PAPN protein mutant, substance enabling pAPN protein to be mutated and application

Abstract

The invention provides a pAPN protein mutant, a substance for mutating pAPN protein and application of the pAPN protein mutant, which are characterized in that on the basis of wild pAPN protein, amino acid of the pAPN protein is mutated into 220 D into G, or / and 918 F into P, or / and 570 V into I, or / and 595 H into Y, and other amino acid residues are not changed, so that TGEV SD / L strain infection can be effectively inhibited; a target spot is provided for obtaining a pig herd which can effectively inhibit TGEV infection and does not affect the growth performance of piglets and the physiological activity function of TGEV.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a pAPN protein mutant and a substance causing the pAPN protein to mutate, and applications related to inhibiting transmissible gastroenteritis virus (TGEV). Background Art

[0002] Transmissible gastroenteritis (TGE) is a highly contagious intestinal disease with severe diarrhea and rapid dehydration leading to death in infected piglets as the main clinical features. It is a pig infectious disease that must be strictly quarantined as required by the World Organization for Animal Health. The pathogen of the disease is TGEV. The mortality rate of piglets under 2 weeks old infected with TGEV is extremely high, especially for piglets under 10 days old, the mortality rate can reach 100%. Therefore, TGE is considered one of the important infectious diseases that endanger the pig farming industry.

[0003] The coding sequence of porcine aminopeptidase N (pAPN) is 2892bp in length, contains 21 exons, and encodes 963 amino acids. It is widely present on the surface of small intestinal epithelial cells and has a wide range of biological functions. Studies have shown that pAPN mediates the infection of TGEV and porcine deltacoronavirus (PDCoV) as the main receptor. Both TGEV and PDCoV are coronaviruses. Infection causes diarrhea and vomiting in piglets, rapid dehydration, and high infection and mortality rates in 2-week-old piglets. Studies have shown that knocking out pAPN at the cellular level and in pigs can significantly inhibit the infection of TGEV and PDCoV, further confirming that pAPN is the main receptor for the invasion of the above two coronaviruses into the host. Some mutants of pAPN do not affect the physiological function of APN, but can significantly inhibit the infection of TGEV and PDCoV, which has important application value. At present, relevant studies all adopt the method of overexpressing wild-type and mutant pAPN, overexpressing them in cell lines and infecting them with TGEV at the same time, and detecting the copy number of TGEV. For example, the patent document with publication number CN116769016A discloses a pAPN protein mutant and a composition and application for site-directed modification of the pAPN gene, and the patent document with publication number CN 116445454A discloses a complete system and application for breeding varieties resistant to TGEV infection, both of which are aimed at mutating the amino acid at position 734 of pAPN infected with TGEV (overexpression mutants were prepared, mutating positions 729, 734 and 735, and ultimately proving that position 734 is effective) and mutating the amino acid at position 737 of pAPN (overexpression mutants were prepared, mutating positions 737 and 739, and ultimately proving that position 737 is effective). However, gene overexpression can express target genes in large quantities in a short time. The artificial amplification of the functions of mutants and wild types leads to relatively large differences in performance. The results of amino acid mutations in cells that normally express the target gene may be inconsistent with the results of overexpression. Therefore, the actual inhibitory effect of pAPN mutations 734 and 737 on the virus is unknown. Amino acid mutations in genes naturally expressed in cells can directly and truly reflect the impact on viral infection. In addition, the ileal cell line IPI-2I is used in the above two patent documents. The ileal cell line IPI-2I itself can be infected with TGEV virus, but it can be seen from the results that the wild-type IPI-2I is not infected with TGEV.

[0004] A single base editor is a gene editing system composed of a fusion expression of Cas9 protein and deaminase (mainly adenine deaminase and cytosine deaminase). The system can accurately and irreversibly achieve the transition from one base pair to another without introducing double-strand DNA breaks and exogenous repair templates. Single base editors currently mainly include adenine base editors (ABEs), cytosine base editors (CBEs), guanine base editors (gGBE), and DdCBEs and TALEDs that can achieve precise editing of mitochondrial genomes. Adenine base editors are fusion proteins composed of nCas9 (D10A) and artificially modified adenine deaminase, which can specifically recognize and bind to the target sequence under the guidance of sgRNA to achieve the conversion from A to G. Due to the high efficiency of base editing and simple operation of the base editing system, various versions of ABEs (mainly including ABE7.10, ABEmax and ABE8e, etc.) have been widely used. The core components of cytosine base editors are nCas9 or dCas9 and cytosine deaminase. Cas9 protein and cytosine deaminase form a fusion protein. When the fusion protein targets genomic DNA under the guidance of sgRNA, it achieves direct replacement of C•G base pairs to T•A base pairs.

