A porcine epidemic diarrhea virus resistance-related protein EIF2A, and related biological materials and applications thereof

Abstract

The present invention discloses a protein EIF2A related to resistance to porcine epidemic diarrhea virus, its related biological materials and applications, which relate to the field of biotechnology. The problem to be solved by this application is how to regulate the resistance of animals to porcine epidemic diarrhea virus, especially the resistance of porcine intestinal epithelial cells to porcine epidemic diarrhea virus. This application discloses the applications of the EIF2A protein, substances that regulate the expression of the coding gene of the EIF2A protein, or substances that regulate the activity or content of the EIF2A protein in any of the following aspects: A1) regulating the resistance of animals to porcine epidemic diarrhea virus; A2) preparing products for regulating the resistance of animals to porcine epidemic diarrhea virus; A3) applications in animal breeding. Substances that regulate the expression of the EIF2A protein can be used for breeding animals resistant to porcine epidemic diarrhea virus or treating porcine epidemic diarrhea virus.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a protein EIF2A related to resistance to porcine epidemic diarrhea virus, and related biological materials and applications thereof. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) is a pathogen of a highly contagious intestinal infectious disease, which mainly causes acute diarrhea, vomiting, and dehydration in piglets, especially leading to a high mortality rate in suckling piglets, causing huge economic losses to the pig industry. At present, the method of preventing and controlling with vaccines has the problem of low immune efficiency and cannot fundamentally prevent the occurrence of this disease. Therefore, exploring functional genes that regulate PEDV from the host perspective has become an urgent problem to be solved. Summary of the Invention

[0003] The technical problem to be solved by the present invention is how to improve the resistance of animals to porcine epidemic diarrhea.

[0004] To solve the above problems, the present application provides the following applications.

[0005] Applications of a protein, a substance that regulates the expression of the coding gene of the protein, or a substance that regulates the activity or content of the protein in any of the following aspects:

[0006] A1) Regulating the resistance of animals to porcine epidemic diarrhea virus;

[0007] A2) Preparing a product for regulating the resistance of animals to porcine epidemic diarrhea virus;

[0008] A3) Applications in animal breeding;

[0009] The protein is any of the following:

[0010] B1) A protein with an amino acid sequence shown in Sequence 2;

[0011] B2) A protein obtained by substitution and / or deletion and / or addition of amino acid residues of the protein in B1), having more than 80% identity with the protein shown in B1) and having the ability to regulate the resistance of animals to porcine epidemic diarrhea virus;

[0012] B3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of B1) or B2).

[0013] Among the above-mentioned proteins, the protein-tag refers to a polypeptide or protein that is expressed by fusion with the target protein using in vitro DNA recombination technology, so as to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein-tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0014] Among the above-mentioned proteins, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0015] Among the above-mentioned proteins, the identity of more than 80% can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0016] Among the above-mentioned proteins, Sequence 2 (SEQ ID No. 2) consists of 585 amino acid residues.

[0017] In the above context, the regulation of the resistance to porcine epidemic diarrhea virus can be up-regulation or enhancement or increase and / or down-regulation or attenuation or decrease of the resistance to porcine epidemic diarrhea.

[0018] In the above context, up-regulation or enhancement or increase of the resistance to porcine epidemic diarrhea can be achieved by up-regulating or enhancing or increasing the expression of the coding gene of the protein in animals.

[0019] In the above context, down-regulation or attenuation or decrease of the resistance to porcine epidemic diarrhea is achieved by down-regulating or attenuating or decreasing the expression of the coding gene of the protein in animals with resistance to porcine epidemic diarrhea.

[0020] In the above-mentioned application, the protein is derived from pigs.

[0021] In this article, the protein is named EIF2A.

[0022] In this article, the regulation of the resistance to porcine epidemic diarrhea virus can be achieved by knocking out or downregulating or weakening or reducing the expression of the EIF2A gene, which can upregulate or enhance or improve the resistance to porcine epidemic diarrhea.

[0023] In this article, the regulation of the resistance to porcine epidemic diarrhea virus can be achieved by upregulating or enhancing or improving the expression of the EIF2A gene, which can downregulate or weaken or reduce the resistance to porcine epidemic diarrhea.

[0024] In this article, the animal can be a pig. The porcine epidemic diarrhea virus can be the porcine epidemic diarrhea virus.

[0025] In this article, the substance that regulates the activity and / or content of the protein can be a substance that regulates gene expression, and the protein encoded by the gene is any one of the following:

[0026] A1) A protein whose amino acid sequence is the sequence shown in Sequence 2;

[0027] A2) A protein obtained by substitution and / or deletion and / or addition of amino acid residues of the protein described in A1), having more than 80% identity with the protein shown in A1) and having the ability to regulate the resistance to porcine epidemic diarrhea virus;

[0028] A3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0029] Above, the substance that regulates gene expression can be a substance that inhibits or reduces or downregulates the expression of the coding gene of the protein. The substance can be a substance that inhibits or reduces or downregulates the expression of the coding gene of the protein by gene knockout. The gene knockout (gene knockout) can inactivate a specific target gene by changing the DNA sequence. The gene knockout can be achieved by the CRISPR / Cas9 system. The CRISPR / Cas9 system includes an expression vector expressing sgRNA (guide RNA). Among them, the target sequence of sgRNA can be 5'-AGACAGCTATCCCATTGTCA-TGG-3' or 5'-CTCATATTGTTCTATTGACA-AGG-3' or 5'-AAATCCATAATTAAAGCTAG-AGG-3' or 5'-TTCTAGGGTAGAAATTGTGG-AGG-3'.

