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Preparation method of anti-African swine fever cloned pig

An African swine fever virus and gene technology, which is applied in the field of preparation of African swine fever-resistant cloned pigs, can solve the problems of disease-resistant breeding, lack of vaccines and treatment methods, and loss of ASF pig industry.

Active Publication Date: 2021-07-06
GUANGDONG OCEAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In August 2018, the first ASF epidemic was confirmed in China, and then ASFV spread rapidly in my country. At present, ASF outbreaks have occurred in 31 provinces, autonomous regions, municipalities directly under the Central Government and Hong Kong Special Administrative Region. Due to the lack of effective ASFV vaccines and treatments, ASF has caused huge losses to my country's pig industry
Therefore, the identity of the receptor involved in the entry of the virus into the cell is still unknown, causing great trouble to the development of disease-resistant breeding

Method used

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  • Preparation method of anti-African swine fever cloned pig
  • Preparation method of anti-African swine fever cloned pig
  • Preparation method of anti-African swine fever cloned pig

Examples

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Comparison scheme
Effect test

Embodiment 1

[0032] The construction of embodiment 1 pig Rosa26 gene donor carrier

[0033] S1. Fusion the sgRNA-Cas9 gene sequence capable of expressing ASFV pE248R with the homologous left arm; the sgRNA-Cas9 gene sequence was synthesized by Shanghai Sangon and verified by sequencing, and then amplified using it as a template, and the primers used for amplification (As shown in SEQ ID NO.3-4) are F5'-GAGGGCCTATTTCCCATGAT-3' and R5'-TTATCGTCCGT ACGACCCCT-3', the PCR products were purified and sequenced to verify correctness. The homologous left arm of the first intron of the porcine Rosa26 gene (as shown in SEQ ID NO.5) was amplified with 3780 bp using the porcine cell genome DNA as a template. The amplification system is: porcine cell genomic DNA, 20 μL; GreenTaq Mix, 25 μL; pRosa26-Lelf-F’ (100 μM) 2 μL; pRosa26-Lelf-R’ (100 μM) 2 μL; ddH 2 O, 1 μL. The amplification program is: 95°C, 3min; 95°C, 15sec, 60°C, 15sec, 72°C, 4min, 35 cycles; 72°C, 5min. The primer pair used for amplific...

Embodiment 2

[0036] Example 2 Construction of CRISPR-Cas9 Targeting Vector

[0037] S1. Use the online design website (https: / / benchling.com / ) to predict the targeting site of the first intron of the pig Rosa26 gene; according to the self-assessment and the scores in the prediction results, the candidate target sites are clocked The one with the highest score is selected, and its sgRNA sequence (as shown in SEQ ID NO.12) is ACTGCTTCTAGACCAACCAA. Complementary paired oligonucleotides were synthesized according to the sgRNA sequence, as shown in Table 1, where the lowercase letters are restriction sites.

[0038] Table 1 Oligonucleotide sequence

[0039] name sequence (5'-3') px459-F (as shown in SEQ ID NO.13) caccGACTGCTTCTAGACCAACCAA px459-R (as shown in SEQ ID NO.14) aaacTTGGTTGGTCTAGAAGCAGTCAGTC

[0040] S2. Construction of targeting vector

[0041] Using the oligonucleotides in Table 1, the construction process is: 94°C, 5min, then 35°C, 10min, and then ...

Embodiment 3

[0050] Example 3 Screening and Identification of Positive Cell Monoclonal

[0051] S1. Screening of Positive Monoclonal Cells

[0052] Digest and collect porcine fibroblasts (approximately 1 × 10 6 1), the targeting vector px459 constructed in Example 2 and the pig Rosa26 gene donor vector constructed in Example 1 were mixed according to the ratio of the amount of substances 1:2, and the total mass was 6 μg, according to the method of S5 in Example 2 After transfection, place in CO 2 In the incubator, cultivate at 37°C. After 48 hours, the confluence of the cells reached 80%-90%. At this time, the cells in one well were evenly divided into eight 10cm culture dishes. After 24 days, the cells adhered to the wall, and the medium was replaced with the fibroblast medium containing G418 (500 μg / mL), and the medium was changed every 3 to 4 days, and the medium was still the fibroblast medium containing G418 (500 μg / mL) . After the cells were cultured for 7-10 days, the formation...

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Abstract

The invention discloses a preparation method of an anti-African swine fever cloned pig and belongs to the field of animal genetic engineering and genetic modification. A CRISPR / Cas9 gene knock-in technology is used, a CRISPR / Cas9 targeting vector and a pig Rosa26 gene donor vector are jointly transferred into a pig fibroblast to obtain a positive cell clone, a first intron of the pig Rosa26 gene in the positive clone is knocked in a gene capable of expressing sgRNA-Cas9 of a specific targeted knockout virus pE248R gene, such that the pE248R gene is knocked out in a targeted manner when viruses are propagated in vivo and a pig has ASFV resistance; a cloned embryo is obtained through a somatic cell nuclear transfer technology; and the cloned embryo are transferred into a pig uterus for pregnancy to obtain a cloned pig. The sgRNA-Cas9 system is knocked into a pig body for the first time and stably expressed to obtain ASFV resistance, such that a foundation is laid for breeding research on African swine fever resistance.

Description

technical field [0001] The invention relates to the fields of animal genetic engineering and genetic modification, in particular to a method for preparing cloned pigs resistant to African swine fever. Background technique [0002] African swine fever (ASF) is an acute, febrile, highly contagious infectious disease caused by African swine fever virus (ASFV), usually showing systemic hemorrhage, neurological symptoms, and dyspnea Clinical symptoms, with a short course of disease, high mortality and other characteristics. ASFV is a single-molecule linear double-stranded DNA virus, belonging to the order Double-stranded DNA Viridae, the African swine fever virus family, and the African swine fever virus genus. It is the only member of the ASFV family and the only arbovirus-borne DNA virus. The main source of infection of ASF is infected pigs and infected pigs. Infected tissues, blood, pig secretions and excreta all contain viruses. The virus can be attached to feed, transport v...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90C12N15/66C12N15/55C12N15/113A01K67/027
CPCC12N15/8509C12N15/907C12N9/22C12N15/113C12N15/1131A01K67/0275C12N2800/107C12N2310/20A01K2217/072A01K2227/108A01K2267/02
Inventor 巨向红雍艳红陈圣威照那木拉
Owner GUANGDONG OCEAN UNIVERSITY
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