[0005] Therefore, precise mutation of key sites of the pAPN gene is used to simultaneously achieve resistance to TGEV infection and maintain normal expression of the pAPN protein, which has important scientific value and practical application significance in pig resistance to TGEV. Summary of the invention

[0006] The purpose of the present invention is to provide a pAPN protein mutant and its related applications for effectively inhibiting TGEV infection without affecting the physiological activity function of pAPN and the growth performance of gene-edited pigs.

[0007] In the first aspect, the present invention provides a pAPN protein mutant that inhibits TGEV infection, wherein the amino acid mutation is based on the wild-type pAPN protein, and the 220th aspartic acid (D) is mutated to glycine (G), or / and the 918th phenylalanine (F) is mutated to proline (P), or / and the 570th valine (V) is mutated to isoleucine (I), or / and the 595th histidine (H) is mutated to tyrosine (Y), the above amino acid mutation sites are all located in the extracellular domain, and other amino acid residues remain unchanged. The amino acid sequence of the wild-type pAPN protein is shown in SEQ ID NO: 1.

[0008] The present invention also provides a nucleic acid molecule encoding the pAPN protein mutant as claimed in claim 1.

[0009] In a second aspect, the present invention provides the use of pAPN protein and nucleic acid molecules and substances that can cause the above-mentioned mutations in pAPN protein without affecting the physiological activity function of pAPN, for: preparing products for preventing or treating porcine transmissible gastroenteritis; or preparing products for improving the resistance of an organism or its cells to porcine transmissible gastroenteritis virus; or preparing cells resistant to porcine transmissible gastroenteritis virus; or preparing pigs resistant to porcine transmissible gastroenteritis virus; or breeding pigs resistant to porcine transmissible gastroenteritis virus; or constructing a porcine transmissible gastroenteritis virus-resistant cell model; or constructing a porcine transmissible gastroenteritis virus-resistant pig model.

[0010] In the application, the organism is a mammal, further a livestock, and further a pig.

[0011] In the application, the substance that causes the pAPN protein to achieve the above mutation is ABEs and / or CBEs and a guide sequence that matches the single-base editor. The guide sequence guides ABEs and / or CBEs to achieve the following single-base editing: ABEs were used to mutate the 60th A in the 3rd exon of the wild-type pAPN gene to G to obtain the pAPN D220G gene; CBEs were used to mutate the 10th and 11th TT in the 21st exon of the wild-type pAPN gene to CC to obtain the pAPN F918P gene; CBEs were used to mutate the 154th G in the 11th exon of the original pAPN gene sequence to A to obtain the pAPN V570I gene; CBEs were used to mutate the 53rd C in the 12th exon of the wild-type pAPN gene to T to obtain the pAPN H595Y gene. The nucleotide sequence of the wild-type pAPN gene is shown in SEQ ID NO: 10, and the nucleotide sequences of the pAPN D220G gene, pAPN F918P gene, pAPN V570I gene, and pAPN H595Y gene obtained after editing are shown in SEQ ID NOs: 11 to 14, respectively. The guide sequences sgRNA-D220G, sgRNA-F918P, sgRNA-V570I and sgRNA-H595Y are shown in SEQ ID NOs: 6 to 9 respectively.

[0012] In a third aspect, the present invention provides a method for constructing a porcine transmissible gastroenteritis virus-resistant cell model, comprising the following steps: causing porcine kidney cells to express a pAPN protein mutant to obtain a porcine transmissible gastroenteritis virus-resistant cell model.

[0013] In a fourth aspect, the present invention provides a method for constructing pAPN protein mutant cells for non-diagnostic and therapeutic purposes, comprising the following steps: mutating the 220th D of the wild-type pAPN protein in pig kidney cells to G, or / and the 918th F to P, or / and the 570th V to I, or / and the 595th H to Y, while leaving other amino acid residues unchanged to obtain pAPN protein mutant cells.

[0014] The wild-type pAPN protein in the pig kidney cells is caused to undergo the above mutation specifically by introducing substances (ABEs and / or CBEs and corresponding sgRNA) that cause the pAPN protein to undergo the above mutation into the pig kidney cells to obtain pAPN protein mutant cells.