[0030] A substance that can also inhibit, reduce, or down-regulate the expression of the encoding gene of the protein through gene silencing. Gene silencing refers to the phenomenon in which a gene is not expressed or is expressed at a low level without damaging the original DNA. Gene silencing occurs without changing the DNA sequence, resulting in the non-expression or low expression of the gene. Gene silencing can occur at two levels. One is transcriptional gene silencing caused by DNA methylation, heterochromatinization, and position effects, etc. The other is post-transcriptional gene silencing, that is, the gene is inactivated by specifically inhibiting the target RNA at the post-transcriptional level of the gene, including antisense RNA, co-suppression, quelling, RNA interference (RNAi), and translation inhibition mediated by microRNA (miRNA), etc.

[0031] In the above text, the substance that regulates gene expression can be a substance that performs at least one of the following six regulations: 1) regulation at the transcriptional level of the gene; 2) regulation after gene transcription (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of the RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) regulation after translation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0032] In the above application, the substance that regulates the expression of the encoding gene of the protein can be any of the following:

[0033] B1) A nucleic acid molecule encoding the above protein;

[0034] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0035] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0036] B4) A nucleic acid molecule that inhibits, reduces, or down-regulates the expression of the encoding gene of the above or the protein described in sequence 2;

[0037] B5) The encoding gene expressing the nucleic acid molecule described in B5);

[0038] B6) An expression cassette containing the gene described in B5);

[0039] B7) A recombinant vector containing the gene described in B5), or a recombinant vector containing the expression cassette described in B6);

[0040] B8), a transgenic animal cell line containing the nucleic acid molecule described in B1) or B4), or a transgenic animal cell line containing the expression cassette described in B2) or B6), or a transgenic animal cell line containing the recombinant vector described in B3) or B7);

[0041] B9), a transgenic animal tissue containing the nucleic acid molecule described in B1) or B4), or a transgenic animal tissue containing the expression cassette described in B2) or B6), or a transgenic animal tissue containing the recombinant vector described in B3) or B7);

[0042] B10), a transgenic animal organ containing the nucleic acid molecule described in B1) or B4), or a transgenic animal organ containing the expression cassette described in B2) or B6), or a transgenic animal organ containing the recombinant vector described in B3) or B7).

[0043] In the nucleic acid molecule described in B1) or B4), those skilled in the art can easily mutate the nucleotide sequence encoding protein EIF2A of the present invention by using known methods, such as directed evolution or point mutation methods. Those artificially modified nucleotides having 80% or more identity with the nucleotide sequence of protein EIF2A isolated from the present invention, as long as they encode protein EIF2A and have the function of protein EIF2A, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0044] The above 80% or more identity can be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0045] In this article, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web page on the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively and performing a search to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0046] Among the above biological materials, the nucleic acid molecule described in B1) can be the coding gene of the said protein. Specifically, the nucleic acid molecule described in B1) can be a DNA molecule whose coding sequence of the coding strand is the DNA molecule shown in Sequence 1.

[0047] In this text, the vectors are well-known to those skilled in the art, including but not limited to: plasmids, phages (such as λ phage or M13 filamentous phage, etc.), cosmids (i.e., cosmid plasmids), Ti plasmids or viral vectors. Specifically, it can be vector pEASY-Blunt and / or pCAMBIA-139.

[0048] Among the above biological materials, the expression cassette described in B2) or B6) refers to DNA that can express the gene in a host cell. This DNA can not only include a promoter that initiates gene transcription, but also include a terminator that terminates gene transcription. Further, the expression cassette can also include enhancer sequences. Promoters that can be used in the present invention include but are not limited to: constitutive promoters, tissue-, organ- and development-specific promoters, and inducible promoters. As a specific example, the promoter can be CMV or TRE.

[0049] In B3) or B7) above, an animal expression vector can be used to construct a recombinant expression vector containing the gene expression cassette. As a specific example, the expression vector can be pEGFP-N1 or.pcDNA3.1.

[0050] In B8) above, the animal cell line can be a small intestinal epithelial cell line. The small intestinal epithelial cell line can be derived from pigs. The small intestinal epithelial cell line derived from pigs can be the IPEC-J2 cell line.

[0051] In B9) above, the animal tissue can be small intestinal epithelial tissue. The small intestinal epithelial tissue can be derived from pigs. The small intestinal epithelial tissue derived from pigs can be composed of the IPEC-J2 cell line.

[0052] In B10) above, the animal organ can be a small intestinal epithelial organ. The small intestinal epithelial organ can be derived from pigs. The small intestinal epithelial organ derived from pigs can be composed of the IPEC-J2 cell line.

[0053] In the above application, the nucleic acid molecule described in B1) is any one of the following:

[0054] C1) A DNA molecule whose nucleotide sequence of the coding strand is Sequence 1;

[0055] C2) A nucleic acid molecule obtained by nucleotide substitution and / or deletion and / or addition of the nucleic acid molecule described in C1), which has more than 80% identity with the nucleic acid molecule shown in C1) and has the ability to regulate the resistance to porcine epidemic diarrhea virus.

