papn protein mutants and substances for mutating papn protein and use thereof

Abstract

The present application provides a pAPN protein mutant and a substance for mutating pAPN protein and application, taking wild type pAPN protein as a basis, the amino acid is mutated to D mutation G at the 220th position, or / and F mutation P at the 918th position, or / and V mutation I at the 570th position, or / and H mutation Y at the 595th position, and other amino acid residues remain unchanged, which can effectively inhibit TGEV SD / L strain infection, and provides a target for obtaining pig population 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 application relates to the field of genetic engineering, in particular to pAPN protein mutants and substances for mutating pAPN protein and related applications for inhibiting transmissible gastroenteritis virus (TGEV). BACKGROUND

[0002] Porcine transmissible gastroenteritis (TGE) is a highly contagious enteric disease with the main clinical features of severe diarrhea and rapid dehydration in infected piglets, and is a porcine infectious disease that must be strictly quarantined by the World Organization for Animal Health. The pathogen of the disease is TGEV, and the mortality rate of small pigs infected with TGEV under 2 weeks of age is extremely high, especially for piglets less than 10 days old, the mortality rate can reach 100%. Therefore, TGE is considered as one of the important infectious diseases that endanger the pig industry.

[0003] Porcine aminopeptidase N (pAPN) encoding sequence is 2892 bp in length, contains 21 exons, encodes 963 amino acids, and is widely present on the surface of small intestinal epithelial cells, with relatively wide biological functions. Studies have shown that pAPN mediates the infection of TGEV and porcine deltacoronavirus (PDCoV) as the main receptor. Both TGEV and PDCoV belong to coronavirus, and infection causes diarrhea and vomiting in piglets, rapid dehydration, and high infection rate and high mortality in 2-week-old piglets. Previous 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, and have important application value. At present, related researches all adopt the method of constructing overexpression of wild type and mutant of pAPN, and overexpressing in cell lines while TGEV infection, 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 pAPN gene, and the patent document with publication number CN116445454A discloses a complete system for breeding TGEV infection-resistant varieties and its application, both of which are for mutant pAPN 734 amino acid (overexpressed mutant is prepared respectively, 729, 734 and 735 sites are mutated, and finally it is proved that 734 site is effective) and mutant pAPN 737 amino acid (overexpressed mutant is prepared respectively, 737 and 739 sites are mutated, and finally it is proved that 737 site is effective). However, gene overexpression can express a large amount of target genes in a short time, and the difference caused by the function of mutant and wild type is relatively large, so the result of amino acid mutation in cells with normal expression of target genes may not be consistent with that of overexpression, and therefore the real inhibitory effect of pAPN mutation 734 and 737 sites on virus is unknown. Amino acid mutation of naturally expressed genes in cells can directly and truly reflect the influence on virus infection. And the two patent documents above use ileal cell line IPI-2I, which can infect TGEV virus, but it can be seen from the results that wild type IPI-2I does not infect TGEV.

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

[0005] Therefore, the precise mutation of the key site of the pAPN gene is used to simultaneously realize 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

[0006] The purpose of the present application is to provide a pAPN protein mutant and its related application for effectively inhibiting TGEV infection, and not affecting the physiological activity function of pAPN and the growth performance of gene edited pigs.

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

[0008] The present application also provides a nucleic acid molecule encoding the pAPN protein mutant according to claim 1.

[0009] In a second aspect, the present application provides the application of pAPN protein and nucleic acid molecules and substances that make pAPN protein realize the above-mentioned mutations without affecting the physiological activity of pAPN, which are: preparing products for preventing or treating porcine transmissible gastroenteritis; or preparing products for improving the resistance of organisms or organism 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 cell model resistant to porcine transmissible gastroenteritis virus; or constructing a pig model resistant to porcine transmissible gastroenteritis virus.

