A pAPN protein mutant and application thereof

Abstract

The application provides a pAPN protein mutant, a substance for mutating pAPN protein, and application thereof; the pAPN protein mutant is based on wild-type pAPN protein, wherein the asparagine at the 783th position is mutated into alanine, and other amino acid residues are unchanged; the application screens and verifies that the N783A mutation of the pAPN protein can significantly reduce cell damage and cell death caused by TGEV infection, and can significantly inhibit the infection or replication of TGEV in cells, and can be applied to the construction of a stable TGEV-resistant pig-derived cell model, and then applied to TGEV receptor mechanism research, antiviral drug screening and anti-swine transmissible gastroenteritis virus pig breeding research and the like.

Description

Technical Field

[0001] This invention relates to a pAPN protein mutant and its applications; it belongs to the field of genetic engineering technology. Background Technology

[0002] Transmissible gastroenteritis (TGE) is an acute, highly contagious intestinal infectious disease caused by the transmissible gastroenteritis virus (TGEV). TGEV belongs to the order Nidovirales, family Coronaviridae, and genus Alphacoronavirus; it is an enveloped, single-stranded, positive-sense RNA virus. Pigs of all breeds and ages are susceptible to TGEV, but suckling piglets under two weeks of age are most susceptible, with a mortality rate that can reach 100%. TGEV virus entry into host cells typically relies on the recognition and binding of its S protein to cell surface receptors. Porcine aminopeptidase N (pAPN, also known as ANPEP) is one of the important receptors for TGEV infection of porcine cells.

[0003] Porcine aminopeptidase N (pAPN) is widely present on the surface of small intestinal epithelial cells and has a wide range of biological functions.

[0004] Existing technologies have reported that some pAPN mutants do not affect the basic physiological functions of pAPN but can significantly inhibit TGEV infection. Based on this, researchers in this field have attempted to modify key binding sites of the viral receptor without significantly disrupting the structure and basic physiological functions of the receptor protein (pAPN) itself, in order to obtain porcine cells or breeding candidates with reduced viral susceptibility. For example, precise mutation screening targeting the pAPN-TGEV S protein interaction interface. Existing technologies have reported on this, with Chinese patents (e.g., CN202310706300) and international patents (e.g., US20250049947) disclosing specific pAPN mutants (e.g., mutations at amino acids 734 or 727) that can resist TGEV infection while preserving its normal physiological functions as much as possible.

[0005] In addition, existing technologies also disclose the use of Prime editing to achieve site-specific modifications at the single-base or codon level at endogenous sites in porcine cells.

[0006] However, further research is needed to verify the resistance of pAPN-mutated cells to TGEV infection.

[0007] There is an urgent need in this field for a fully verifiable technical solution to discover and verify the key pAPN sites that affect TGEV infection, obtain precise mutants of the key pAPN gene sites, achieve resistance to TGEV infection, and be applied to the construction of stable TGEV-resistant porcine cell models and the breeding of pigs resistant to porcine transmissible gastroenteritis virus. Summary of the Invention

[0008] To address the aforementioned technical problems, the first aspect of this invention provides a pAPN protein mutant, wherein: the pAPN protein mutant is based on the wild-type pAPN protein, with asparagine at position 783 mutated to alanine, while other amino acid residues remain unchanged; the amino acid sequence of the wild-type pAPN protein is shown in SEQ ID NO: 2.

[0009] A second aspect of the present invention provides a nucleic acid molecule encoding the above-mentioned pAPN protein mutant.

[0010] A third aspect of the present invention provides an expression cassette containing the aforementioned nucleic acid molecules.

[0011] A fourth aspect of the present invention provides a recombinant vector containing the above-mentioned nucleic acid molecules.

[0012] The fifth aspect of the present invention provides recombinant cells containing the above-mentioned nucleic acid molecules.