[0056] To solve the above problems, the present application also provides a method for upregulating or enhancing or increasing the resistance of animals to porcine epidemic diarrhea virus.

[0057] The method includes upregulating, enhancing, or improving the resistance of the target animal to porcine epidemic diarrhea virus by knocking out, downregulating, weakening, or reducing the expression level of the coding gene of the above-mentioned protein in the target animal, and / or the activity and / or content of the above-mentioned protein.

[0058] To solve the above problems, the present application also provides a method for preparing animal cells resistant to porcine epidemic diarrhea virus.

[0059] The method includes knocking out, downregulating, weakening, or reducing the expression level of the coding gene of the above-mentioned protein in the target animal cells, and / or the activity and / or content of the above-mentioned protein, to obtain animal cells resistant to porcine epidemic diarrhea virus, and the animal cells resistant to porcine epidemic diarrhea virus have higher resistance to porcine epidemic diarrhea virus than the target animal cells.

[0060] In the above method, the animal is a pig, and the method for knocking out, downregulating, weakening, or reducing the expression level of the coding gene of the above-mentioned protein in the target animal, and / or the activity and / or content of the above-mentioned protein is to perform the following mutation on the protein coding gene in the pig genome: nucleotides at positions 23702-33082 of Sequence 3 in the sequence listing are deleted in the animal genome.

[0061] To solve the above problems, the present application also provides a method for breeding animals resistant to porcine epidemic diarrhea virus.

[0062] The method includes using the above method to prepare animal cells resistant to porcine epidemic diarrhea virus, and then using the animal cells resistant to porcine epidemic diarrhea virus to breed animals resistant to porcine epidemic diarrhea virus.

[0063] In this article, the regulation of the resistance of animals to porcine epidemic diarrhea virus can be upregulating, enhancing, or improving and / or downregulating, weakening, or reducing the resistance to porcine epidemic diarrhea.

[0064] In this article, the regulation of the resistance of animals to porcine epidemic diarrhea virus can be knocking out, downregulating, weakening, or reducing the expression of the protein coding gene to upregulate, enhance, or improve the resistance to porcine epidemic diarrhea.

[0065] In this article, the regulation of the resistance of animals to porcine epidemic diarrhea virus can be upregulating, enhancing, or improving the expression of the protein coding gene to downregulate, weaken, or reduce the resistance to porcine epidemic diarrhea.

[0066] In the above text, the protein is any one of the following:

[0067] B1) a protein with an amino acid sequence shown in Sequence 2;

[0068] B2) a protein obtained by substitution and / or deletion and / or addition of amino acid residues of the protein described in B1), having more than 80% identity with the protein shown in B1) and having the ability to regulate the resistance of animals to porcine epidemic diarrhea virus;

[0069] A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of B1) or B2).

[0070] In the above, the protein-coding gene may be any of the following:

[0071] R1) DNA with a nucleotide sequence shown in Sequence 1;

[0072] R2) DNA obtained by substitution and / or deletion and / or addition of nucleotide residues of the DNA described in R1), having an identity of more than 80% with the DNA shown in R1) and having the ability to regulate the resistance to porcine epidemic diarrhea virus;

[0073] R3) A fusion DNA obtained by linking a protein tag-coding gene to the 5'-end and / or 3'-end of R1) or R2).

[0074] In the above, the expression of the protein-coding gene in the target animal is regulated by any of the following methods:

[0075] E1) When the regulation of porcine epidemic diarrhea virus resistance is to up-regulate or enhance or improve the resistance to porcine epidemic diarrhea, knockout or down-regulate or weaken or reduce the protein-coding gene of the protein described in Claim 1 in the target animal;

[0076] E2) Introduce the coding gene of any of the following proteins into an animal in which the expression of the above protein-coding gene has been knocked out or down-regulated or weakened or reduced:

[0077] M1) A protein with an amino acid sequence shown in Sequence 2;

[0078] M2) A protein obtained by substitution and / or deletion and / or addition of amino acid residues of the protein of M1), having an identity of more than 80% with the protein shown in M1) and having the ability to regulate the resistance to porcine epidemic diarrhea virus;

[0079] M3) A fusion protein with the same function obtained by linking a tag to the N-terminus and / or C-terminus of M1) or M2).

[0080] In the above, the animal in which the expression of the above protein-coding gene has been knocked out or down-regulated or weakened or reduced in E2) may be the animal obtained by the method of E1).

[0081] In the above, the method of knockout or down-regulation or weakening or reduction in E2) may be gene knockout.

[0082] In the above text, the nucleic acid molecule that inhibits, reduces, or downregulates the expression of the encoding gene of the protein can be achieved by gene knockout or gene silencing. The gene knockout (gene knockout) inactivates a specific target gene through changes in the DNA sequence. The gene knockout can be achieved through the CRISPR / Cas9 system. The CRISPR / Cas9 system includes an expression vector that expresses sgRNA (guide RNA). Among them, the target sequence of sgRNA can be 5'-AGACAGCTATCCCATTGTCA-TGG-3' or 5'-CTCATATTGTTCTATTGACA-AGG-3' or 5'-AAATCCATAATTAAAGCTAG-AGG-3' or 5'-TTCTAGGGTAGAAATTGTGG-AGG-3'.