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

[0011] In the application, the substance that makes pAPN protein realize the above-mentioned mutations is ABEs and / or CBEs and a guide sequence matched with a single-base editor. The guide sequence guides ABEs and / or CBEs to realize the following single-base editing:

[0012] The A at the 60th position in the 3rd exon of the wild-type pAPN gene is mutated to G by ABEs to obtain a pAPN D220G gene; the TT at the 10th and 11th positions in the 21st exon of the wild-type pAPN gene is mutated to CC by CBEs to obtain a pAPN F918P gene; the G at the 154th position in the 11th exon in the sequence of the original pAPN gene is mutated to A by CBEs to obtain a pAPN V570I gene; the C at the 53rd position in the 12th exon of the wild-type pAPN gene is mutated to T by CBEs to obtain a 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 edited pAPN D220G gene, pAPN F918P gene, pAPN V570I gene and pAPN H595Y gene are shown in SEQ ID NO: 11-14, respectively. The guide sequences sgRNA-D220G, sgRNA-F918P, sgRNA-V570I and sgRNA-H595Y are shown in SEQ ID NO: 6-9, respectively.

[0013] In a third aspect, the present application provides a method for constructing a cell model resistant to porcine transmissible gastroenteritis virus, comprising the following steps: making a porcine kidney cell express a pAPN protein mutant to obtain a cell model resistant to porcine transmissible gastroenteritis.

[0014] In a fourth aspect, the present application provides a method for constructing a pAPN protein mutant cell for non-diagnostic and therapeutic purposes, comprising the following steps: mutating D at position 220 of wild-type pAPN protein to G, or / and mutating F at position 918 to P, or / and mutating V at position 570 to I, or / and mutating H at position 595 to Y in a pig kidney cell, and keeping other amino acid residues unchanged to obtain a pAPN protein mutant cell.

[0015] The mutation of wild-type pAPN protein in the pig kidney cell is specifically introducing a substance (ABEs and / or CBEs and corresponding sgRNA) that causes the mutation of pAPN protein into the pig kidney cell to obtain a pAPN protein mutant cell.

[0016] In a fifth aspect, the present application provides a method for constructing a pAPN protein mutant gene edited pig for non-diagnostic and therapeutic purposes, which is method 1 or method 2 as follows:

[0017] The method 1 comprises the following steps:

[0018] Mutating D at position 220 of pAPN protein to G, or / and mutating F at position 918 to P, or / and mutating V at position 570 to I, or / and mutating H at position 595 to Y in an ex vivo pig kidney cell, and keeping other amino acid residues unchanged to obtain a pAPN protein mutant cell;

[0019] The pAPN protein mutant cell is used as a nuclear transfer donor cell to be transplanted into a sow by somatic cell nuclear transfer to produce offspring, which is a pAPN protein mutant gene edited pig;

[0020] The method 2 comprises the following steps:

[0021] The above-mentioned substance that causes the mutation of pAPN protein is microinjected into a pig zygote embryo to obtain a pAPN gene modified embryo, and the pAPN gene modified embryo is transplanted into a mother to be pregnant to obtain a pAPN protein mutant gene edited pig.

[0022] The pAPN protein mutant gene editing pig is a pAPN protein coding gene for the pAPN protein coding gene on the genome of the pig. The present application does not limit whether the pAPN before mutation also contains other mutation sites, so the precursor of the pAPN protein mutant provided by the present application can be wild type pAPN, or a pAPN protein mutant that has been mutated at other sites based on wild type pAPN. The precursor of the pAPN protein mutant is the pAPN protein as defined according to the general definition in the art. In an alternative embodiment, the wild type pAPN protein amino acid sequence is shown in SEQ ID NO: 1 or an amino acid sequence comprising at least 80% identity with SEQ ID NO: 1, for example, but not limited to, an amino acid sequence comprising at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID NO: 1.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] The present application finds that four mutation sites of the extracellular domain of the pAPN protein can effectively inhibit TGEV infection independently or in combination. The pAPN protein mutant provided by the present application can maintain normal expression of pAPN itself, while reducing specific binding of the host expressing the pAPN protein mutant to TGEV.