[0013] The sixth aspect of the present invention provides a substance that mutates the 783rd amino acid residue of the wild-type pAPN protein, wherein the substance is a Prime editing plasmid system that edits the 783rd codon of pAPN from AAC to GCT; the amino acid sequence of the wild-type pAPN protein is shown in SEQ ID NO: 2; The plasmid system includes a pegRNA targeting the pAPN N783 site and an ngRNA that assists in editing. The pegRNA sequence is shown in SEQ ID NO:3; the ngRNA sequence for editing assistance is shown in SEQ ID NO:6.

[0014] The seventh aspect of this invention provides the use of a substance that mutates amino acid residue 783 of the wild-type pAPN protein in any of the following: 1) Construct cell lines for site-directed modification of the pAPN gene for non-diagnostic and non-therapeutic purposes; 2) Preparation of cells resistant to porcine transmissible gastroenteritis virus; 3) Prepare pigs resistant to porcine transmissible gastroenteritis virus; 4) Breeding pigs resistant to transmissible gastroenteritis virus (TGEV); 5) Prepare products for the prevention or treatment of transmissible gastroenteritis in pigs; The wild-type pAPN protein has a mutation at amino acid residue 783, which replaces asparagine with alanine; the wild-type pAPN protein is the protein encoded by SEQ ID NO: 2.

[0015] In one specific embodiment of the present invention, the substance that causes a mutation at amino acid residue 783 of the wild-type pAPN protein is the substance provided in the sixth aspect above.

[0016] The eighth aspect of this invention provides a method for constructing a pAPN protein mutant cell model or a porcine transmissible gastroenteritis virus (TGEV) resistant cell model for non-diagnostic and therapeutic purposes. The method involves mutating asparagine at position 783 of the wild-type pAPN protein in the target cells to alanine, while keeping other amino acid residues unchanged, to obtain the pAPN protein mutant cells. These pAPN protein mutant cells also constitute the porcine TGEV resistant cell model. The amino acid sequence of the wild-type pAPN protein is shown in SEQ ID NO: 2. The substance provided in the sixth aspect above is used to mutate asparagine at position 783 of the wild-type pAPN protein in the target cell to alanine.

[0017] The ninth aspect of this invention provides a method for constructing pAPN protein mutant gene-edited pigs for non-diagnostic and therapeutic purposes, wherein, 1) Mutate the asparagine at position 783 of the pAPN protein in isolated porcine fibroblasts to alanine, while keeping other amino acid residues unchanged, to obtain pAPN protein mutant cells; 2) The pAPN protein mutant cells are used as nuclear transfer donor cells and are transplanted into sows via somatic cell nuclear transfer. The offspring produced are pAPN protein mutant gene-edited pigs. The amino acid sequence of the wild-type pAPN protein is shown in SEQ ID NO: 2; The substance provided in the sixth aspect above is used to mutate asparagine at position 783 of the wild-type pAPN protein in the target cell to alanine.

[0018] Starting from the interaction interface between the TGEV S protein receptor-binding region and pAPN, the inventors of this application screened and verified the pAPN N783A receptor mutation site, which can resist TGEV infection. This invention uses prime editing to achieve precise site-specific editing of the endogenous pAPN site, avoiding the interference of traditional random mutation or exogenous overexpression models on receptor expression levels and cellular background. More importantly, the inventors of this application verified that the N783A mutation can significantly reduce cell damage and cell death caused by TGEV infection, and can also significantly inhibit TGEV infection or replication in cells. It can be applied to construct stable TGEV-resistant porcine cell models, and further applied to TGEV receptor mechanism research, antiviral drug screening, and porcine transmissible gastroenteritis virus (TGEV) resistant pig breeding research. Attached Figure Description

