function of MAGED2 will increase the infection of SARS-CoV-2 to cells; NSP5 causes microvascular brain lesions by cleaving NEMO in brain endothelial cells. In addition, NSP5 can effectively cleave the selective autophagy receptor p62, and p62 specifically binds to the ubiquitinated SARS-CoV-2 M protein and promotes its autophagic degradation. In the presence of NSP5, the autophagic degradation of the M protein regulated by p62 is inhibited, thereby accelerating the potential strategy of the virus to evade autophagy to clear proteins. These studies have revealed that the NSP5 protein of the novel coronavirus has similar and unique pathological mechanisms in causing the high pathogenicity and transmission of the novel coronavirus. Therefore, establishing an in vivo animal research model for elucidating the pathogenic mechanism of the novel coronavirus NSP5 protein is of great significance for the development of therapeutic drugs and means.

[0004] Caenorhabditis elegans (hereinafter referred to as "nematode") is a non - pathogenic and independently viable worm. It feeds on bacteria and inhabits moist soil in nature. The nematode reaches adulthood 3 days after hatching from the embryo, and the stage from embryo to full adulthood can be easily divided into four stages (L1 - L4 stages). The nematode is a multicellular organism with a simple anatomical structure of tissues and organs, which has strong advantages for its use as a research model. In addition to the above characteristics, the nematode also has some features that strengthen its use as an animal model, such as small size (about 1 mm), convenient for operation, large number of offspring (about 300 offspring can be produced), low maintenance cost, etc. The simple nematode helps to understand complex cell signaling pathways in mammals, such as the process of neurodegeneration. The nematode has only 302 neurons, and most of the functional components of synaptic transmission in mammals are conserved. Combining the above advantages, the nematode is widely used in many research fields such as genetics, development, metabolism, neurodegeneration and aging. As the first multicellular organism to complete whole - genome sequencing, the nematode contains more than 60% of genes homologous to human disease genes. Therefore, the biochemical pathways of the nematode are similar to those of mammals, and basic biological mechanisms related to metabolism, nutrient sensing and fat storage in organisms can be studied. In addition, the nematode has perfect gene screening and gene manipulation tools, and reverse genetic techniques such as RNAi and CRISPR / Cas9 can be conveniently applied, or transgenic mutants can be constructed by microinjection, which accelerates the identification of potential targets. Summary of the Invention

[0005] The technical problem to be solved by the present invention is: to provide a method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons and its application.

[0006] To solve the above - mentioned technical problem, the technical solution adopted by the present invention is: A method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons, comprising: Optimizing the amino acid residue sequence of NSP5 protein encoded by the novel coronavirus shown in SEQ ID No. 1 to obtain the nsp5 DNA sequence of the gene shown in SEQ ID No. 2; Knocking the DNA sequence of the nsp5 gene into the genome of the nematode through the CRISPR / Cas9 gene editing system to obtain a nematode model.

[0007] Preferably, further optimizing the DNA sequence of the nsp5 gene shown in SEQ ID No. 2 includes: using a public online tool for nematode codon adaptation to optimize the nsp5 codons, and at the same time nsp5Three synthetic introns were inserted into the cDNA to obtain the DNA sequence of the gene shown in SEQ ID No. 2. nsp5 The DNA sequence of the gene.

[0008] Preferably, integrating the DNA sequence of the nsp5 gene into the genome of nematodes further includes: constructing a repair template plasmid, and the construction of the expression plasmid includes: Constructing a control group repair template plasmid: The control group nematode repair plasmid includes a left homology arm (Left Homology Arm, LHA), a fluorescent expression sequence to be inserted, and a right homology arm (Right Homology Arm, RHA). The fluorescent expression sequence includes a promoter for pan-neuronal expression in nematodes shown in SEQ ID No. 3, rab-3 a gfp expression sequence, and the 3' untranslated region of the unc-54 gene in nematodes, and finally is represented as shown in SEQ ID No. 4; Constructing a model group repair template plasmid: The model group nematode repair plasmid includes LHA, the nsp5 ::SL2 sequence to be inserted and RHA. Among them, the trans-splicing sequence SL2 shown in SEQ ID No. 5 is used to nsp5 form an operon with a green fluorescent protein coding sequence gfp and is finally represented as shown in SEQ ID No. 6.

[0009] Preferably, the first round of gene editing: Microinjecting the control group repair template plasmid into the gonads of nematodes to obtain control group nematodes; The second round of gene editing: Microinjecting the model group repair template plasmid into the gonads of the control group nematodes obtained in the first round to obtain model group nematodes.