[0025] The present application uses single base editor to precisely edit the pAPN gene, successfully prepares PK-15-APN-D220G cells with D220G mutation, PK-15-APN-F918P cells with F918P mutation, PK-15-APN-V570I cells with V570I mutation and PK-15-APN-H595Y cells with H595Y mutation. At the same time, it can avoid destroying or changing the normal expression of the remaining amino acids of pAPN, thus retaining the physiological activity function of pAPN protein to the maximum extent on the basis of resisting TGEV infection, and has the advantages of wide application range, high gene editing efficiency, etc. The above-mentioned cells can effectively inhibit the replication of TGEV, and provide a theoretical basis for obtaining pigs that can effectively inhibit TGEV infection and do 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, and provide a new idea for the prevention and control of TGEV.

[0026] The preparation method of the gene editing pig obtained by using the pAPN mutant cell has the advantages of convenient operation and strong universality, and the amino acid mutant gene editing pig prepared has good TGEV resistance while retaining normal expression of pAPN protein. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 A is sgRNAs enriched by screening APN under-saturating mutation library on PK-15-ABE8 cells, B is sgRNAs enriched by screening APN under-saturating mutation library on PK-15-AncBE4max cells.

[0028] Figure 2 A is sequencing peak chart for recombinant vector construction, B is sequencing peak chart for evaluating sgRNA editing efficiency of D220G, F918P, V570I, H595Y mutations;

[0029] Figure 3 A is sequencing peak chart for evaluating PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, PK-15-APN-H595Y cell line construction;

[0030] Figure 4 A is qPCR detection result of PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, PK-15-APN-H595Y cell lines infected with TGEV-SD / L;

[0031] Figure 5 A is qPCR detection result of PK-15-APN-KO cells complemented with APN D220G, APN F918P, APN V570I, APN H595Y mutants after infection with TGEV SD / L;

[0032] Figure 6 A is qPCR detection result of PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, PK-15-APN-H595Y cells inhibiting TGEV-SD / L proliferation mechanism, wherein A is TGEV adsorption experiment, B is TGEV entry experiment. DETAILED DESCRIPTION

[0033] The technical solutions of the present application will be described below in conjunction with the embodiments, obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. Unless otherwise specified, the professional and scientific terms used in this paper have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content can also be applied to the present application.

[0034] In addition, when a specific technical operation step or condition is not specified in the embodiments, it is performed according to the general technology or condition described in the literature in the art or according to the product manual. When the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained on the market.

[0035] The application provides a pAPN protein mutant for inhibiting TGEV infection, which is based on a wild-type pAPN protein, and the amino acid of the wild-type pAPN protein is mutated to D at the 220th position, F at the 918th position, V at the 570th position, H at the 595th position, or a combination thereof, and the amino acid mutation sites are located in the extracellular domain, and other amino acid residues are unchanged. The amino acid sequences are shown in SEQ ID NOs: 2-5.

