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Composition for fixed-point modification of pAPN-gene 16 exon and application of composition

A technology of site-directed modification and composition, which can be applied to cells modified by the introduction of foreign genetic material, genetically modified cells, and the introduction of foreign genetic material using vectors, which can solve problems such as affecting the physiological functions of the body.

Pending Publication Date: 2021-11-05
中农种源(深圳)科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, directly knocking out pAPN may affect other physiological functions of the body

Method used

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  • Composition for fixed-point modification of pAPN-gene 16 exon and application of composition
  • Composition for fixed-point modification of pAPN-gene 16 exon and application of composition
  • Composition for fixed-point modification of pAPN-gene 16 exon and application of composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Construction of recombinant plasmid pX458-pAPN-sgRNA vector and double-stranded donor sequence design

[0082]1. First lock the sequence around the 736th amino acid of the porcine pAPN gene, and use the sgRNA analysis tool CRISPOR ( crispor.tefor.net ) sgRNA selects a targeting site sgRNA close to the N736 site and has a higher score, and its sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2. Complementary paired oligonucleotide sequences as shown in SEQ ID NO: 4 and SEQ ID NO: 5; SEQ ID NO: 6 and SEQ ID NO: 7 were synthesized according to the sgRNA sequence. At the same time, according to the sgRNA sequence, this embodiment also designed a pAPN-dsODN sequence such as the dsODN sequence shown in SEQ ID NO: 3 as the double-stranded donor sequence. When the double-stranded donor sequence replaced the wild-type sequence, N736 was successfully replaced by A736. The single amino acid precise mutation pattern of porcine pAPN gene N736 is shown in the figure fig...

Embodiment 2

[0084] Example 2 Establishment and functional verification of monoclonal porcine ileal epithelial cells with precise modification of the 736th amino acid of pAPN gene

[0085] 1. Establishment of porcine ileal epithelial cells by precisely modifying the 736th amino acid of pAPN gene

[0086] The wild-type porcine ileal epithelial cells (Immortal Pig Intestinal-2I wild type, IPI-2I-WT) were resuscitated into a 10 cm plate two days in advance, and the cells could be transfected when the cells reached 70-80% confluence. 5 μg pX458-pAPN-sgRNA-1 plasmid, 5 μg pX458-pAPN-sgRNA-2 plasmid and 5 μg pAPN-dsODN were co-transfected into IPI-2I-WT cells, and the transfection steps were strictly in accordance with the Basic Primary Nucleofector Kit (Lonza) kit manual to operate.

[0087] After 36 hours of electroporation, the cells were collected, and a single positive cell was sorted into a 96-well plate by a flow sorter and cultured, and the culture medium was changed every 3 days. Afte...

Embodiment 3

[0094] Example 3 Establishment of Monoclonal Porcine Fibroblasts Precisely Modified at the 736th Amino Acid of pAPN Gene

[0095] 1. Preparation of Porcine Fetal Fibroblasts

[0096] Remove the head, tail, limbs, viscera and bones from the 35-day-old pig embryo, and clean up the blood. Continue to cut the fetus with elbow ophthalmic scissors for 30 minutes to ensure that it is fully shredded. Pipette the shredded fetal tissue into a 15mL centrifuge tube with the blue tip of the scissors, add 5mL of complete medium, and remove the above solution after natural sedimentation for several minutes. And add a few drops of fetal calf serum to the lower tissue block, suck it out with a 15cm glass pasteur tube bent at the tip of 1cm, spread it in two T75 culture flasks, place the bottom of the bottle upward, and add 15mL full culture to the opposite side After 6-8 hours, carefully turn over the culture bottle, immerse the tissue pieces in the culture solution, change the solution every...

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Abstract

The invention relates to the technical field of biology and particularly provides a composition for fixed-point modification of a pAPN-gene 16 exon and application of the composition. The composition for the fixed-point modification of the pAPN-gene 16 exon, provided by the invention, comprises a targeted enzyme digestion vector 1, a targeted enzyme digestion vector 2 and a double-stranded donor sequence. A modified pAPN gene can effectively resist TGEV infection, meanwhile, the protein physiological activity function of the modified pAPN gene is not affected, and the modified pAPN gene has the advantages of being wide in application range, high in gene editing efficiency and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a composition for site-directed modification of exon 16 of pAPN gene and its application. Background technique [0002] Transmissible gastroenteritis of pigs (TGE) is an acute, highly contagious gastrointestinal infectious disease caused by transmissible gastroenteritis virus. Pigs of all ages can be infected, piglets within ten days of age are most susceptible to infection, and piglets within seven days of age are infected with a mortality rate of up to 100%. [0003] pAPN protein is considered to be the key receptor for TGEV (transmissible gastroenteritis virus) to enter cells. So far, several units have successfully produced pAPN gene knockout pigs, and challenge experiments have confirmed that pAPN gene knockout can completely resist TGEV infection. However, in addition to playing an important role in mediating TGEV invasion, pAPN also plays a role in hydrolyzing amide bonds in...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/113C12N15/11C12N5/10C12N15/877A01K67/027
CPCC12N15/85C12N15/1138C07K14/70596C12N9/485C12N5/0656C12N15/8778A01K67/0273A01K67/0275C12Y304/11002C12N2310/20C12N2800/107C12N2510/00A01K2217/07A01K2227/108A01K2267/02
Inventor 李奎牟玉莲徐长江刘志国
Owner 中农种源(深圳)科技有限公司
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