[0083] In the above text, gene silencing refers to the phenomenon in which a gene is not expressed or is expressed at a low level without damaging the original DNA. Gene silencing enables a gene to be not expressed or expressed at a low level on the premise of not changing the DNA sequence. Gene silencing can occur at two levels. One is transcriptional gene silencing caused by DNA methylation, heterochromatinization, and position effects, etc. The other is post-transcriptional gene silencing, that is, at the post-transcriptional level of the gene, the gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, quelling, RNA interference (RNAi), and translation inhibition mediated by microRNA (miRNA), etc.

[0084] In the above text, the gene silencing can be achieved through the BSMV-VIGS technology. The silencing target sequence can be designed in Sequence 1 (the CDS sequence of the EIF2A gene). Specifically, it can be GCUGGAUUUGGAAAUCUAATT / CCUAAAGUUGCAACAGCUUTT.

[0085] In the above text, the animal can be a pig. The pig can be the small intestine of a pig. The small intestine of a pig can be the epithelial cells of the small intestine of a pig. The epithelial cells of the small intestine of a pig can be IPEC-J2.

[0086] In the above-mentioned protein, the above-mentioned application, or any of the above-mentioned methods, the animal is an animal body and / or an ex vivo animal organ and / or an ex vivo animal tissue and / or an ex vivo animal cell.

[0087] The animal is any one of the following:

[0088] G1) Mammalia;

[0089] G2) Artiodactyla;

[0090] G3) Suidae;

[0091] G4) Pig;

[0092] G5) Pig small intestine;

[0093] G6) Porcine intestinal epithelial cells.

[0094] The porcine intestinal epithelial cells may be IPEC-J2.

[0095] To solve the above problems, the present application also provides a substance.

[0096] The substance is a substance that regulates the expression of the coding gene of the protein.

[0097] In the above text, the application of the above protein as a target in the preparation of a drug for treating or preventing epidemic diarrhea.

[0098] In the above text, the drug may be a substance that knocks out or down-regulates or weakens or reduces the expression of the protein coding gene. The substance that knocks out or down-regulates or weakens or reduces the expression of the protein coding gene may be as described above.

[0099] The purpose of the above application and method may be non-therapeutic purposes. For example, the purpose of the above application and method is animal breeding, and the purpose of the animal breeding includes breeding animals resistant to porcine epidemic diarrhea virus.

[0100] Beneficial effects

[0101] The present invention constructs an EIF2A gene knockout vector, introduces it into the porcine intestinal epithelial cell (IPEC-J2) line, and obtains a porcine intestinal epithelial cell line with EIF2A gene deletion, named IPEC-J2-EIF2A-KO.

[0102] The present invention also constructs an EIF2A gene complementation vector, introduces it into IPEC-J2-EIF2A-KO cells, and obtains EIF2A-RESCUE3.

[0103] The porcine intestinal epithelial cell line with EIF2A gene knockout (IPEC-J2-EIF2A-KO) and the porcine intestinal epithelial cell complementation cell line with EIF2A gene deletion (EIF2A-RESCUE3) are inoculated with PEDV. The control group of the porcine intestinal epithelial cell line with EIF2A gene knockout is the wild-type porcine intestinal epithelial cells, and the control group of the porcine intestinal epithelial cell complementation cell line with EIF2A gene deletion is the porcine intestinal epithelial cell line transfected with an empty vector. The differences in PEDV proliferation between the control group and the experimental group are compared.

[0104] The results showed that, compared with the wild-type control, the porcine intestinal epithelial cells with the EIF2A gene knocked out significantly inhibited the proliferation of PEDV, while the porcine intestinal epithelial cells with the re-expression of the EIF2A gene significantly increased the proliferation of PEDV, indicating that EIF2A strongly negatively regulates the resistance of porcine intestinal epithelial cell lines to PEDV. Regulating porcine intestinal epithelial cells to change their resistance to porcine epidemic diarrhea has certain value and can be used in industrial applications. Brief Description of the Drawings

[0105] Figure 1 To provide evidence for the gene knockout cells obtained in this patent and show the effect of EIF2A gene knockout in gene-edited cells.

[0106] Figure 2 To provide evidence that after the EIF2A gene is knocked out in this patent, the expression of viral genes and proteins is inhibited. Figure 2 A: The expression level of the viral M gene was detected by fluorescence quantitative PCR. GAPDH was selected as the internal reference gene, and the Vero cells and wild-type IPEC-J2 cells after virus inoculation were used as positive controls. The results showed that the expression level of the viral M gene in the positive control Vero cells increased by more than 7000 times after infection, while the expression level of the viral M gene in the control wild-type IPEC-J2 cell line increased by about 60 times after infection. The EIF2A-KO cell line strongly inhibited the viral M gene. Figure 2 B: Further, immunofluorescence detection of the viral N protein was performed on EIF2A-KO cells 24 hours after PEDV infection. The N protein was labeled with green fluorescence and the nucleus was labeled with blue fluorescence. The results showed that the protein expression level of the virus in the EIF2A knockout line was significantly lower than that in wild-type cells, that is, the expression of the viral protein was significantly inhibited.

