Kit for breeding a TGEV infection resistant pig and use thereof
A kit for breeding a pig breed with resistance to the porcine transmissible gastroenteritis virus infection and application thereof. The systems includes a genetically edited protein, pAPN-sgRNA-1, pAPN-sgRNA-2, and a donor DNA. Effectively enzymatic cleavage can be made in two target sites of a pAPN gene by the gene editing protein. By replacing the fragment to be site-directed modified located between two target sites with donor DNA, a codon encoding tryptophan at position 737 in pAPN protein can be mutated to a codon encoding alanine, thereby achieving precise mutation of tryptophan to alanine at position 737 in a pAPN protein. The systems can avoid disruption or alteration of the normal expression of other amino acids in pAPN protein, therefore, the present invention maximally retains the physiological activity function of pAPN protein on the basis of resisting TGEV infection.
1. A method for constructing a cell with site-directed modification of a porcine aminopeptidase N (pAPN) gene, which comprises the following steps: introducing a kit comprising a genetically edited protein of Cas9, pAPN-sgRNA-1,pAPN-sgRNA-2, and a donor DNA, wherein the pAPN-sgRNA-1 and pAPN-sgRNA-2 target two different target sequences of the pAPN gene set forth in SEQ ID NO. 1 and SEQ ID NO. 2; and a fragment between the two different target sequences of SEQ ID NO. 1 and SEQ ID NO. 2 is designated as a fragment to be site-directed modified, which comprises a codon encoding tryptophan at position 737 in a pAPN protein; and the donor DNA contains a fragment with site-directed modification, which is obtained by mutating a codon encoding tryptophan at position 737 in the pAPN protein to a codon encoding alanine in the fragment to be site-directed modified or a set of vectors comprising a vector expressing the kit into porcine ileal epithelial cells or porcine fibroblasts to obtain the cell with site-directed modification of the pAPN gene; wherein the donor DNA is double stranded DNA set forth in SEQ ID NO. 3.
2. The method according to claim 1 , wherein the set of vectors consists of a vector for expressing the genetically edited protein of Cas9 and the pAPN-sgRNA-1, a vector for expressing the genetically edited protein of Cas9 and the pAPN-sgRNA-2, and a vector containing the donor DNA.
3. The method according to claim 2 , wherein the vector for expressing the genetically edited protein of Cas9 and the pAPN-sgRNA-1 is obtained by connecting annealed double stranded DNA fragments from single stranded DNAs set forth in SEQ ID NO. 5 and SEQ ID NO. 6 into a genetically edited backbone vector; alternatively, the vector for expressing the genetically edited protein of Cas9 and the pAPN-sgRNA-2 is obtained by connecting annealed double stranded DNA fragments from single stranded DNAs set forth in SEQ ID NO. 7 and SEQ ID NO. 8 into a genetically edited backbone vector.