[0036] MAKGFYISKALGILGILLGVAAVATIIALSVVYAQEKNKNAEHVPQAPTSPTITTTAAITLDQSKPWNRYRLPTTLLPDSYFVTLRPYLTPNADGLYIFKGKSIVRLLCQEPTDVIIIHSKKLNYTTQGHMVVLRGVGDSQVPEIDRTELVELTEYLVVHLKGSLQPGHMYEMESEFQGELADDLAGFYRSEYMEGNVKKVLATTQMQSTDARKSFPCFDEPAMKATFNITLIHPNNLTALSNMPPKGSSTPLAEDPNWSVTEFETTPVMSTYLLAYIVSEFQSVNETAQNGVLIRIWARPNAIAEGHGMYALNVTGPILNFFANHYNTSYPLPKSDQIALPDFNAGAMENWGLVTYRENALLFDPQSSSISNKERVVTVIAHELAHQWFGNLVTLAWWNDLWLNEGFASYVEYLGADHAEPTWNLKDLIVPGDVYRVMAVDALASSHPLTTPAEEVNTPAQISEMFDSISYSKGASVIRMLSNFLTEDLFKEGLASYLHAFAYQNTTYLDLWEHLQKAVDAQTSIRLPDTVRAIMDRWTLQMGFPVITVDTKTGNISQKHFLLDSESNVTRSSAFDYLWIVPISSIKNGVMQDHYWLRDVSQAQNDLFKTASDDWVLLNVNVTGYFQVNYDEDNWRMIQHQLQTNLSVIPVINRAQVIYDSFNLATAHMVPVTLALDNTLFLNGEKEYMPWQAALSSLSYFSLMFDRSEVYGPMKKYLRKQVEPLFQHFETLTKNWTERPENLMDQYSEINAISTACSNGLPQCENLAKTLFDQWMSDPENNPIHPNLRSTIYCNAIAQGGQDQWDFAWGQLQQAQLVNEADKLRSALACSNEVWLLNRYLGYTLNPDLIRKQDATSTINSIASNVIGQPLAWDFVQSNWKKLFQDYGGGSFSFSNLIQGVTRRFSSEFELQQLEQFKKNNMDVGFGSGTRALEQALEKTKANIKWVKENKEVVLNWFIEHS

[0037] The present application provides nucleic acid molecules encoding the pAPN protein mutants as claimed in claim 1. The "nucleic acid molecule" referred to in the present application refers to a polymer form of nucleotides of any length, and the nucleic acid molecule includes ribonucleotides and / or deoxyribonucleotides. Examples of the nucleic acid molecule include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrid, or a polymer containing purine and pyrimidine bases or other natural, chemical or biochemical modified, unnatural or derived nucleotide bases. The polynucleotide encodes the pAPN protein mutants as described above, and the encoding can be optionally a sense strand or an antisense strand. The nucleic acid molecule can be naturally occurring, synthetic, recombinant or any combination thereof.

[0038] The application of the pAPN protein mutants, the nucleic acid molecules and the substances for mutating the pAPN protein in the present application is specifically for preparing products for preventing or treating porcine transmissible gastroenteritis; or preparing products for improving the resistance of an organism or cells of the organism 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 cell model resistant to porcine transmissible gastroenteritis virus; or constructing a pig model resistant to porcine transmissible gastroenteritis virus.

[0039] The substances for mutating the pAPN protein are ABEs and / or CBEs and guide sequences cooperating with the ABEs and / or CBEs. The guide sequence cooperating with the ABEs for mutating the A at the 60th position of the 3rd exon of the wild-type pAPN gene (the 659th position of the pAPN gene), the guide sequence cooperating with the CBEs for mutating the TT at the 10th and 11th positions in the 21st exon of the wild-type pAPN gene (the 2752th and 2753th positions of the pAPN gene), the guide sequence cooperating with the CBEs for mutating the G at the 154th position in the 11th exon of the wild-type pAPN gene (the 1708th position of the pAPN gene), and the guide sequence cooperating with the CBEs for mutating the C at the 53rd position in the 12th exon of the wild-type pAPN gene (the 1783th position of the pAPN gene) are sequentially shown in SEQ ID NOs: 6-9. The single base editing is implemented as follows in the present application: mutating the A at the 60th position of the 3rd exon of the wild-type pAPN gene to G with the ABEs; mutating the TT at the 10th and 11th positions in the 21st exon of the wild-type pAPN gene to CC with the CBEs; mutating the G at the 154th position in the 11th exon of the wild-type pAPN gene to A with the CBEs; and mutating the C at the 53rd position in the 12th exon of the wild-type pAPN gene to T with the CBEs, and the nucleotide sequence of the wild-type pAPN gene is shown in SEQ ID NO: 10.