[0015] In a fifth aspect, the present invention provides a method for constructing a pAPN protein mutation gene-edited pig for non-diagnostic and therapeutic purposes, which is the following method 1 or method 2: The method 1 comprises the following steps: The pAPN protein mutant cells are obtained by mutating the 220th D to G, or / and the 918th F to P, or / and the 570th V to I, or / and the 595th H to Y of the pAPN protein in the isolated pig kidney, while other amino acid residues remain unchanged; The pAPN protein mutant cells are used as nuclear transplant donor cells and are transplanted into sows through somatic cell nuclear transplantation, and the offspring produced are pAPN protein mutant gene-edited pigs; The method 2 comprises the following steps: The substance causing the above mutation in the pAPN protein is microinjected into pig zygotic embryos to obtain pAPN gene-modified embryos, and then the pAPN gene-modified embryos are transplanted into the mother's body for pregnancy to obtain pAPN protein mutation gene-edited pigs.

[0016] The pAPN protein mutant gene-edited pig is a pig in which the pAPN protein coding gene on the pig genome is a coding gene for the pAPN mutant protein. The present invention does not limit whether the pAPN before mutation contains other mutation sites, so the precursor of the pAPN protein mutant provided by the present invention can be a wild-type pAPN, or a pAPN protein mutant that has mutated other sites based on the wild-type pAPN. The precursor of the pAPN protein mutant is a pAPN protein considered according to the general definition in the art. In an optional embodiment, the wild-type pAPN protein amino acid sequence is shown in SEQ ID NO: 1 or contains an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, for example, it can be but is not limited to an amino acid sequence that is at least 80%, 85%, 90%, 95% or 98% identical to SEQ ID NO: 1.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention finds that four mutation sites in the extracellular domain of the pAPN protein can effectively inhibit TGEV infection independently or in combination. The pAPN protein mutant provided by the present invention can maintain the normal expression of pAPN itself, while reducing the specific binding between the host expressing the pAPN protein mutant and TGEV.

[0018] The present invention uses a single base editor to accurately edit the pAPN gene, and successfully prepares PK-15-APN-D220G cells with D220G mutations, PK-15-APN-F918P cells with F918P mutations, PK-15-APN-V570I cells with V570I mutations, and PK-15-APN-H595Y cells with H595Y mutations. At the same time, it can avoid destroying or changing the normal expression of the remaining amino acids of pAPN, so on the basis of resisting TGEV infection, the physiological activity function of the pAPN protein is retained to the greatest extent, and it has the advantages of a wide range of applications and high gene editing efficiency. The above-mentioned cells can effectively inhibit the replication of TGEV, provide a theoretical basis for obtaining a pig herd that can effectively inhibit TGEV infection and does not affect the growth performance of piglets and the physiological function of pAPN, and minimize the impact of pAPN gene modification on the normal physiological function of pAPN and the growth performance of piglets, providing a new idea for the prevention and control research of TGEV.

[0019] The method for preparing gene-edited pigs using pAPN mutated cells has the advantages of easy operation and strong universality. The prepared amino acid mutation gene-edited pigs have good TGEV resistance while retaining normal expression of pAPN protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A in the middle shows sgRNAs enriched by APN subsaturation mutation library screening on PK-15-ABE8 cells, and B shows sgRNAs enriched by APN subsaturation mutation library screening on PK-15-AncBE4max cells.

[0021] Figure 2 A is a sequencing peak graph for constructing the recombinant vector, and B is a sequencing peak graph for evaluating the sgRNA editing efficiency of D220G, F9180, V570I, and H595Y mutations; Figure 3 Sequencing peak profiles constructed for evaluation of PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, and PK-15-APN-H595Y cell lines; Figure 4Results of qPCR detection of PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, and PK-15-APN-H595Y cells infected with TGEV-SD / L; Figure 5 The results of qPCR detection of PK-15-APN-KO cells infected with TGEV SD / L after complementation with APN D220G, APN F918P, APNV570I, and APNH595Y mutants; Figure 6 The results of qPCR detection of the mechanism of PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, and PK-15-APN-H595Y cells inhibiting the proliferation of TGEV-SD / L. A is the TGEV adsorption experiment, and B is the TGEV entry experiment. DETAILED DESCRIPTION

[0022] The technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. Unless otherwise specified, the professional and scientific terms used in this article have the same meanings as those familiar to those skilled in the art. In addition, any method or material similar or equal to the recorded content can also be applied to the present invention.

[0023] In addition, if no specific technical operation steps or conditions are specified in the examples, they are all carried out according to the general techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained through commercial purchase.