[0010] Preferably, the microinjection in the first round of gene editing further includes: mixing the control group repair template plasmid, 40 ng / μL; CRISPR / Cas9 expression plasmid, 50 ng / μL; sgRNA expression plasmid, 25 ng / μL; red fluorescent reporter plasmid, 25 ng / μL; deionized water to form an injection system and microinjecting it into the gonads of nematodes to obtain control group nematodes with the P rab-3 :: gfp :: unc-54 3' UTR sequence inserted.

[0011] Preferably, the microinjection of the second round of gene editing further includes: the repair template plasmid of the model group, 40 ng / μL; the CRISPR / Cas9 expression plasmid, 50 ng / μL; the sgRNA expression plasmid, 25 ng / μL; the red fluorescent reporter plasmid, 25 ng / μL; after being configured with deionized water to form an injection system, it is microinjected into the gonads of the control group nematodes obtained in the first round to obtain the model group nematodes with the P rab-3 :: nsp5 ::SL2:: gfp :: unc-54 3’ UTR sequence inserted.

[0012] Preferably, the red fluorescent reporter plasmid is used to screen potential single-copy gene knock-in nematodes, and the target gene fragments of the control group and the model group are verified to be inserted into the nematode genome by PCR.

[0013] Preferably, wild-type male worms are backcrossed with hermaphrodite nematodes of the control group and the model group respectively, male worms are selected from the first-generation offspring and then hybridized with wild-type hermaphrodite nematodes, and then homozygous nematodes are screened from the fourth-generation offspring to complete two backcrosses; Repeat the above process again to complete four backcrosses.

[0014] Preferably, the wild-type nematode is Bristol N2.

[0015] To solve the above technical problems, another technical solution adopted by the present invention is: An application of the method for establishing an NSP5 protein research model in Caenorhabditis elegans neurons described in any one of the above, characterized in that the application is selected from at least one of the following applications: application in drug tests using the new coronavirus NSP5 protein nematode model as a carrier, application in low-throughput or high-throughput drug screening using the new coronavirus NSP5 protein nematode model as a carrier, and application in analyzing its neurotoxicity mechanism using the new coronavirus NSP5 protein nematode model as a carrier.

[0016] The beneficial effect of the present invention is that: the construction method of a nematode model expressing the new coronavirus NSP5 protein in neurons provided by the present invention uses the CRISPR / Cas9 gene editing system to knock in the optimized nsp5 expression sequence into the genome of nematodes in a site-directed manner by homologous recombination, which not only fills the blank of the existing NSP5 protein nematode research model, but also can better study the mechanism of action of NSP5 protein on cells by studying the toxic effects and mechanisms shown by NSP5 protein in the nematode nervous system, which will greatly promote the research on its mechanism of action in the human body and the screening work of related targeted drugs. Description of the Drawings

[0017] Figure 1Construction process of the NSP5 protein-related nematode model, which is a method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons in a specific embodiment of the present invention; Figure 2 Schematic diagram of homologous recombination of the control group and the model group in a method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons in a specific embodiment of the present invention; Figure 3 Plasmid map of the repaired template of the control group constructed in a method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons in a specific embodiment of the present invention; Figure 4 Plasmid map of the repaired template of the model group constructed in a method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons in a specific embodiment of the present invention; Figure 5 Green fluorescence images of nematodes in the model group (left) and the control group (right) in a method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons in a specific embodiment of the present invention; Figure 6 Comparison of body lengths of nematodes in the model group and the control group in a method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons in a specific embodiment of the present invention; Figure 7 Comparison of the number of body bends of nematodes in the model group and the control group in a method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons in a specific embodiment of the present invention; Figure 8 Comparison of the number of offspring of nematodes in the model group and the control group in a method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons in a specific embodiment of the present invention; Figure 9 Comparison of the paralysis rates of nematodes in the model group and the control group in a method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons in a specific embodiment of the present invention; Figure 10 Analysis of the enrichment of differential gene phenotypes of nematodes in the model group and the control group in a method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons in a specific embodiment of the present invention; Figure 11 Gene ontology analysis of nematodes in the model group and the control group in a method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons in a specific embodiment of the present invention. Specific embodiments

[0018] To illustrate the technical content, achieved objectives and effects of the present invention in detail, the following is described in conjunction with the embodiments and accompanied by drawings.