[0019] Figure 1 A schematic diagram showing the predicted structure and interaction sites of the TGEV S protein receptor-binding region and the pAPN complex; Figure 2 The results of morphological observation of PE7-PK15 and N783A PE7-PK15 cells after infection with TGEV at different MOIs; Figure 3-5 The results show the CCK-8 cell viability assays for TGEV-infected PE7-PK15 and N783A PE7-PK15 cells at 24 h, 36 h, and 48 h, respectively. Figure 6 Plots showing the LDH release levels in PE7-PK15 and N783A PE7-PK15 cells 24 h after infection with TGEV at different MOIs; Figure 7 The image shows the fluorescence staining results of dead cells of N783A PE7-PK15 infected with TGEV at different MOIs 24 h later. Figure 8 qPCR images of TGEV N gene RNA levels in PE7-PK15 and N783A PE7-PK15 cells 24 h after infection with TGEV at different MOIs; Figure 9 A graph showing the sequencing results used to evaluate the construction of the N783A PE7-PK15 monoclonal cell line; Figure 10 The results of Western blot analysis were used to evaluate APN protein expression in the N783 PE7-PK15 monoclonal cell line. Detailed Implementation

[0020] The following provides some definitions that can be used to explain the “Implementation Scheme” section below.

[0021] As used herein, “about,” “approximately,” and “substantially” should be understood to refer to a number within a certain numerical range, such as -10% to +10% of the mentioned number, preferably -5% to +5% of the mentioned number, more preferably -1% to +1% of the mentioned number, and most preferably -0.1% to +0.1% of the mentioned number.

[0022] Furthermore, all numerical ranges herein should be understood to include all integers or fractions within that range (including endpoint values). Additionally, these numerical ranges should be understood to support claims that involve any number or subset of numbers within that range. For example, disclosures of 1 to 10 should be understood to support ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0023] As used herein and in the appended claims, the singular form of a word includes the plural form unless the context clearly specifies otherwise. Therefore, references to “an,” “a,” and “the” generally include the plural form of the corresponding term. For example, references to “component” or “method” include references to multiple such components or methods. The term “and / or” used in the context of “X and / or Y” should be interpreted as “X” or “Y” or “X and Y.” Similarly, “at least one of X or Y” should be interpreted as “X” or “Y” or “both X and Y.”

[0024] Similarly, the terms "including / comprises / contains" are used to interpret them inclusively rather than exclusively. Likewise, the terms "including / comprises / contains" and "or" should be considered inclusive unless the context explicitly prohibits this interpretation. However, the embodiments provided in this disclosure may not contain any elements not expressly disclosed herein. Therefore, a disclosure of one embodiment defined by the term "including / comprises / contains" is also a disclosure of multiple embodiments that are "substantially composed of the disclosed components" and "composed of the disclosed components."

[0025] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0026] The term "example / implementation" used herein is for illustrative purposes only and should not be considered exclusive or comprehensive. Any implementation disclosed herein may be combined with any other implementation disclosed herein unless expressly indicated otherwise.

[0027] Implementation Plan Example 1: Obtaining and predicting the sequence and structure of the TGEV S protein receptor-binding region and pAPN. The TGEV S protein receptor-binding region gene sequence was amplified in vitro and then subjected to next-generation sequencing to obtain a complete and accurate TGEV S protein receptor-binding region sequence (SEQ ID NO:1): LPSFYTHTIVNITIGLGMKRSGYGQPIASTLSNITLPMQDHNTDVYCIRSDQFSVYVHSTCKSALWDNIFKRNCTDVLDATAVIKTGTCPFSFDKLNNYLTFNKFCFSLSPVGANCKFDVAARTRTNEQVVRSLYVIYEEGDNIVGVPSD.