[0040] The application discloses a method for constructing a pAPN protein mutant cell for non-diagnostic and therapeutic purposes, which comprises the following steps: mutating D at the 220th position of a wild-type pAPN protein into G, or / and mutating F at the 918th position into P, or / and mutating V at the 570th position into I, or / and mutating H at the 595th position into Y in a pig kidney cell, and keeping other amino acid residues unchanged to obtain a pAPN protein mutant cell.

[0041] The application discloses a method for constructing a pAPN protein mutant gene edited pig for non-diagnostic and therapeutic purposes, which is as follows:

[0042] The first method comprises the following steps: mutating D at the 220th position of a pAPN protein into G, or / and mutating F at the 918th position into P, or / and mutating V at the 570th position into I, or / and mutating H at the 595th position into Y in an ex vivo pig kidney to obtain a pAPN protein mutant cell; and transplanting the pAPN protein mutant cell as a nuclear transfer donor cell into a sow in vivo through somatic cell nuclear transfer to produce a pAPN protein mutant gene edited pig as a progeny;

[0043] The second method comprises the following steps: introducing a substance for mutating the pAPN protein into a pig blastocyst embryo to obtain a pAPN gene modified embryo; and transplanting the pAPN gene modified embryo into a mother to obtain a pAPN protein mutant gene edited pig through pregnancy.

[0044] Example 1, screening of an APN subsaturation mutant library and selection of D220G, F9180, V570I and H595Y mutation sites.

[0045] A series of sgRNAs targeting pAPN mutants are designed, synthesized and connected on a Lenti-puro vector to form a pAPN subsaturation mutant sgRNA library, and then the library is packaged into a lentivirus, transfected into a PK-15-ABE8e cell line expressing NG-ABE8e or a PK-15-AncBE4max cell line expressing NG-AncBE4max, and subjected to three rounds of screening at an MOI of 0.0003 TGEV-SD / L, and high-throughput sequencing is performed on the surviving cells.

[0046] As shown in FIG. Figure 1 A, sgRNA-D220G targeting mutation of D at the 220th position of a pig APN protein into G (APN D220G) and sgRNA-F918P targeting mutation of F at the 918th position of a pig APN protein into P (APN F918P) are enriched on a PK-15-ABE8e cell line.

[0047] As shown in FIG. Figure 1B, sgRNA-V570I and sgRNA-H595Y were analyzed on PK-15-AncBE4max cell line, which were enriched for the mutation of V amino acid at position 570 of the pig APN protein to I (APN V570I) and the mutation of H amino acid at position 595 of the pig APN protein to Y (APN H595Y).

[0048] The sgRNA-D220G action site DNA sequence is 5'-CTTTGACGAGCCAGCCATGA-3' (SEQ ID NO: 6);

[0049] The sgRNA-F918P action site DNA sequence is 5'-CTTGAACTGCTCCAGCTGCC-3' (SEQ ID NO: 7);

[0050] The sgRNA-V570I action site DNA sequence is 5'-GGTGACGTTGGATTCGGAGT-3' (SEQ ID NO: 8);

[0051] The sgRNA-H595Y action site DNA sequence is 5'-TGCAGGATCACTACTGGCTG-3' (SEQ ID NO: 9).

[0052] Example 2, Construction of sgRNA lentiviral expression vector and verification of point mutation efficiency.

[0053] The pig APN gene mutant was obtained by using the sgRNA-D220G sequence in Example 1 and NG-ABE8e to realize the conversion of A to G. The DNA sequences of two single-stranded sgRNAs were annealed to form an oligonucleotide chain targeting the pig APN D220G mutation; then the oligonucleotide chain was connected to the Lenti-puro plasmid vector, and the expression vector was successfully constructed by sanger sequencing, see Figure 2 A Recombinant vector construction sequencing peak map. The sgRNA-D220G-Lenti-puro was packaged into lentivirus, and was transfected into the PK-15-ABE8e cell line expressing ABE8e at a MOI of 1. After 48 hours of transfection, 5 μg / mL puromycin was used for screening for 7 days, and then 1 / 2 cells were collected to extract the cell genome. Specific mutation identification primers were used for PCR reaction, and the point mutation efficiency was evaluated by analyzing the sequencing peak map after sequencing the PCR product, see Figure 2 B Sequencing peak map for evaluating the editing efficiency of sgRNA for D220G mutation.