[0019] The following is combined with specific examples and appendicesFigures 1-11 , for a further detailed description of the present invention, the protection scope 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 conceived by those skilled in the art are included in the present invention, and the scope of protection is defined by the appended claims. The processes, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the specifically mentioned content below, are all common knowledge and common sense in this research field. The present invention has no special restrictive content, such as as described in "Molecular Cloning: A Laboratory Manual (Fourth Edition)" by M.R. Green and J. Sambrook, WormBook, or according to the recommended conditions provided by the product supplier. Example 1

[0020] A method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons, comprising: Step 1, nsp5 Nucleic acid sequence optimization In the present invention, the amino acid residue sequence of NSP5 protein is from the virus strain isolated from Wuhan-Hu-1, and its accession number in NCBI-Protein is: YP_009725301. Through analysis by an online bioinformatics tool, it is found that the original nsp5 nucleic acid sequence of the novel coronavirus (GenBank: NC_045512.2) has poor transcription and translation effects in C. elegans. Therefore, the present invention first needs to nsp5 optimize the codon sequence encoding it, that is, perform synonymous mutations on the codons encoding the amino acid residues of NSP5 protein by using the degeneracy of amino acid codons, and improve nsp5 the mRNA stability and the overall expression level. The present invention uses the public online tool for C. elegans codon adaptation (C.elegans Codon adapter, https: / / worm.mpi-cbg.de / codons / cgi-bin / optimize.py) to nsp5 optimize the codons, and at the same time insert three synthetic introns (represented by lowercase letters) into the nsp5 cDNA as shown in SEQ ID No. 2, which can further enhance nsp5 the transcription and translation. The optimized nucleic acid coding sequence is analyzed by a bioinformatics tool, indicating that it can significantly improve nsp5 the expression level of the nucleic acid sequence and does not change its amino acid residue sequence as shown in SEQ ID No. 1.

[0021] Step 2, constructing a repair template plasmid The present invention uses the CRISPR / Cas9 gene editing system to integrate the target fragment into the nematode genome using the homologous recombination method. In addition, the present invention uses a two-round editing method, that is, the first round first constructs the control group nematodes, and the second round uses the control group nematodes obtained in the first round of editing as the background to construct the model group nematodes. In order to construct the repair template plasmid of the control group, the pan-neuronal expression of rab-3 The promoter sequence is as shown in SEQ ID No. 3, gfp Expression sequence, unc-54 The 3'UTR sequence and the left and right recombination arm sequences (LHA-1 and RHA-1) were connected to the pSL1190 backbone plasmid. The above sequences were verified to be completely correct by sequencing. The final complete repair template plasmid sequence is shown in SEQ ID No. 4; the plasmid map is shown in Figure 3 shown. gfp The GFP protein finally expressed by the expression sequence is green fluorescent protein.

[0022] In order to observe the transcriptional expression of NSP5 protein and its damage to neurons in the model group nematodes, when constructing the expression plasmid of the model group, the optimized intron-containing plasmid was synthesized from Beijing Qingke Biotechnology Co., Ltd. nsp5 The full gene is shown in SEQ ID No. 2, the product name is NSP5(opt) plasmid, and the product order number is XM0026027-2. From the synthesized NSP5(opt) plasmid, nsp5 The full gene was then fused to SL2 ( gpd-2 / gpd-3 The intergenic sequence between the two) is shown in SEQ ID No. 5 to form a new fragment nsp5 ::SL2 and insert it into rab-3 Promoter and gfp between nsp5 Green fluorescent protein coding sequence gfp An operon was formed, and then the left and right recombination arm sequences (LHA-2 and RHA-2) were connected to the pSL1190 backbone plasmid using the Gibson assembly method. The above sequence was verified to be completely correct by sequencing. The final complete template group repair template plasmid is shown in SEQ ID No.6, and the plasmid map is shown in Figure 4 shown.

[0023] Step 3: C. elegans gene editing The CRISPR / Cas9 gene editing system was used to insert the target fragment P into the control group and the model group by homologous recombination. rab-3 :: gfp :: unc-54 3'UTR and Prab-3 :: nsp5 ::SL2:: gfp :: unc-54 The 3’ UTR was respectively integrated into the position between 8420158..8420158 on chromosome II of C. elegans.