[0028] The pAPN gene cDNA sequence of porcine PK15 cells was amplified in vitro and subjected to next-generation sequencing to obtain a complete and error-free pAPN gene cDNA sequence; specifically, the amino acid sequence of porcine pAPN protein (SEQ ID NO:2): MAKGFYISKALGILGILLGVAAVATIIALSVVYAQEKNKNAEHVPQAPTSPTITTTAAITLDQSKPWNRYRLPTTLLPDSYNVTLRPYLTPNADGLYIFKGKSIVRFICQESTDVIIIHSKKLNYTTQGHMVVLRGVGDSQVPEIDRTELVELTEYLVVHLKGSLQPGHMYEMESEFQGELADDLAGFYRSEYMEGNVKKVLATTQMQSTDARKSFPCFDEPAMKATFNITLIHPNNLTALSNMPPKGSSTPLAEDPNWSVTEFETTPVMSTYLLAYIVSEFQSVNETAQNGVLIRIWARPNAIAEGHGMYALNVTGPILNFFANHYNTSYPLPKSDQIALPDFNAGAMENWGLVTYRENALLFDPQSSSISNKERVVTVIAHELAHQWFGNLVTLAWWNDLWLNEGFASYVEYLGADHAEPTWNLKDLIVPGDVYRVMAVDALASSHPLTTPAEEVNTPAQISEMFDSISYSKGASVIRMLSNFLTEDLFKEGLASYLHAFAYQNTTYLDLWEHLQKAVDAQTSIRLPDTVRAIMDRWTLQMGFPVITVDTKTGNISQKHFLLDSESNVTRSSAFDYLWIVPISSIKNGVMQDHYWLRDVSQAQNDLFKTASDDWVLLNVNVTGYFQVNYDEDNWRMIQHQLQTNLSVIPVINRAQVIYDSFNLATAHMVPVTLALDNTLFLNGEKEYMPWQAALSSLSYFSLMFDRSEVYGPMKKYLRKQVEPLFQHFETLTKNWTERPENLMDQYSEINAISTACSNGLPQCENLAKTLFDQWMSDPENNPIHPNLRSTIYCNAIAQGGQDQWDFAWGQLQQAQLVNEADKLRSALACSNEVWLLNRYLGYTLNPDLIRKQDATSTINSIASNVIGQPLAWDFVQSNWKKLFQDYGGGSFSFSNLIQGVTRRFSSEFELQQLEQFKKNNMDVGFGSGTRALEQALEKTKANIKWVKENKEVVLNWFIEHS。

[0029] The TGEV S-RBD and pAPN sequences were input into AlphaFold3 for complex conformation prediction. The prediction results showed that the ipTM value of the TGEV S-RBD / pAPN protein complex was 0.46 and the pTM value was 0.8, indicating that the complex conformation prediction results had good reliability.

[0030] Further HDOCK docking analysis was performed on the TGEV S-RBD and pAPN protein data. The results showed that the HDOCK docking score was -260.33, which is lower than -200, suggesting a high probability of interaction between TGEV S-RBD and pAPN.

[0031] Example 2: Identification of TGEV S-RBD-pAPN Interaction Sites Structural interaction analysis was performed on the TGEV S-RBD-pAPN complex to screen for potential key amino acid sites with close spatial distances between TGEV S-RBD and pAPN. The results of the interaction analysis are shown in Table 1 below.

[0032] Table 1

[0033] See Figure 1 Schematic diagram of the predicted structure and interaction sites of the TGEV S-RBD-pAPN complex, where TGEV S-RBD is represented in green, pAPN in blue, and Trp571 and Asn783 are represented by rod-shaped models.

[0034] from Figure 1 As can be seen, Trp571 of TGEV S-RBD is located near pAPN Asn783, with a distance of about 3.30 Å between them, suggesting that pAPN Asn783 may be involved in the interaction between TGEV S-RBD and pAPN, and may be an important amino acid site affecting their binding.

[0035] Example 3: Screening for candidate mutation sites using FoldX alanine scanning Using the FoldX alanine scanning method, alanine-by-alanine mutations were performed on the amino acids related to the pAPN protein-protein interaction interface. The changes in free energy of the TGEV S-RBD-pAPN complex and the pAPN receptor itself before and after the mutations were calculated. Considering both the spatial interaction between the mutation site and TGEV S-RBD and the impact of the mutation on the structural stability of pAPN, candidate mutation sites that might affect viral receptor binding without significantly disrupting pAPN structural stability were screened. The screening results are shown in Table 2 below.