[0107] Figure 3 To provide evidence for the porcine intestinal epithelial cells overexpressing EIF2A obtained in this patent and show the changes in virus expression after overexpression of the EIF2A gene. After re-expressing EIF2A, the expression level of the PEDV N protein in the knockout cells increased significantly. The above results indicate that the inhibitory effect of EIF2A-KO cells on PEDV is specifically due to the insufficient expression of EIF2A. Detailed Description of the Invention

[0108] The present invention will be further described in detail below in conjunction with the specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0109] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0110] Animal virus: The CV777 strain is stored in our laboratory (described in the following literature: Hu Z, Li Y, Du H, et al. Transcriptome analysis reveals modulation of the STAT family in PEDV-infected IPEC-J2 cells[J]. BMC Genomics, 2020, 21(1).)

[0111] Porcine intestinal epithelial cells IPEC-J2: IPEC-J2 was purchased from DSMZ and is currently stored in our laboratory (described in the following literature: Hu Z, Li Y, Du H, et al. Transcriptome analysis reveals modulation of the STAT family in PEDV-infected IPEC-J2 cells[J]. BMC Genomics, 2020, 21(1).)

[0112] The public can obtain the biological material from the applicant in accordance with the relevant regulations of national biosafety. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes such as plasmids.

[0113] Experimental materials:

[0114] Gene knockout vector: pSpCas9(BB)-2A-GFP, purchased from addgene, catalog number 48138.

[0115] Gene complementation vector: Using Tkara's Snap Assembly Master Mix (CodeNo. 638948) kit, the EIF2A CDS sequence was cloned into the pcDNA 3.1 vector.

[0116] Culture reagent: The complete medium for the IPEC-J2 cell line is: DMEM containing 10% fetal bovine serum.

[0117] PEDV virus: The porcine epidemic diarrhea virus CV777 strain was provided by the College of Veterinary Medicine, China Agricultural University.

[0118] Example 1. Improving the resistance of porcine cells to porcine epidemic diarrhea virus

[0119] The nucleotide sequence of the genomic gene of porcine EIF2A is Sequence 3 in the sequence listing, the nucleotide sequence of the cDNA gene of porcine EIF2A is Sequence 1 in the sequence listing, the coding sequence (CDS) is Sequence 1 in the sequence listing, and it encodes a protein with the amino acid sequence of Sequence 2 in the sequence listing.

[0120] The CDS (Sequence 1) of the said EIF2A is specifically as follows

[0121] Atggcgccgtccgcgccgctcctgacagtccgaggatcagaaggactgtacctggtgaacggaccacc

[0122] acattttacggaaagcacagtgtttccaagagaatctgggaaaaactgcaaagcttatacctttagta

[0123] aggatgggaccttatttgcctggggcaatggagaaagaataaatgtcatcaatgtcactaccaaggaa

[0124] ctactgcactccttcgaccttccaaaggcagtttgccttgaattctcaccaaaaaacactatcctggc

[0125] aacatggcagccttacactacttctaaagatggcgcagctgggatacccaacctacaactttatgaca

[0126] tgaaaactgggacatgtttgaagtctttcatccagaaaaaaatgcaaaattggtgtccatcctggtca

[0127] gaagatgaaactctttgtgcccgaaatgttaacaatgaagttcacttctttgaaaacaacaattttaa

[0128] cacaattgcaaataaattgcacttgaaaaaaattaatgattttgtattatcacctggaccccagccct

[0129] acaaggtggctgtctatgttccagggagtaagggtgcaccttcatttgttagattgtatcagtacccc

[0130] aactttgatggacctcatgcagctttagccaataaaagtttctttaaggctgataaggttacaatgct

[0131] atggaataaaaaagctactgctgtgttggtcatagctagtacagaagttgacaagacaggagcgtcct

[0132] actatggggaacaaacactgcactacattgcaacaaatggagaaagtgctgtggtgcaattaccaaaa

[0133] aatggccccatttatgatgtagtttggaattctagttctactgagttttgtgctgtttatggcttcat

[0134] gccagccaaagcaacaattttcaacctgaaatgtgatcctgtgtttgactttggaactggtcctcgta

[0135] atgcagcctactacagccctcatggacatatattagtactagctggatttggaaatctaagaggacaa

[0136] atggaagtgtgggatgttaaaaactacaaacttatttctaaaccagtggcctctgattctacatattt

[0137] tgcttggtgcccagatggtgagcatattttaacagccacatgtgctcccagattacgtgttaataatg

[0138] ggtacaagatttggcattatactggctctgtcctgcacaagtatgatgtaccatcaaatgcagaatta

[0139] tggcaagtttcttggcagccattcctagatggaatctttccagaaaaatcgataacttaccaagcagt

[0140] tccaagtgatgtacccagtgaagaacctaaagttgcaacagcttacagacccccggctttaagaaata

[0141] aaccagtcactaattccaaactgcatgaggaggaaccaccacagaatatgaaaccacaaccaggaaat

[0142] gagaaaccgttatcaaaaactgcccttaaaaatcaaaggaagcatgaggctaagaaagctgcaaagca

[0143] ggaagcgagaagtgacaagagtccagatatggcaccttctcctgccccacagaacacaccacgaaata

[0144] ctgtctctcattcaacttctggagaccctgagatagacaaaaaaatcaagaacctaaagaagaaactg

[0145] aaagcaatcgaacaactgaaagaacaagcagcaactggaaaacagctagaaaaaaatcagttggagaaaattcagaaagaaaaagcccttctccaggagctggaagatttggaactgggtatttaa, denoted as SEQ ID No 1.