[0024] In the first round of gene editing, the constructed repair template plasmid was mixed with the reporter plasmid, CRISPR / Cas9 expression plasmid and sgRNA expression plasmid to prepare a nematode injection mixture, which was injected into the gonads of young adult wild nematodes through a microinjection platform. The control repair template plasmid was 40 ng / μL; the CRISPR / Cas9 expression plasmid was 50 ng / μL; the sgRNA expression plasmid was 25 ng / μL; the red fluorescent reporter plasmid was 25 ng / μL; deionized water was used to prepare an injection system and microinjected into the gonads of nematodes to obtain control nematodes. The selected reporter plasmid can express red fluorescent protein, which is beneficial to screening potential gene knock-in nematodes. The ribonucleoprotein complex formed by the products of the CRISPR / Cas9 expression plasmid and the sgRNA expression plasmid is mainly responsible for cleaving the nematode genome to initiate intracellular DNA repair. The repair template plasmid provides a homologous repair template during the DNA repair process.

[0025] Preferably, the microinjection in the second round of gene editing further includes: the model group repair template plasmid was 40 ng / μL; the CRISPR / Cas9 expression plasmid was 50 ng / μL; the sgRNA expression plasmid was 25 ng / μL; the red fluorescent reporter plasmid was 25 ng / μL; deionized water was used to prepare an injection system and microinjected into the gonads of the control nematodes obtained in the first round to obtain model group nematodes. The selected reporter plasmid can express red fluorescent protein, which is beneficial to screening potential integrated nematodes. The ribonucleoprotein complex formed by the products of the CRISPR / Cas9 expression plasmid and the sgRNA expression plasmid is mainly responsible for cleaving the nematode genome to initiate intracellular DNA repair. The repair template plasmid provides a homologous repair template during the DNA repair process.

[0026] Step 4. Screen gene knock-in edited nematodes Pick the first-generation nematodes expressing red fluorescence and culture these nematodes individually on NGM nematode culture dishes with Escherichia coli OP50. After culturing for three to four days, a large number of second-generation nematode populations were propagated in each culture dish. Randomly pick second-generation nematodes and lyse them for PCR verification to clarify whether these nematode strains have successfully undergone homologous recombination integration.

[0027] Step 5. Backcross CRISPR / Cas9 gene editing may cause off-target editing of genes other than the target site, resulting in other unknown mutations. To exclude the effects of these unknown mutations on the phenotype of C. elegans and directly exhibit the true transgenic phenotype of C. elegans, the usual practice is to backcross with wild-type C. elegans of Bristol N2. Wild-type male worms were used to backcross with hermaphrodite worms in the control group and the model group respectively, and male worms were selected from the first-generation offspring and backcrossed with wild-type hermaphrodite worms again. Then, homozygous worms were screened in the fourth-generation offspring, completing two backcrosses. Repeating the above process again completes four backcrosses.

[0028] Step 6. Phenotype analysis The nervous system function of C. elegans has a wide range of effects on many aspects such as its growth and development. To verify that the NSP5 protein expressed in the nervous system of C. elegans has strong neurotoxicity, a series of phenotype evaluation tests were conducted on the C. elegans obtained by backcrossing the model group and the control group, including developmental ability, locomotor ability, reproductive ability, and nerve function. The effect of the nervous system of C. elegans on developmental ability is shown by comparing body lengths as Figure 6 shown, the effect of the nervous system of C. elegans on locomotor ability is shown by comparing the number of body bends as Figure 7 shown, the effect of the nervous system of C. elegans on reproductive ability is shown by the number of offspring as Figure 8 shown, and the paralysis rate caused by the nervous system of C. elegans is shown as Figure 9 shown. The results showed significant differences between the two groups, indicating that the expression of NSP5 protein in the pan-neurons of C. elegans can cause significant neurotoxicity, and also proving that the nematode nervous system model of the SARS-CoV-2 NSP5 protein was successfully constructed in this invention.