[0054] The pig APN gene mutant was obtained by using sgRNA-F918P and NG-ABE8e in Example 1 to realize the conversion of A to G. After the DNA sequences of the two single-stranded sgRNAs were annealed, an oligonucleotide chain targeting the sgRNA of the F918P mutation of the pig APN was formed; then the oligonucleotide chain was connected into the Lenti-puro plasmid vector, and the successful construction of the expression vector was verified by sanger sequencing, see Figure 2 A Recombinant vector construction sequencing peak map. Using sgRNA-F918P-Lenti-puro to package into lentivirus, transfect into PK-15-ABE8e cell line expressing ABE8e at MOI = 1, 48 hours after transfection, use 5 μg / mL puromycin for screening for 7 days, then collect 1 / 2 cells to extract the cell genome, use specific mutation identification primers for PCR reaction, after sequencing the PCR product, evaluate the point mutation efficiency by analyzing the sequencing peak map, see Figure 2 B Sequencing peak map for evaluating the sgRNA editing efficiency of F918P mutation.

[0055] The pig APN gene mutant was obtained by using sgRNA-V570I and NG-AncBE4max in Example 1 to realize the conversion of C to T. After the DNA sequences of the two single-stranded sgRNAs were annealed, an oligonucleotide chain targeting the sgRNA of the V570I mutation of the pig APN was formed; then the oligonucleotide chain was connected into the Lenti-puro plasmid vector, and the successful construction of the expression vector was verified by sanger sequencing, see Figure 2 A Recombinant vector construction sequencing peak map. Using sgRNA-V570I-Lenti-puro to package into lentivirus, transfect into PK-15-AncBE4max cell line expressing AncBE4max at MOI = 1, 48 hours after transfection, use 5 μg / mL puromycin for screening for 7 days, then collect 1 / 2 cells to extract the cell genome, use specific mutation identification primers for PCR reaction, after sequencing the PCR product, evaluate the point mutation efficiency by analyzing the sequencing peak map, see Figure 2 B Sequencing peak map for evaluating the sgRNA cleavage efficiency of V570I mutation.

[0056] The C to T conversion was achieved using sgRNA-H595Y and NG-AncBE4max in Example 1 to obtain a pig APN gene mutant. The DNA sequences of two single-stranded sgRNAs were annealed to form an oligonucleotide chain targeting the pig APN H595Y mutant sgRNA; then the oligonucleotide chain was ligated into the Lenti-puro plasmid vector, and the expression vector was verified to be successfully constructed by sanger sequencing, and then packaged into lentivirus to transfect the PK-15-AncBE4max cell line expressing AncBE4max, see Figure 2 A recombinant vector construction sequencing peak map. Using sgRNA-H595Y-Lenti-puro to package lentivirus, transfecting the PK-15-AncBE4max cell line expressing AncBE4max at MOI=1, 48 hours after transfection, using 5 μg / mL puromycin for screening at 7 dpi, then collecting 1 / 2 cells to extract the cell genome, using specific mutation identification primers for PCR reaction, and after sequencing the PCR product, the point mutation efficiency was evaluated by analyzing the sequencing peak map, see Figure 2 B Sequencing peak map for evaluating the sgRNA cleavage efficiency of H595Y mutation.

[0057] Example 3, obtaining of PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, and PK-15-APN-H595Y mutant cells.