[0146] The amino acid sequence of EIF2A protein (Sequence 2) is as follows:

[0147] MAPSAPLLTVRGSEGLYLVNGPPHFTESTVFPRESGKNCKAYTFSKDGTLFAW

[0148] GNGERINVINVTTKELLHSFDLPKAVCLEFSPKNTILATWQPYTTSKDGAAGIP

[0149] NLQLYDMKTGTCLKSFIQKKMQNWCPSWSEDETLCARNVNNEVHFFENNNF

[0150] NTIANKLHLKKINDFVLSPGPQPYKVAVYVPGSKGAPSFVRLYQYPNFDGPHA

[0151] ALANKSFFKADKVTMLWNKKATAVLVIASTEVDKTGASYYGEQTLHYIATNG

[0152] ESAVVQLPKNGPIYDVVWNSSSTEFCAVYGFMPAKATIFNLKCDPVFDFGTGP

[0153] RNAAYYSPHGHILVLAGFGNLRGQMEVWDVKNYKLISKPVASDSTYFAWCP

[0154] DGEHILTATCAPRLRVNNGYKIWHYTGSVLHKYDVPSNAELWQVSWQPFLD

[0155] GIFPEKSITYQAVPSDVPSEEPKVATAYRPPALRNKPVTNSKLHEEEPPQNMKP

[0156] QPGNEKPLSKTALKNQRKHEAKKAAKQEARSDKSPDMAPSPAPQNTPRNTV

[0157] SHSTSGDPEIDKKIKNLKKKLKAIEQLKEQAATGKQLEKNQLEKIQKEKALLQELEDLELGI, denoted as SEQ ID No 2.

[0158] I. Construction of a porcine intestinal epithelial cell line with EIF2A gene deletion

[0159] 1. Construction of the EIF2A gene knockout vector

[0160] Design a gene knockout vector according to the EIF2A gene to knockout the EIF2A gene

[0161] (1) Design the sgRNA of EIF2A and clone it into the knockout vector (vector name pSpCas9(BB)-2A-GFP, purchased from addgene, catalog number 48138). The specific sgRNA sequences are as follows:

[0162] gRNA-A1: AGACAGCTATCCCATTGTCA;

[0163] gRNA-A2: CTCATATTGTTCTATTGACA;

[0164] gRNA-B1: AAATCCATAATTAAAGCTAG;

[0165] gRNA-B2: TTCTAGGGTAGAAATTGTGG。

[0166] Construction of the vector: The pSpCas9(BB)-2A-GFP vector was used, and the sequence of Insert 1 (gRNA-A1-U6>gRNA-A2-U6>gRNA-B1-U6>gRNA-B2) was inserted as follows:

[0167] Construction of the pSpCas9(BB)-2A-GFP-1 recombinant vector: Insert 1 with the nucleotide sequence of Sequence 4 was inserted between the recognition sites of the restriction endonuclease BbSI of the pSpCas9(BB)-2A-GFP vector (Addgene), and the other nucleotide sequences of the pSpCas9(BB)-2A-GFP vector were kept unchanged to obtain the pSpCas9(BB)-2A-GFP-1 recombinant vector.

[0168] The specific nucleotide sequence of Sequence 4 is as follows:

[0169] caccAGACAGCTATCCCATTGTCAgagggcctatttcccatgattccttcatatttgcatatacgata

[0170] caaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgt

[0171] gacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcat

[0172] atgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggaCTCATATT

[0173] GTTCTATTGACAgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttag

[0174] agagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagt

[0175] aataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgta

[0176] acttgaaagtatttcgatttcttggctttatatatcttgtggaaaggaAAATCCATAATTAAAGCTAG

[0177] gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattgg

[0178] aattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttg

[0179] ggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggaTTCTAGGGTAGAAATTGTGG, denoted as SEQ ID No 4.

[0180] 2. Culture of porcine intestinal epithelial cell (IPEC-J2) line

[0181] The porcine intestinal epithelial cell IPEC-J2 purchased from DSMZ was cultured: The IPEC-J2 cells cryopreserved in liquid nitrogen were quickly thawed in a 37 °C water bath. The cell suspension was transferred to a 15 mL centrifuge tube, and an equal volume of complete medium was added and gently pipetted to mix evenly. After mixing, it was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and an appropriate amount of complete medium was added again. After gently pipetting to mix evenly, cell plating was carried out. The cells were cultured in a constant temperature incubator at 37 °C, 95% humidity, and 5% CO2.

[0182] 3. Obtaining of porcine intestinal epithelial cell line with EIF2A gene deletion

[0183] The constructed pSpCas9(BB)-2A-GFP-1 recombinant vector was introduced into the above-cultured porcine intestinal epithelial cells by electroporation (Lonza 2B electroporator, electroporation program U-023). After electroporation, single colonies were picked and verified by sequencing, and homozygous cells with porcine EIF2A gene knockout were successfully obtained. It was named IPEC-J2-EIF2A-KO cell line.

[0184] Compared with the wild-type porcine intestinal epithelial cell line IPEC-J2, the same mutation occurred in the porcine EIF2A gene on two homologous chromosomes. In the genomic sequence table, nucleotides at positions 20411 to 29621 in region 3 of sequence 3 were deleted, resulting in a large fragment knockout of the porcine EIF2A gene.