[0029] Explanation of potential mechanism To further verify the potential mechanism of the effect of NSP5 protein on the neurotoxicity of C. elegans, transcriptome sequencing was performed on the model group and the control group respectively, and the phenotype enrichment analysis (Phenotype Enrichment Analysis) of the differentially expressed genes in the two groups was carried out using the enrichment analysis tool in the C. elegans database (https: / / wormbase.org / tools / enrichment / tea / tea.cgi). The related phenotypes involved in the genes affected by the NSP5 viral protein were shown as: nematode phenotype, larval physiology phenotype, dauer metabolism phenotype, and increased fat content, etc., multiple phenotypes related to the physiological metabolism of C. elegans as Figure 10As shown below. 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 related to Molecular Function (MF), Biological Process (BP), and Cellular Component (CC), so as to better understand how the changes in gene expression patterns caused by the expression of NSP5 in nematode neurons affect the function and structure of organisms, and how these changes are associated with disease states. The results of the GO analysis are shown as Figure 11 below. For example, among molecular functions, the differentially expressed genes mainly involve functions such as phosphoprotein phosphatase activity, protein serine / threonine kinase activity, structural constituent of cuticle, and non-membrane spanning protein tyrosine kinase activity; among biological processes, they mainly involve processes such as phosphorus metabolic process, response to stimulus, and proteolysis; among cellular components, they mainly cover the cell membrane and cytoskeleton, etc. The above analysis results indicate that the expression of NSP5 protein in neurons has a wide range of potential impact mechanisms on the physiological and metabolic functions and cell structure of nematodes. Example 2

[0030] Application in a drug test using a nematode model of SARS-CoV-2 NSP5 protein as a vector, including: First, prepare potential candidate drugs, which may affect the nematode model of the model group of nematodes obtained in Example 1, that is, the NSP5 protein nematode model, according to different mechanisms of action. In the drug test, it is divided into an experimental group and a control group. The NSP5 protein nematode model is exposed to the experimental group containing the drug, and the control group is not treated or exposed to an inactive solvent. After a period of time, by evaluating the differences in related indicators such as the motility, fecundity, and body size of the NSP5 protein nematode model in the experimental group and the control group, it is finally confirmed whether the potential drug has an impact on the neurotoxicity of NSP5 protein to nematodes. Example 3

[0031] An application in low-throughput or high-throughput drug screening using a nematode model of the SARS-CoV-2 NSP5 protein as a carrier, including: First, determine the research objective and the scope of drug screening, and prepare the corresponding drug library. These drugs may affect the nematode model of the model group obtained in Example 1, that is, the NSP5 protein nematode model, according to different mechanisms of action. For low-throughput screening: Select the NSP5 protein nematode model and expose it to a small number of representative drugs for individual testing; for high-throughput screening: Use automated equipment and software, and use the microplate technology. Place different drugs in each well, and use an automated sorting device to expose the NSP5 protein nematode model to different drug micro-wells for rapid screening of a large number of drugs or compounds. 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 differences in the locomotor ability indexes of the NSP5 protein nematode model in the experimental group and the control group, finally confirm whether the potential drugs screened by the throughput have an impact on the neurotoxicity of the NSP5 protein to nematodes. Example 4

[0032] An application in analyzing the neurotoxicity mechanism using a nematode model of the SARS-CoV-2 NSP5 protein as a carrier, including: First, perform forward genetic screening (such as EMS mutagenesis) or reverse genetic screening (such as RNAi, CRISPR / Cas9 gene editing) on the nematode model of the model group obtained in Example 1, that is, the NSP5 protein nematode model. Then select the nematode strains with significantly different phenotypes (compared with the NSP5 protein nematode model) obtained by screening. Through subsequent analysis methods, such as transcriptome sequencing, proteomics and other technologies, analyze the changes in the gene expression profile and protein expression profile in the nematodes, and then combine bioinformatics analysis to predict and verify the targets and signaling pathways related to neurotoxicity.

[0033] SEQ ID No.1 MSGFRKMAFPSGKVEGCMVQVTCGTTTLNGLWLDDVVYCPRHVICTSEDMLNPNYEDLLIRKSNHNFLVQAGNVQLRVIGHSMQNCVLKLKVDTANPKTPKYKFVRIQPGQTFSVLACYNGSPSGVYQCAMRPNFTIKGSFLNGSCGSVGFNIDYDCVSFCYMHHMELPTGVHAGTDLEGNFYGPFVDRQTAQAAGTDTTITVNVLAWLYAAVINGDRWFLNRFTTTLNDFNLVAMKYNYEPLTQDHVDILGPLSAQTGIAVLDMCASLKELLQNGMNGRTILGSALLEDEFTPFDVVRQCSGVTFQ SEQ ID No. 2 SEQ ID No. 3 SEQ ID No. 4 SEQ ID No. 5 ggatcccgctgtctcatcctactttcacctagttaactgcttgtcttaaaatctatgcttctctttagtatctaaaattttcctagaagcttacaagtatataaatggtctcttctcaataaaggttgtatatttattcatcttattgaatctgccatttcctcgtttttgcgagtttatataccttccaattttctttctattgtattttcaacttctaattttaattcagggaaactgcttcaacgcatc SEQ ID No. 6 The above are only embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent transformation made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in related technical fields, shall be similarly included in the patent protection scope of the present invention.