[0024] The present invention provides a pAPN protein mutant that inhibits TGEV infection, based on the wild-type pAPN protein, wherein the amino acid is mutated from D at position 220 to G, or / and F at position 918 to P, or / and V at position 570 to I, or / and H at position 595 to Y, and the above amino acid mutation sites are all located in the extracellular domain, and other amino acid residues remain unchanged. The amino acid sequences are shown in SEQ ID NOs: 2-5.

[0025] The amino acid sequence of the wild-type pAPN protein is as follows: MAKGFYISKALGILGILLGVAAVATIIALSVVYAQEKNKNAEHVPQAPTSPTITTTAAITLDQSKPWNRYRLPTTLLPDSYFVTLRPYLTPNADGLYIFKGKSIVRLLCQEPTDVIIIHSKKLNYTTQGHMVVLRGVGDSQVPEIDRTELVELTEYLVVHLKGSLQPGHMYEMESEFQGELADDLAGFYRSEYMEGNVKKVLATTQMQSTDARKSFPCFDEPAMKATFNITLIHPNNLTALSNMPPKGSSTPLAEDPNWSVTEFETTPVMSTYLLAYIVSEFQSVNETAQNGVLIRIWARPNAIAEGHGMYALNVTGPILNFFANHYNTSYPLPKSDQIALPDFNAGAMENWGLVTYRENALLFDPQSSSISNKERVVTVIAHELAHQWFGNLVTLAWWNDLWLNEGFASYVEYLGADHAEPTWNLKDLIVPGDVYRVMAVDALASSHPLTTPAEEVNTPAQISEMFDSISYSKGASVIRMLSNFLTEDLFKEGLASYLHAFAYQNTTYLDLWEHLQKAVDAQTSIRLPDTVRAIMDRWTLQMGFPVITVDTKTGNISQKHFLLDSESNVTRSSAFDYLWIVPISSIKNGVMQDHYWLRDVSQAQNDLFKTASDDWVLLNVNVTGYFQVNYDEDNWRMIQHQLQTNLSVIPVINRAQVIYDSFNLATAHMVPVTLALDNTLFLNGEKEYMPWQAALSSLSYFSLMFDRSEVYGPMKKYLRKQVEPLFQHFETLTKNWTERPENLMDQYSEINAISTACSNGLPQCENLAKTLFDQWMSDPENNPIHPNLRSTIYCNAIAQGGQDQWDFAWGQLQQAQLVNEADKLRSALACSNEVWLLNRYLGYTLNPDLIRKQDATSTINSIASNVIGQPLAWDFVQSNWKKLFQDYGGGSFSFSNLIQGVTRRFSSEFELQQLEQFKKNNMDVGFGSGTRALEQALEKTKANIKWVKENKEVVLNWFIEHS* The present invention provides a nucleic acid molecule encoding the pAPN protein mutant as described in claim 1. The "nucleic acid molecule" involved in the present invention refers to a polymer form of nucleotides of any length, and the nucleic acid molecule includes ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acid molecules include but are not limited to single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The polynucleotide encodes the above-mentioned pAPN protein mutant, and the encoding can optionally encode the sense strand or the antisense strand. The nucleic acid molecule can be naturally occurring, synthetic, recombinant, or any combination thereof.

[0026] The pAPN protein mutant, the nucleic acid molecule and the substance that causes the pAPN protein to achieve the mutation provided in the present invention are specifically used for: preparing products for preventing or treating porcine transmissible gastroenteritis; or preparing products for improving the resistance of an organism or its cells to porcine transmissible gastroenteritis virus; or preparing cells resistant to porcine transmissible gastroenteritis virus; or preparing porcine transmissible gastroenteritis virus-resistant pigs; or breeding porcine transmissible gastroenteritis virus-resistant pigs; or constructing a porcine transmissible gastroenteritis virus-resistant cell model; or constructing a porcine transmissible gastroenteritis virus-resistant pig model.