[0058] The above lentivirus-infected cells were uniformly plated in 100 mm cell culture dishes at 600 cells / dish by limiting dilution method, and the cell culture medium was replaced once every 2-3 dpi. After 8-10 days, when the cell clones grew, the cell clones were labeled under a microscope, and these labeled clones were picked into 24-well cell culture plates for culture. After 2-3 dpi, when the cells in the 24-well plates grew to 80% confluence, 1 / 4 cells were extracted with NP40 lysis buffer to obtain genomic DNA, which was used as a template for PCR identification. After electrophoresis and sequencing, the sequencing peak map was analyzed to verify whether the pig APN D220G, APN F918P, APN V570I, and APN H595Y were successfully mutated. The sequencing peak for evaluating the construction of the PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, and PK-15-APN-H595Y cell lines is shown in FIG. 3, which shows that the pig APN D220G, APN F918P, APN V570I, and APN H595Y were successfully mutated.

[0059] Example 4, qPCR detection of PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, PK-15-APN-H595Y cell infection with TGEV SD / L.

[0060] The above constructed PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, PK-15-APN-H595Y cells and PK-15-APN cells were inoculated in equal amounts in 6-well cell culture plates at a certain density. After the cells adhered, the cells were incubated with a virus suspension at MOI = 0.003 for 1 hour, and 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 the total RNA of the cells. 1 μg of RNA was used as a template for reverse transcription, and cDNA was used as a template for qPCR to detect the copy number of TGEV. By comparing the quantitative results with the control group PK-15-APN cells, the anti-TGEV effect of PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, PK-15-APN-H595Y cells was determined, see Figure 4 The qPCR detection of PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, PK-15-APN-H595Y cell infection with TGEV-SD / L was excellent.

[0061] Example 5, qPCR detection of PK-15-APN-KO cell infection with TGEV SD / L after complementation of APN D220G, APN F918P, APN V570I, APN H595Y mutants.

[0062] The CDS region fragment of the APN gene was obtained from the cDNA of PK-15-APN using PCR and was connected to the pIRES plasmid to obtain the APN expression vector APN-pIRES, and using the point mutation kit (Tiangen) to construct the APN D220G, APN F918P, APN V570I, APN H595Y expression vectors APN D220G-pIRES, APN F918P-pIRES, APN V570I-pIRES, APN H595Y-pIRES using the template.

[0063] PK-15-APN-KO cells were inoculated in 6-well cell culture plates at equal amount, and 2 μg of APN D220G-pIRES, APN F918P-pIRES, APN V570I-pIRES, APN H595Y-pIRES and APN-pIRES expression vectors were transfected using jetOPTIMUS® transfection reagent (Polyplus), respectively. At 24 hpi, the cells were inoculated with TGEV SD / L at MOI = 0.3, and the cells were collected at 24 hpi, and 1 mL of TRNzol was added to extract total RNA from the cells. Reverse transcription was performed using 1 μg of RNA as a template, and qPCR was performed using cDNA as a template to detect the TGEV copy number. The anti-TGEV effects of APN-D220G, APN-F918P, APN-V570I and APN-H595Y mutants were determined by comparison with the quantitative results of the control group of PK-15-APN-KO cells complemented with APN H595Y-pIRES, see Figure 5 The PK-15-APN-KO cells complemented with APN-D220G, APN-F918P, APN-V570I and APN-H595Y had excellent effects on TGEV-SD / L infection.

[0064] Example 6, 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 detected by qPCR.

[0065] PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I, PK-15-APN-H595Y cells and PK-15-APN cells were inoculated in 6-well cell culture plates at equal amount. The cells were inoculated with TGEV SD / L at MOI = 20 to ensure that there was at least one virion on the surface of each cell. After the adsorption and invasion experiment, total RNA was extracted from the adsorbed and invaded cells, and reverse transcription PCR and qPCR were performed to detect the relative expression level of TGEV mRNA to reflect the adsorption and invasion of virions, see Figure 6 The PK-15-APN-D220G, PK-15-APN-F918P, PK-15-APN-V570I and PK-15-APN-H595Y cells had excellent effects on the mechanism of inhibiting the proliferation of TGEV-SD / L.