[0185] II. Complementation of the porcine intestinal epithelial cell line with EIF2A gene deletion

[0186] 1. Construction of the EIF2A gene complementation vector

[0187] Using the software Snapgene, primers were designed outside the CDS region of the porcine EIF2A gene sequence (accession number XM_021069600.1), and homology arms were added to the 5' ends of the upstream and downstream primers. The primer sequences were F: CAGTGTGGTGGAATTatggcgccgtccgc; R: GATATCTGCAGAATTttaaatacccagttccaaatcttccagct. Using TransGen High Fidelity (HiFi) PCR SuperMix II (-dye) to amplify the CDS region of the target gene. The pcDNA3.1(+) plasmid was digested with EcoRI. After linearizing the pcDNA3.1(+) vector (Invitrogen, catalog number: V79020), the EIF2A amplification product and the linearized pcDNA3.1(+) were purified using a gel extraction kit. Then, In-Fusion Snap Assembly Master Mix was used for ligation. The ligation reaction system is shown in the following table. After that, it was transferred to DH5α competent cells, and single colonies after shaking the bacteria were selected to determine their positive rates. Finally, the recombinant vector pcDNA3.1-EIF2A was obtained through sequencing and plasmid extraction.

[0188] Ligation reaction system

[0189]

[0190] 2. Culture of the porcine intestinal epithelial cell line with EIF2A gene deletion

[0191] Take the above porcine intestinal epithelial cell line with EIF2A gene deletion (IPEC-J2-EIF2A-KO cell line) for culture. Set aside for use.

[0192] 3. Obtaining the complemented cell line of porcine intestinal epithelial cells with EIF2A gene deletion

[0193] The constructed recombinant vector pcDNA3.1-EIF2A was transfected into the above-cultured porcine intestinal epithelial cell line with EIF2A gene deletion (IPEC-J2-EIF2A-KO cell line) by liposome transfection method to obtain a rescued cell line, which was named EIF2A-RESCUE3 cell line. After 36 hours of transfection, the mRNA level of EIF2A gene was detected by fluorescence quantitative method. EIF2A-RESCUE cells were used for subsequent experiments.

[0194] III. Study on the resistance of IPEC-J2-EIF2A-KO cell line and EIF2A-RESCUE3 cell line to PEDV Culture of IPEC-J2-EIF2A-KO cell line:

[0195] Take the cells cryopreserved in liquid nitrogen and quickly thaw them in a 37°C water bath. Transfer the cell suspension to a 15 mL centrifuge tube, add an equal volume of complete medium, and gently pipette to mix evenly. After mixing, centrifuge at 1000 rpm for 5 min, discard the supernatant, add an appropriate amount of complete medium again, gently pipette to mix evenly, and then plate the cells. The cells were cultured in a constant temperature incubator at 37°C, 95% humidity, and 5% CO2. The complete medium for the cell line was DMEM containing 10% fetal bovine serum.

[0196] When the cell density reached over 95%, passage was carried out. Discard the medium, wash twice with PBS, add 1 mL of 0.25% trypsin and digest for 2 - 3 min, then add 1 mL of complete medium to terminate digestion. The digested cells were collected in a centrifuge tube, centrifuged at 1000 rpm for 5 min, discard the supernatant, wash with PBS and centrifuge again, and then resuspend with complete medium and replate.

[0197] Culture of EIF2A-RESCUE3 cell line: The same as above.

[0198] Culture of IPEC-J2 cell line: The same as above.

[0199] Culture of Vero cell line: The same as above.

[0200] Culture of virus: When the Vero cell monolayer grew to 90% in a T25 cell culture dish, discard the complete medium. After gently washing with PBS, add 100 μL of virus stock solution to the T25 culture flask and make up to 1 mL with serum-free DMEM. Adsorb for 1 h, gently shake 2 - 3 times during this period. Discard the virus solution, add 5 mL of DMEM and continue to culture. After 80% of the cells showed cytopathic effect, take out the culture flask, freeze-thaw 3 times repeatedly, then aspirate the mixture, centrifuge at 8000 rpm for 10 min, and take the supernatant as the virus solution.

[0201] Preparation of PEDV inoculum: When the cell monolayer was in a 10 cm 2When the cells grew to 80% confluence in the cell culture dish, the complete medium was discarded. After gently washing with PBS, the cells were incubated in serum-free DMEM and inoculated with CV777 at an MOI of 1. After 1 h of adsorption, the serum-free DMEM was replaced with the medium.

[0202] The above-cultured porcine intestinal epithelial cell line with EIF2A gene knockout (IPEC-J2-EIF2A-KO cell line), the rescued cell line of porcine intestinal epithelial cells with EIF2A gene knockout (EIF2A-RESCUE3 cell line), the wild-type cell line of porcine intestinal epithelial cells IPEC-J2 cell line, and Vero cell line were inoculated with the above-cultured PEDV at a ratio of MOI = 1. The cells were cultured in a constant temperature incubator at 37 °C, 95% humidity, and 5% CO2. The control group of the porcine intestinal epithelial cell line with EIF2A gene knockout was wild-type porcine intestinal epithelial cells, and the control group of the rescued cell line of porcine intestinal epithelial cells with EIF2A gene knockout was the porcine intestinal epithelial cell line transfected with an empty vector. The differences in PEDV proliferation between the control group and the experimental group were compared.