[0036] Table 2

[0037] Based on the combined protein-protein interaction interface analysis and FoldX alanine scanning results, Asn783 is likely an important candidate site involved in the binding of TGEV S-RBD to pAPN. The N783A mutation is not expected to significantly disrupt the structural stability of pAPN itself; therefore, pAPN N783A is identified as a candidate mutation site for subsequent cellular-level functional validation.

[0038] Example 4: Construction of N783A PE7-PK15 cells using Prime editing In a single-clonal cell line (PE7-PK15 cells) stably transfecting PE7 protein in PK15 cells, a Prime editing plasmid system was designed and constructed to edit codon 783 of pAPN from AAC to GCT.

[0039] The system includes a pegRNA targeting the pAPN N783 site and an ngRNA that assists in editing.

[0040] The pegRNA sequence of the N783A mutant plasmid (SEQ ID NO: 3): 5'-GGAACAAAGGACACACTCACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAAATGCTCCGTGAGTGTGTCCTTTGCGGTAGAACGCGGTTCTATCTAGTTACGCGTTAAAACCAACTAGAA-3'.

[0041] The pegRNA spacer and PAM sequence (SEQ ID NO:4) is: 5'-GGAACAAAGGACACACTCACGGG-3'; The pegRNA RT-PBS sequence (SEQ ID NO:5) is: 5'-AAATGCTCCGTGAGTGTGTCCTTTG-3'; The helper ngRNA contains the following sequence (SEQ ID NO:6): 5′-ATTGCCTCAATGTGAGAATCTGG-3′.

[0042] The above plasmid system was introduced into PE7-PK15 cells. After culture, screening and sequencing, heterozygous cells carrying the pAPNN783A mutation (mutation mode AAC→GCT, corresponding to the mutation of asparagine to alanine at position 783 of the pAPN protein) were obtained and named N783A PE7-PK15.

[0043] See Figure 9 Sequencing results used to evaluate the construction of the N783A PE7-PK15 monoclonal cell line. Figure 9 The data shows two peaks here, GCT and AAC, indicating that the mutation was successful and the mutation is heterozygous.

[0044] See Figure 10 Western blot analysis of APN protein expression in the N783 PE7-PK15 monoclonal cell line showed that APN protein bands could be detected in both cell groups, and the overall APN protein expression levels were similar. The internal control bands were also basically consistent, suggesting that the N783A mutation does not significantly affect the expression level of APN protein.

[0045] Example 5: Validation of TGEV challenge in N783A PE7-PK15 cells (1) Cell viability detection PE7-PK15 cells (control group) and N783A PE7-PK15 cells (experimental group) were simultaneously plated, and the cell confluence was approximately 70% after 24 h. Subsequently, they were challenged with TGEV, and the MOI (Multiplicity of Infection) was set to 0, 0.01, 0.1, and 1.

[0046] After virus adsorption for 1 hour, the cells were washed and replaced with fresh culture medium. Cell morphology was observed and CCK-8 assay was performed at 24 hpi, 36 hpi, and 48 hpi.

[0047] See Figure 2 Cell morphology observation results of PE7-PK15 and N783A PE7-PK15 cells at different time points after infection with TGEV at different MOIs; See Figure 3-5 The results show the CCK-8 cell viability assays for TGEV-infected PE7-PK15 and N783A PE7-PK15 cells at 24 h, 36 h, and 48 h, respectively; regarding... Figure 3-5 The explanation of the vertical axis “Cellular activity of 24 hpi, 36 hpi, 48 hpi (% of control)” is as follows: the relative cell viability of the experimental group at different MOIs at the time points of 24h, 36h, and 48h, respectively, and normalized with the cell viability of the corresponding uninfected group (MOI=0) as 100%.

[0048] from Figure 2The results showed that with prolonged infection time and increased MOI, PE7-PK15 cells exhibited significant cell damage and shedding, while N783A PE7-PK15 cells maintained good cell morphology and survival status overall. Figure 3-5 In the study, the overall viability of PE7-PK15 cells decreased with prolonged TGEV infection time and increased MOI; in contrast, N783APE7-PK15 cells maintained high cell viability at multiple MOIs and time points. The difference in cell viability between the two groups was particularly pronounced under conditions of higher MOI and longer infection time.