[0066] In some embodiments of the present application, the above-mentioned mutations of the wild-type pAPN protein in the pig kidney cells are specifically introducing a substance (ABEs and / or CBEs and one or more corresponding sgRNAs) into the pig kidney cells to cause the above-mentioned mutations of the pAPN protein, thereby obtaining the pAPN protein mutant cells.

[0067] Since the coronavirus recognizes and binds to the receptor molecules on the host cell membrane to initiate the invasion and fusion process, and finally releases the viral genome into the infected cell, APN V570I, H595Y, D220G and F918P are important sites for TGEV binding, and can inhibit viral infection by affecting the binding of TGEV. By integrating two or more of the above-mentioned amino acid mutation sites, the same or better anti-TGEV effect can be achieved.

[0068] Although the embodiments of the present application have been shown and described above, it should be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-mentioned embodiments within the scope of the present application.

Claims

1. A mutant of pAPN protein, characterized in that: The wild type pAPN protein is used as a basis, and the amino acid is mutated to D at position 220, or F at position 918, or V at position 570, or H at position 595, and other amino acid residues remain unchanged, the pAPN protein mutant has an inhibitory effect on TGEV infection, and the wild type pAPN protein amino acid sequence is shown in SEQ ID NO:

1.

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

3. Use of the pAPN protein mutant according to claim 1 and of the nucleic acid molecule according to claim 2 and of the agent that mutates the pAPN protein according to claim 1 for non-diagnostic and therapeutic purposes, characterized in that: Preparation of a product for preventing or treating porcine transmissible gastroenteritis; or preparation of a product for improving the resistance of an organism or cells of the organism to porcine transmissible gastroenteritis virus; or preparation of cells resistant to porcine transmissible gastroenteritis virus; or breeding of pigs resistant to porcine transmissible gastroenteritis virus; or construction of a cell model resistant to porcine transmissible gastroenteritis virus; the organism is a pig; The substance for mutating the pAPN protein is ABEs and / or CBEs and a guide sequence cooperating with the ABEs and / or CBEs.

4. Use according to claim 3, wherein: The guide sequence guides the ABEs and / or CBEs to achieve the following single base editing: The A at position 60 of the 3rd exon of the wild type pAPN gene is mutated to G in cooperation with ABEs; 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; and the C at position 53 in the 12th exon of the wild type pAPN gene is mutated to T in cooperation with CBEs, and the nucleotide sequence of the wild type pAPN gene is shown in SEQ ID NO:

10.

5. A method for constructing a pAPN protein mutant cell for non-diagnostic and therapeutic purposes, comprising the following steps: mutating the wild type pAPN protein at position 220 to G, or at position 918 to P, or at position 570 to I, or at position 595 to Y, and other amino acid residues remain unchanged to obtain a pAPN protein mutant cell, and the wild type pAPN protein amino acid sequence is shown in SEQ ID NO:

1.

6. A method for constructing a pAPN protein mutant gene edited pig for non-diagnostic and therapeutic purposes, which is method 1 or method 2 as follows: The method 1 comprises the following steps: Mutating the pAPN protein at position 220 to G, or at position 918 to P, or at position 570 to I, or at position 595 to Y, and other amino acid residues remain unchanged to obtain a pAPN protein mutant cell in an isolated pig kidney, and the wild type pAPN protein amino acid sequence is shown in SEQ ID NO: 1; The pAPN protein mutant cell is used as a nuclear transfer donor cell to be transplanted into a sow by somatic cell nuclear transfer to produce offspring, which are pAPN protein mutant gene edited pigs; The method 2 comprises the following steps: Introducing a substance for mutating the pAPN protein into a pig blastocyst embryo to obtain a pAPN gene modified embryo, and then transplanting the pAPN gene modified embryo into a mother to obtain a pAPN protein mutant gene edited pig through pregnancy; The substance that mutates the pAPN protein is ABEs and / or CBEs and a leader sequence that cooperates with the ABEs and / or CBEs.

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