[0027] The substances that cause the pAPN protein to achieve the mutation are ABEs and / or CBEs and guide sequences that cooperate with ABEs and / or CBEs. The guide sequence that cooperates with ABEs to mutate the 60th position (659th position of pAPN gene) of the 3rd exon of the wild-type pAPN gene to G, the guide sequence that cooperates with CBE to mutate the 10th and 11th positions (2752nd and 2753rd positions of pAPN gene) of the 21st exon of the wild-type pAPN gene to CC, the guide sequence that cooperates with CBEs to mutate the 154th position (1708th position of pAPN gene) of the 11th exon of the wild-type pAPN gene to A, and the guide sequence that cooperates with CBEs to mutate the 53rd position (1783rd position of pAPN gene) of the 12th exon of the wild-type pAPN gene to T are shown in SEQ ID NOs: 6 to 9, respectively. The present invention realizes the following single base editing: in cooperation with ABEs, the A at position 60 of the 3rd exon of the wild-type pAPN gene is mutated to G; in cooperation with CBE, the TT at positions 10 and 11 in the 21st exon of the wild-type pAPN gene is mutated to CC; in cooperation with CBEs, the G at position 154 in the 11th exon of the wild-type pAPN gene is mutated to A; in cooperation with CBEs, the C at position 53 in the 12th exon of the wild-type pAPN gene is mutated to T. The nucleotide sequence of the wild-type pAPN gene is shown in SEQ ID NO: 10.

[0028] The method for constructing a pAPN protein mutant cell for non-diagnostic and non-therapeutic purposes in the present invention comprises the following steps: mutating the 220th D of the wild-type pAPN protein in pig kidney cells to G, or / and the 918th F to P, or / and the 570th V to I, or / and the 595th H to Y, while keeping other amino acid residues unchanged to obtain the pAPN protein mutant cell.

[0029] The present invention provides two methods for constructing a pAPN protein mutation gene-edited pig for non-diagnostic and non-therapeutic purposes: The first method comprises the following steps: mutating the 220th D of the pAPN protein in the isolated pig kidney to G, or / and the 918th F to P, or / and the 570th V to I, or / and the 595th H to Y, while keeping other amino acid residues unchanged to obtain pAPN protein mutant cells; using the pAPN protein mutant cells as nuclear transplant donor cells into sows through somatic cell nuclear transplantation, and the produced offspring are pAPN protein mutant gene-edited pigs; The second method includes the following steps: introducing the substance that causes the above-mentioned mutation in the pAPN protein into a pig zygotic embryo to obtain a pAPN gene-modified embryo, and then transplanting the pAPN gene-modified embryo into the mother's body for pregnancy to obtain a pAPN protein mutant gene-edited pig.

[0030] Example 1. Screening of APN subsaturation mutation library and selection of D220G, F9180, V570I, and H595Y mutation sites.

[0031] A series of sgRNAs targeting pAPN mutants were designed, synthesized and ligated to the Lenti-puro vector to construct a pAPN subsaturation mutant sgRNA library, which was then packaged into lentivirus and transfected into the PK-15-ABE8e cell line expressing NG-ABE8e or the PK-15-AncBE4max cell line expressing NG-AncBE4max. After three rounds of screening at MOI=0.0003 TGEV-SD / L, high-throughput sequencing was performed on the surviving cells.

[0032] like Figure 1 As shown in A, sgRNA-D220G targeting the mutation of amino acid D to G at position 220 of porcine APN protein (APN D220G) and sgRNA-F918P targeting the mutation of amino acid F to P at position 918 of porcine APN protein (APN F918P) were enriched in the PK-15-ABE8e cell line.

[0033] like Figure 1As shown in B, sgRNA-V570I targeting the V amino acid mutation to I at position 570 of porcine APN protein (APN V570I) and sgRNA-H595Y targeting the H amino acid mutation to Y at position 595 of porcine APN protein (APN H595Y) were enriched in the PK-15-AncBE4max cell line.

[0034] The DNA sequence of the sgRNA-D220G action site is: 5′-CTTTGACGAGCCAGCCATGA-3′ (SEQ ID NO: 6); The DNA sequence of the sgRNA-F918P action site is: 5′-CTTGAACTGCTCCAGCTGCC-3′ (SEQ ID NO: 7); The DNA sequence of the sgRNA-V570I action site is: 5′-GGTGACGTTGGATTCGGAGT-3′ (SEQ ID NO: 8); The DNA sequence of the sgRNA-H595Y action site is: 5'-TGCAGGATCACTACTGGCTG-3' (SEQ ID NO: 9).

[0035] Example 2: Construction of sgRNA lentiviral expression vector and verification of point mutation efficiency.