[0049] The above results indicate that the N783A mutation can alleviate the decline in cell viability caused by TGEV infection and enable cells to maintain a better survival status after TGEV infection, suggesting that the N783A mutation can enhance the tolerance of PK15 cells to TGEV infection.

[0050] (2) Detection of cell damage and death PE7-PK15 cells (control group) and N783A PE7-PK15 cells (experimental group) were simultaneously plated, and the cell confluence was approximately 70% after 24 h. Subsequently, they were challenged with TGEV, with MOIs set to 0, 0.01, 0.1, and 1.

[0051] After virus adsorption for 1 h, the cells were washed and replaced with fresh culture medium. At 24 hpi, the supernatant was collected to detect the release level of lactate dehydrogenase (LDH) to evaluate the degree of cell membrane damage. At the same time, dead cells were stained with green fluorescent dye to observe cell death after TGEV infection.

[0052] See Figure 6 The graph shows the LDH release levels of PE7-PK15 and N783A PE7-PK15 cells 24 h after infection with TGEV at different MOIs; regarding Figure 6 The ordinate LDH release of 24hpi (% of control) is interpreted as follows: the relative degree of cell membrane damage caused by different MOIs in the experimental groups 24 hours after infection, normalized to 100% with the corresponding LDH release level of the uninfected group (MOI=0).

[0053] See Figure 7 The image shows the fluorescence staining results of dead cells of N783A PE7-PK15 cells infected with TGEV at different MOIs for 24 hours; among them, PE7-PK15 cells carry red fluorescence, and green fluorescence represents dead cells.

[0054] from Figure 6As can be seen, with the increase of TGEV infection dose, the LDH release level of PE7-PK15 cells increased significantly, while the LDH release level of N783A PE7-PK15 cells was relatively low, especially at MOI=0.1 and 1, indicating that N783A mutation can alleviate cell damage caused by TGEV infection.

[0055] from Figure 7 As can be seen, with the increase of TGEV infection dose, the green fluorescence signal of dead cells in PE7-PK15 cells is significantly enhanced, while the green fluorescence signal of N783A PE7-PK15 cells is relatively weak, indicating that N783A mutation can reduce cell death induced by TGEV infection.

[0056] The above results indicate that the N783A mutation can significantly reduce cell damage and cell death caused by TGEV infection, thereby enhancing the cell's tolerance to TGEV infection.

[0057] (3) Detection of TGEV N gene RNA level PE7-PK15 cells and N783A PE7-PK15 cells were simultaneously plated, and the cell confluence was approximately 70% after 24 h. Subsequently, the cells were challenged with TGEV, and the MOI was set to 0, 0.01, 0.1 and 1.

[0058] After virus adsorption for 1 hour, the cells were washed and the culture medium was replaced with fresh medium. After 24 hours of infection, the cells were collected and total RNA was extracted. The RNA was reverse transcribed into cDNA and then detected by RT-qPCR to evaluate the viral RNA accumulation of TGEV in different cells.

[0059] See Figure 8 qPCR diagram of TGEV N gene RNA levels in cells infected with TGEV at different MOIs for 24 h after PE7-PK15 and N783A PE7-PK15. Figure 8 The vertical axis “Relative TGEV N RNA of 24hpi” represents the relative level of TGEV N gene RNA in cells 24 h after infection.

[0060] from Figure 8 As can be seen, under TGEV infection conditions with different MOIs, the TGEV N gene RNA level in N783A PE7-PK15 cells was significantly lower than that in PE7-PK15 cells, indicating that the N783A mutation can significantly reduce the viral RNA level in cells after TGEV infection.

[0061] The above results indicate that the N783A mutation can significantly reduce the accumulation of viral RNA after TGEV infection, suggesting that it can inhibit TGEV infection or replication in PK15 cells.