[0203] Detection of the expression level of the M gene: The gene expression level was detected by a method based on the fluorescent dye SYBR Green. Using cDNA as a template, it was detected with a Bio-Rad CFX96 quantitative instrument. The porcine housekeeping gene β-actin gene was used as an internal reference, and the relative expression level was calculated by the 2-△△Ct method. Specific primers for the gene were designed using the NCBI Primers-BLAST online program (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) and synthesized by Beijing Bioengineering Co., Ltd. GAPDH primer F: TCGGAGTGAACGGATTTGGC; R: TGACAAGCTTCCCGTTCTCC. Fluorescent quantitative primer for the viral M gene F: GGTTCTATTCCCGTTGATGAGGT; R: AACACAAGAGGCCAAAGTATCCAT.

[0204] Fluorescent quantitative reaction system: 2×SYBR Green Mix 10 μL; upstream primer 1 μL; downstream primer 1 μL; template 2 μL; double-distilled water 6 μL. A total of 20 μL.

[0205] The fluorescent quantitative reaction conditions are as follows:

[0206]

[0207] Detection of immunofluorescence:

[0208] 1) Place a sterile coverslip in a petri dish and inoculate the cells into this petri dish during subculture. When the cell density reaches 60%, perform virus inoculation. After 24 h, take out the cell slides and wash them twice with PBS.

[0209] 2) Fix with a fixing solution containing 4% paraformaldehyde at room temperature for 20 min.

[0210] 3) Discard the fixing solution and wash three times with PBS for 5 min each time.

[0211] 4) Permeabilize with 0.1% Triton at room temperature for 10 min.

[0212] 5) Discard the permeabilization solution and wash three times with PBS for 5 min each time.

[0213] 6) Block with goat serum diluted 10-fold at 37 °C for 30 min.

[0214] 7) Discard the blocking solution, blot the liquid with filter paper, and incubate with the primary antibody at 4 °C overnight.

[0215] 8) Wash three times with PBS for 5 - 10 min each time.

[0216] 9) Add a suitable secondary antibody with fluorescent label, incubate in the dark at room temperature, and shake on a shaker for 1 h. It should be noted that all operations after this step should pay attention to avoiding light.

[0217] 10) Discard the secondary antibody and wash three times with PBS for 10 min each time.

[0218] 11) Add DAPI and incubate in the dark at room temperature for 5 - 10 min.

[0219] 12) Wash three times with PBS for 10 min each time.

[0220] 13) Blot the PBS and use filter paper to dry the cell slides. Drop an appropriate amount of anti-fluorescence quencher onto the glass slide, invert the cell slides so that the anti-fluorescence quencher fills the cell slides, and fix the slides with clear nail polish. Place in a light-proof and humid box and detect using a confocal microscope.

[0221] The results show that: Figure 2Provide evidence that after knocking out the EIF2A gene in this patent, the expression of viral genes and proteins is inhibited. Use fluorescence quantitative PCR to detect the expression level of the viral M gene. Select GAPDH as the internal reference gene, and use Vero cells and wild-type IPEC-J2 cells after virus inoculation as positive controls. The results show that the expression level of the viral M gene in the positive control Vero cells increased by more than 7000 times after infection, while the expression level of the viral M gene in the wild-type IPEC-J2 cell line of the control group increased by about 60 times after infection. The EIF2A-KO cell line strongly inhibits the viral M gene. Further, immunofluorescence detection of the viral N protein was performed on EIF2A-KO cells 24 hours after PEDV infection. The N protein was labeled with green fluorescence and the cell nucleus was labeled with blue fluorescence. The results show that the protein expression level of the virus in the EIF2A knockout line is significantly lower than that of wild-type cells, that is, the expression of viral proteins is significantly inhibited.

[0222] Figure 3 Provide evidence of the overexpressed EIF2A porcine intestinal epithelial cells obtained in this patent, showing the changes in virus expression after overexpression of the EIF2A gene. After re-expressing EIF2A, the expression level of PEDV N protein in the knockout cells increased significantly. The above results indicate that the inhibitory effect of EIF2A-KO cells on PEDV is specifically due to the insufficient expression of EIF2A.

[0223] The above details the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses or improvements to the present invention, including those that deviate from the scope disclosed in this application and are made with conventional techniques known in the art. Some basic features can be applied according to the scope of the appended claims below.

Claims

1. Use of sgRNA for knocking out porcine EIF2A gene in the preparation of products against porcine epidemic diarrhea virus, characterized in that, The cDNA sequence of the porcine EIF2A gene is shown as Sequence 1 in the Sequence Listing; the sgRNA sequences are shown as follows: gRNA-A1: AGACAGCTATCCCATTGTCA, gRNA-A2: CTCATATTGTTCTATTGACA, gRNA-B1: AAATCCATAATTAAAGCTAG, gRNA-B2: TTCTAGGGTAGAAATTGTGG; The product is a transgenic animal cell line, a transgenic animal tissue, or a transgenic animal organ, and the animal is a pig.

2. A method for preparing an animal cell resistant to porcine epidemic diarrhea virus, comprising knocking out the EIF2A gene in a target animal cell with the sgRNA according to Claim 1 to obtain an animal cell resistant to porcine epidemic diarrhea virus, wherein the target animal is a pig, and the cDNA sequence of the EIF2A gene is shown as Sequence 1 in the Sequence Listing.

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