[0036] The porcine APN gene mutant was obtained by using the sgRNA-D220G sequence and NG-ABE8e in Example 1 to achieve the conversion from A to G. The two single-stranded sgRNA DNA sequences were annealed to form an oligonucleotide chain of sgRNA targeting the porcine APN D220G mutation; the oligonucleotide chain was then connected to the Lenti-puro plasmid vector, and the expression vector was successfully constructed by Sanger sequencing, see Figure 2 A. Sequencing peak diagram of recombinant vector construction. sgRNA-D220G-Lenti-puro was packaged into lentivirus and transfected into PK-15-ABE8e cell line expressing ABE8e at MOI=1. After 48 hours of transfection, 5μg / mL puromycin was used for selection for 7 days. Then 1 / 2 of the cells were collected to extract the cell genome. PCR reaction was performed using specific mutation identification primers. After PCR product sequencing, the efficiency of point mutation was evaluated by analyzing the sequencing peak diagram. See Figure 2 B Sequencing peak plot evaluating the sgRNA editing efficiency of the D220G mutation.

[0037] The porcine APN gene mutant was obtained by using sgRNA-F918P and NG-ABE8e in Example 1 to achieve the conversion from A to G. The two single-stranded sgRNA DNA sequences were annealed to form an oligonucleotide chain of sgRNA targeting the porcine APN F918P mutation; the oligonucleotide chain was then connected to the Lenti-puro plasmid vector, and the expression vector was successfully constructed by Sanger sequencing, see Figure 2 A. Sequencing peak diagram of recombinant vector construction. sgRNA-F918P-Lenti-puro was packaged into lentivirus and transfected into PK-15-ABE8e cell line expressing ABE8e at MOI=1. After 48 hours of transfection, 5μg / mL puromycin was used for selection for 7 days. Then 1 / 2 of the cells were collected to extract the cell genome. PCR reaction was performed using specific mutation identification primers. After PCR product sequencing, the efficiency of point mutation was evaluated by analyzing the sequencing peak diagram. See Figure 2 B Sequencing peak plot evaluating the sgRNA editing efficiency of the F918P mutation.

[0038] The porcine APN gene mutant was obtained by converting C to T using sgRNA-V570I and NG-AncBE4max in Example 1. The two single-stranded sgRNA DNA sequences were annealed to form an oligonucleotide chain of sgRNA targeting the porcine APN V570I mutation; the oligonucleotide chain was then connected to the Lenti-puro plasmid vector, and the expression vector was successfully constructed by Sanger sequencing, and then packaged into a lentivirus and transfected into the PK-15-AncBE4max cell line expressing AncBE4max, see Figure 2 A. Sequencing peak diagram of recombinant vector construction. sgRNA-V570I-Lenti-puro was packaged into lentivirus and transfected into PK-15-AncBE4max cell line expressing AncBE4max at MOI=1. After 48 hours of transfection, 5μg / mL puromycin was used for selection for 7 days. Then 1 / 2 of the cells were collected to extract the cell genome. PCR reaction was performed using specific mutation identification primers. After PCR product sequencing, the efficiency of point mutation was evaluated by analyzing the sequencing peak diagram. See Figure 2 B Sequencing peak plot evaluating the sgRNA cleavage efficiency of the V570I mutation.

[0039] The porcine APN gene mutant was obtained by using sgRNA-H595Y and NG-AncBE4max in Example 1 to achieve the conversion from C to T. The two single-stranded sgRNA DNA sequences were annealed to form an oligonucleotide chain of sgRNA targeting the porcine APN H595Y mutation; the oligonucleotide chain was then connected to the Lenti-puro plasmid vector, and the successful construction of the expression vector was verified by Sanger sequencing, and then packaged into a lentivirus and transfected into the PK-15-AncBE4max cell line expressing AncBE4max, see Figure 2 A. Recombinant vector construction sequencing peak graph. sgRNA-H595Y-Lenti-puro was packaged into lentivirus and transfected into the PK-15-AncBE4max cell line expressing AncBE4max at MOI=1. After 48 hours of transfection, 5μg / mL puromycin was used for selection at 7dpi. After that, 1 / 2 of the cells were collected to extract the cell genome. PCR reaction was performed using specific mutation identification primers. After PCR product sequencing, the efficiency of point mutation was evaluated by analyzing the sequencing peak graph. See Figure 2 B Sequencing peak plot evaluating the sgRNA cleavage efficiency of the H595Y mutation.

[0040] Example 3. Acquisition of PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, and PK-15-APN-H595Y mutant cells.

