Method for preparing foot-and-mouth disease virus-resistant RNAi transgenic livestock

An anti-foot-and-mouth disease virus and transgenic technology, applied in the field of cell engineering, can solve the problems of difficult control and eradication of FMD, multiple serotypes, rapid mutation, etc., and achieve high practical value, lower infection rate, and broad market prospects

Inactive Publication Date: 2011-02-23
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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Problems solved by technology

FMDV mutates rapidly, has multiple serotypes, and there is no cross-protection between vaccines of diffe

Method used

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  • Method for preparing foot-and-mouth disease virus-resistant RNAi transgenic livestock
  • Method for preparing foot-and-mouth disease virus-resistant RNAi transgenic livestock
  • Method for preparing foot-and-mouth disease virus-resistant RNAi transgenic livestock

Examples

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Embodiment 1

[0047] Example 1: Production and molecular biological detection of somatic cell clone anti-foot-and-mouth disease virus RNAi transgenic cows

[0048] The production process of somatic cell cloning anti-foot-and-mouth disease virus RNAi heterozygous transgenic cows is as follows: figure 1 As shown, the specific process includes the following steps:

[0049] 1. Construction of shRNA recombinant H1 expression plasmid

[0050] After the chemically synthesized RNAi-3B11 and RNAi-3B12, RNAi-3D11 and RNAi-3D12, RNAi-3D21 and RNAi-3D22 (see Table 1) were annealed respectively, they were combined with the H1Lenti-virus vector digested with XbaI and AgeI. The obtained 12.5kb and the 1.5kb fragment ( figure 2 ) according to the following system: 12.5kb vector fragment, 100ng; 1.5kb vector fragment, 50ng; shRNA oligo, 50ng; T4 DNA ligase, 0.5μL; 10×T4DNA ligase buffer, 2μL, supplemented with H 2 O Bring the system to 20 μL and ligate overnight at 16°C. The competent cells of Escheric...

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Abstract

The invention discloses a method for preparing foot-and-mouth disease virus-resistant (FMDV) transgenic livestock by expressing FMDV-resistant ribonucleic acid interference (RNAi), which comprises the following steps of: linearizing an FMDV-resistant RNAi recombinant Lenti-virus vector which carries a short hairpin RNA (shRNA) base sequence and a green fluorescent protein (GFP) reporter gene and transfecting fetus fibroblasts of normal livestock; sieving transgenic nuclear donor cells by using the GFP reporter gene and a flow cell sorter and introducing nuclear donor nuclei into denucleated oocytes of the livestock to obtain a reconstructed embryo; and immigrating the reconstructed embryo into the uterus of surrogate livestock to obtain an offspring, namely the FMDV-resistant RNAi heterozygote transgenic livestock. Heterozygote transgenic male livestock and heterozygote transgenic female livestock are normally mated or reproduced by artificial insemination so as to obtain homozygote transgenic livestock. The transgenic livestock prepared by the method can express the FMDV-resistant RNAi, obviously reduces the FMDV infection rate and has the capability of resisting foot-and-mouth disease (FMD).

Description

technical field [0001] The invention relates to an animal transgenic technology method for obtaining anti-foot-and-mouth disease virus RNAi transgenic livestock, and belongs to the field of cell engineering. Background technique [0002] Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious infection caused by foot-and-mouth disease virus (FMDV) in cattle, sheep, pigs and other cloven-hoofed animals. The World Organization for Animal Health lists it as the first class A severe infectious disease. Once an outbreak occurs, the infected and contact infected animals must be culled. In 2001, the outbreak of foot-and-mouth disease in the United Kingdom cost about 9 billion pounds, accounting for 1.1% of its GDP; in 2005, Asia I type FMD broke out in some parts of my country, and in early 2009, A type FMD occurred in many provinces, which not only caused huge losses. Direct economic losses, and seriously endanger the healthy development of animal husbandry and foreig...

Claims

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Application Information

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IPC IPC(8): A61D19/02C12N15/113A01K67/027C12N15/877
CPCY02A40/70
Inventor 何洪彬王洪梅刘晓武建明杨宏军宋玲玲高运东侯明海武刚仲跻峰
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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