Claims

1. A method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons, characterized in that, Including: The amino acid residue sequence of the NSP5 protein encoded by the novel coronavirus as shown in SEQ ID No. 1 was optimized to obtain the nsp5 DNA sequence of the gene; Express nsp5 The DNA sequence of the gene was knocked into the genome of C. elegans through the CRISPR / Cas9 gene editing system to obtain a C. elegans model.

2. The method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons according to claim 1, wherein Optimizing to obtain the nsp5 DNA sequence of the gene shown in SEQ ID No. 2 further includes: using a common online tool for nematode codon adaptation to nsp5 optimize the codons, and at the same time nsp5 inserting three synthetic introns into the nsp5 cDNA to obtain the DNA sequence of the gene shown in SEQ ID No.

2.

3. The method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons according to claim 2, wherein Expressing nsp5 Knocking the DNA sequence of the gene into the genome of the nematode further includes: constructing a repair template plasmid, and the constructed repair plasmid includes: Construct a control group repair template plasmid: The control group nematode repair plasmid includes LHA, the fluorescence expression sequence to be inserted, and RHA. The fluorescence expression sequence includes the rab-3 promoter, gfp sequence, and the 3' non-coding region of the unc-54 gene in nematodes, and finally is represented as shown in SEQ ID No. 4; Construct a model group to repair the template plasmid: The model group repair plasmid includes LHA, the nsp5 ::SL2 expression sequence and RHA, in which the trans-splicing sequence SL2 shown in SEQ ID No. 5 is used to nsp5 form an operon with a green fluorescent protein coding sequence gfp and finally represented as shown in SEQ ID No.

6.

4. The method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons according to claim 3, wherein The first round of gene editing: Inject the repair template plasmid of the control group into the gonads of wild-type nematodes to obtain control nematodes with the P rab-3 :: gfp :: unc-54 3’ UTR sequence inserted; Second-round gene editing: Inject the repair template plasmid of the model group into the gonads of the control nematodes obtained from the first-round editing to obtain model group nematodes with the P rab-3 :: nsp5 ::SL2:: gfp :: unc-54 3’ UTR sequence inserted.

5. The method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons according to claim 4, wherein The microinjection for the first-round gene editing further includes: forming an injection system by mixing a control group repair template plasmid at 40 ng / μL, a CRISPR / Cas9 expression plasmid at 50 ng / μL, an sgRNA expression plasmid at 25 ng / μL, a red fluorescent reporter plasmid at 25 ng / μL, and deionized water, and performing microinjection into the gonads of nematodes to obtain control group nematodes.

6. The method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons according to claim 4, characterized in that, The microinjection for the second-round gene editing further includes: forming an injection system by mixing a model group repair template plasmid at 40 ng / μL, a CRISPR / Cas9 expression plasmid at 50 ng / μL, an sgRNA expression plasmid at 25 ng / μL, a red fluorescent reporter plasmid at 25 ng / μL, and deionized water, and performing microinjection into the gonads of some of the control group nematodes obtained in the first round to obtain model group nematodes.

7. The method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons according to claim 4, wherein Use the red fluorescent reporter plasmid to screen potential single-copy inserted nematodes, and verify by PCR that the target gene fragments of the control group and the model group are integrated into the nematode genome.

8. The method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons according to claim 7, characterized in that, Use wild-type male worms to backcross with control group and model group hermaphrodite nematodes respectively. Pick male worms in the first filial generation and backcross them with wild-type hermaphrodite nematodes again. Then screen for homozygous control group and model group nematodes in the fourth filial generation to complete two backcrosses; Repeat the above process again to complete four backcrosses.

9. The method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons according to claim 8, wherein The wild-type nematode is Bristol N2.

10. Use of a method for establishing a research model of NSP5 protein in Caenorhabditis elegans neurons as described in any one of claims 1-9, characterized in that, The application is selected from at least one of the following applications: application in drug tests using the nematode model of the SARS-CoV-2 NSP5 protein as a carrier, application in low-throughput or high-throughput drug screening using the nematode model of the SARS-CoV-2 NSP5 protein as a carrier, and application in analyzing its neurotoxicity mechanism using the nematode model of the SARS-CoV-2 NSP5 protein as a carrier.

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