[0041] The above-mentioned lentivirus-infected cells were evenly spread in a 100 mm cell culture dish at 600 cells / dish by the limiting dilution method, and the cell culture medium was replaced every 2-3 dpi. After 8-10 days, when the cell clones have grown, the cell clones are marked under a microscope, and these marked clones are picked into a 24-well cell culture plate for further culture. After 2-3 dpi, when the cells in the 24-well plate grow to 80% confluence, 1 / 4 of the cells are taken to extract genomic DNA with NP40 lysis buffer, which is used as a template for PCR identification. After electrophoresis sequencing, the sequencing peak graph can be analyzed to verify whether the pig APN D220G, APN F918P, APNV570I, and APNH595Y have been successfully mutated. The sequencing peaks used to evaluate the construction of PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, and PK-15-APN-H595Y cell lines are shown in Figure 3 , and it can be seen that porcine APN D220G, APN F918P, APN V570I, and APN H595Y were successfully mutated.

[0042] Example 4: qPCR detection of PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, and PK-15-APN-H595Y cells infected with TGEV SD / L.

[0043] The PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, PK-15-APN-H595Y cells and PK-15-APN cells constructed above were inoculated into 6-well cell culture plates at a certain density. After the cells adhered to the wall, the cells were incubated with a virus suspension at MOI = 0.003 for 1 hour. After the virus suspension was removed, 1 mL of DMEM culture medium containing 5% fetal bovine serum was added. The cells were collected at 72 hpi and 1 mL of TRNzol was added to extract total cell RNA. Reverse transcription was performed using 1 μg RNA as a template, and qPCR was performed using cDNA as a template to detect the number of TGEV copies. The anti-TGEV effects of PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, and PK-15-APN-H595Y cells were determined by comparing the quantitative results with those of the control group PK-15-APN cells. Figure 4 qPCR detected that PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, and PK-15-APN-H595Y cells were infected with TGEV-SD / L with excellent results.

[0044] Example 5: qPCR detection of PK-15-APN-KO cells infected with TGEV SD / L after complementation with APN D220G, APN F918P, APNV570I, and APNH595Y mutants.

[0045] The CDS region fragment of the APN gene was obtained from the cDNA of PK-15-APN by PCR and ligated into the pIRES plasmid to obtain the APN expression vector APN-pIRES. Using this as a template, the APN D220G, APN F918P, APN V570I, and APNH595Y expression vectors APN D220G-pIRES, APN F918P-pIRES, APNV570I-pIRES, and APN H595Y-pIRES were constructed using a point mutagenesis kit (Tiangen).

[0046] The APN knockout PK-15 cell line PK-15-APN-KO was inoculated in equal amounts in a 6-well cell culture plate, and 2 μg of APN D220G-pIRES, APN F918P-pIRES, APN V570I-pIRES, APN H595Y-pIRES, and APN-pIRES expression vectors were transfected using the liposome transfection reagent jetOPTIMUS® transfection reagent (Polyplus). At 24 hpi after transfection, the cells were inoculated with TGEV SD / L at MOI=0.3, and the cells were harvested at 24 hpi, and 1 mL of TRNzol was added to extract total cell RNA. Reverse transcription was performed using 1 μg RNA as a template, and qPCR was performed using cDNA as a template to detect the number of TGEV copies. The anti-TGEV effects of APN-D220G, APN-F918P, APN-V570I, and APN-H595Y mutants were determined by comparing the quantitative results of the control group PK-15-APN-KO complemented APN H595Y-pIRES cells, see Figure 5 PK-15-APN-KO complemented APN-D220G, APN-F918P, APN-V570I, and APN-H595Y cells infected with TGEV-SD / L with excellent results.

[0047] Example 6. qPCR detection of the mechanism by which PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, and PK-15-APN-H595Y cells inhibit the proliferation of TGEV-SD / L.

[0048] PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, PK-15-APN-H595Y and PK-15-APN cells were inoculated in equal amounts in 6-well cell culture plates. The cells were inoculated with TGEV SD / L at an MOI of 20 to ensure that there was at least one virus particle on the surface of each cell. After adsorption and invasion experiments, the total RNA of the adsorbed and invaded cells was extracted, and the relative expression level of TGEV mRNA was detected by reverse transcription PCR and qPCR to reflect the adsorption and invasion of virus particles, see Figure 6 PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, and PK-15-APN-H595Y cells have excellent effects in inhibiting the proliferation of TGEV-SD / L.

[0049] In some embodiments of the present invention, the wild-type pAPN protein in pig kidney cells is caused to undergo the above mutations by introducing substances (ABEs and / or CBEs and one or more corresponding sgRNAs) that cause the pAPN protein to undergo the above mutations into pig kidney cells to obtain pAPN protein mutant cells.