[0062] In summary, from Figure 3-5 and Figure 6-8 The results show that the N783A mutation in PE7-PK15 cells can significantly reduce cell damage and cell death caused by TGEV infection, thereby enhancing the cells' tolerance to TGEV infection. Furthermore, it can also significantly inhibit TGEV infection or replication in PK15 cells.

[0063] It should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0064] The detailed descriptions listed above are merely specific descriptions of feasible embodiments of the present invention, and are not intended to limit the scope of protection of the present invention. All equivalent embodiments or modifications made without departing from the spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pAPN protein mutant, characterized in that: The pAPN protein mutant is based on the wild-type pAPN protein, with asparagine at position 783 mutated to alanine, while other amino acid residues remain unchanged; the amino acid sequence of the wild-type pAPN protein is shown in SEQ ID NO:

2.

2. A nucleic acid molecule encoding the pAPN protein mutant of claim 1.

3. An expression cassette containing the nucleic acid molecule of claim 2.

4. A recombinant vector containing the nucleic acid molecule of claim 3.

5. A recombinant cell containing the nucleic acid molecule of claim 4.

6. A substance that causes a mutation at amino acid residue 783 of the wild-type pAPN protein, characterized in that: The substance is a Prime editing plasmid system in which the 783rd codon of pAPN is edited from AAC to GCT; the amino acid sequence of the wild-type pAPN protein is shown in SEQ ID NO: 2; The plasmid system includes a pegRNA targeting the pAPN N783 site and an ngRNA that assists in editing. The pegRNA sequence is shown in SEQ ID NO:3; the ngRNA sequence for editing assistance is shown in SEQ ID NO:

6.

7. The use of a substance that mutates amino acid residue 783 of the wild-type pAPN protein in any of the following: 1) Construct cell lines for site-directed modification of the pAPN gene for non-diagnostic and non-therapeutic purposes; 2) Preparation of cells resistant to porcine transmissible gastroenteritis virus; 3) Prepare pigs resistant to porcine transmissible gastroenteritis virus; 4) Breeding pigs resistant to transmissible gastroenteritis virus (TGEV); 5) Prepare products for the prevention or treatment of transmissible gastroenteritis in pigs; The wild-type pAPN protein has a mutation at amino acid residue 783, which replaces asparagine with alanine; the wild-type pAPN protein is the protein encoded by SEQ ID NO:

2.

8. The application as described in claim 7, characterized in that, The substance that causes a mutation at amino acid residue 783 of the wild-type pAPN protein is the substance as described in claim 6.

9. A method for constructing a pAPN protein mutant cell model or a porcine transmissible gastroenteritis virus (TGEV) resistant cell model for non-diagnostic and non-therapeutic purposes, characterized in that: The wild-type pAPN protein in the target cells was mutated at position 783 by replacing asparagine with alanine, while other amino acid residues remained unchanged, to obtain the pAPN protein mutant cells. The pAPN protein mutant cells are also the porcine transmissible gastroenteritis virus resistant cell model. The amino acid sequence of the wild-type pAPN protein is shown in SEQ ID NO:

2. The substance used to mutate asparagine at position 783 of the wild-type pAPN protein in the target cells to alanine is the substance described in claim 6.

10. A method for constructing a pAPN protein mutant gene-edited pig for non-diagnostic and therapeutic purposes, characterized in that, 1) Mutate the asparagine at position 783 of the pAPN protein in isolated porcine fibroblasts to alanine, while keeping other amino acid residues unchanged, to obtain pAPN protein mutant cells; 2) The pAPN protein mutant cells are used as nuclear transfer donor cells and are transplanted into sows via somatic cell nuclear transfer. The offspring produced are pAPN protein mutant gene-edited pigs. The amino acid sequence of the wild-type pAPN protein is shown in SEQ ID NO: 2; The substance used to mutate asparagine at position 783 of the wild-type pAPN protein in the target cells to alanine is the substance described in claim 6.

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