[0050] Since coronaviruses initiate the invasion and fusion process by recognizing and binding to receptor molecules on the host cell membrane, and eventually release the viral genome into infected cells, APN V570I, H595Y, D220G, and F918P inhibit viral infection by affecting the binding to TGEV, and these sites are important sites for TGEV binding. By integrating the above two or more amino acid mutation sites, the same or better anti-TGEV effect can be obtained.

[0051] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A pAPN protein mutant, characterized in that: Taking the wild-type pAPN protein as the basis, its amino acid mutation occurs from D at position 220 to G, or / and F at position 918 to P, or / and V at position 570 to I, or / and H at position 595 to Y, and other amino acid residues remain unchanged. The pAPN protein mutant has an inhibitory effect on TGEV infection.

2. A nucleic acid molecule encoding the pAPN protein mutant as claimed in claim 1.

3. The use of the pAPN protein mutant according to claim 1, the nucleic acid molecule according to claim 2, and the substance that causes the pAPN protein to achieve the mutation according to claim 1, characterized in that: Preparation of products for preventing or treating porcine transmissible gastroenteritis; or preparation of products for improving the resistance of the body or body cells to porcine transmissible gastroenteritis virus; or preparation of cells resistant to porcine transmissible gastroenteritis virus; or preparation of pigs resistant to porcine transmissible gastroenteritis virus; or breeding of pigs resistant to porcine transmissible gastroenteritis virus; or construction of a porcine transmissible gastroenteritis virus-resistant cell model; or construction of a porcine transmissible gastroenteritis virus-resistant pig model.

4. The use according to claim 3, characterized in that: The organism is a mammal.

5. The use according to claim 4, characterized in that: The body is a pig.

6. The use according to claim 3, characterized in that: The substance that causes the pAPN protein to achieve the mutation as claimed in claim 1 is ABEs and / or CBEs and a guide sequence that matches ABEs and / or CBEs.

7. The use according to claim 6, characterized in that: The guide sequence guides ABEs and / or CBEs to achieve the following single base editing: In cooperation with ABEs, the A at position 60 in exon 3 of the wild-type pAPN gene is mutated to G; in cooperation with CBE, the TT at positions 10 and 11 in exon 21 of the wild-type pAPN gene is mutated to CC; in cooperation with CBEs, the G at position 154 in exon 11 of the wild-type pAPN gene is mutated to A; in cooperation with CBEs, the C at position 53 in exon 12 of the wild-type pAPN gene is mutated to T. The nucleotide sequence of the wild-type pAPN gene is shown in SEQ ID NO:

10.

8. The use according to claim 7, characterized in that: The DNA sequences of the guiding sequences that cooperate with ABEs to mutate the 60th A in the 3rd exon of the wild-type pAPN gene to G, the guiding sequences that cooperate with CBEs to mutate the 10th and 11th TT in the 21st exon of the wild-type pAPN gene to CC, the guiding sequences that cooperate with CBEs to mutate the 154th G in the 11th exon of the wild-type pAPN gene to A, and the guiding sequences that cooperate with CBEs to mutate the 53rd C in the 12th exon of the wild-type pAPN gene to T are shown in SEQ ID NOs: 6 to 9, respectively.

9. A method for constructing a pAPN protein mutant cell for non-diagnostic and non-therapeutic purposes, comprising the following steps: mutating the 220th D of the wild-type pAPN protein in pig kidney cells to G, or / and the 918th F to P, or / and the 570th V to I, or / and the 595th H to Y, while leaving other amino acid residues unchanged to obtain the pAPN protein mutant cell.

10. A method for constructing a pAPN protein mutation gene-edited pig for non-diagnostic and non-therapeutic purposes, which is the following method 1 or method 2: The method 1 comprises the following steps: The pAPN protein mutant cells are obtained by mutating the 220th D to G, or / and the 918th F to P, or / and the 570th V to I, or / and the 595th H to Y of the pAPN protein in the isolated pig kidney, while other amino acid residues remain unchanged; The pAPN protein mutant cells are used as nuclear transplant donor cells and are transplanted into sows through somatic cell nuclear transplantation, and the offspring produced are pAPN protein mutant gene-edited pigs; The method 2 comprises the following steps: The substance causing the above mutation of the pAPN protein is introduced into pig zygotic embryos to obtain pAPN gene-modified embryos, and the pAPN gene-modified embryos are then transplanted into the mother's body for pregnancy to obtain pAPN protein mutation gene-edited pigs.

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