Method for breeding cattle capable of producing hypoallergenic milk and application of method

A low-sensitization and sensitization technology, applied in the direction of biochemical equipment and methods, applications, chemical instruments and methods, etc., can solve the problem that the sensitization of β-LG cannot be completely eliminated, there is no effective and feasible technical method, and the nutritional effect of milk Lowering and other issues

Inactive Publication Date: 2019-02-12
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods reduce the allergenicity of β-LG to a certain extent, the structure and function of other proteins in milk are destroyed, the nutritional effect of milk is greatly reduced, and the sensitization of β-LG cannot be completely eliminated.
How to effectively reduce the sensitization of β-LG while maintaining the original structure and function of the protein as much as possible, there is currently no effective and feasible technical method at home and abroad

Method used

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  • Method for breeding cattle capable of producing hypoallergenic milk and application of method
  • Method for breeding cattle capable of producing hypoallergenic milk and application of method
  • Method for breeding cattle capable of producing hypoallergenic milk and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Breeding β-LG Biallelic Knockout Cattle Using Zinc Finger Nuclease

[0044] Cattle are diploid animals. When zinc finger nucleases play a role, both alleles on two homologous chromosomes in the same cell may be edited, resulting in the same type or different types of mutations. The β-LG biallelic knockout cattle (β-LG- / -Bovine) The β-LG genes of the two homologous chromosomes were mutated in different forms. The inventors of the present invention use zinc finger nuclease to breed β-LG biallelic knockout cattle. Considering the later industrialization and avoiding biological safety problems, they do not use screening marker genes and use ZFNs mRNA at the same time, so that they can breed without β-LG double-allelic knockout cattle with exogenous DNA integration, see technical route for details figure 1 .

[0045] 1. Synthesis of ZFN plasmids

[0046] According to the sequence information of the bovine β-LG gene (shown as sequence 1 in the sequence listin...

Embodiment 2

[0080] Example 2. Detecting the presence of β-LG protein in the milk of β-LG biallelic knockout cows

[0081] 1. SDS-PAGE detection of β-LG protein in the milk of β-LG biallelic knockout cows

[0082] 1. Extract the total protein from the milk produced on the first day, the third day or the fifth day of lactation of the cow to be tested (beta-LG double-allelic knockout cow or wild Holstein cow).

[0083] 2. Take the total protein of the cow to be tested and the β-LG protein standard (product of Sigma Company) obtained in step 1 respectively, and carry out SDS-PAGE.

[0084] See the experimental results Figure 4 Middle A (M is protein marker, β-LG is β-LG protein standard). The results showed that the expression of β-LG protein was not detected in the total protein of β-LG bi-allelic knockout cows, and there was no β-LG protein in the milk produced by β-LG bi-allelic knockout cows.

[0085] 2. Detection of β-LG protein in the milk of β-LG biallelic knockout cattle by Wester...

Embodiment 3

[0090] Example 3, Allergenicity Detection of Milk of β-LG Biallelic Knockout Cows

[0091] The milk produced by the cow to be tested (beta-LG double-allelic knockout cow or wild Holstein cow) was made into a dry powder, which was named as the milk powder to be tested.

[0092] 1. Simulated gastric juice digestion experiment

[0093] A natural protein needs to maintain its own allergenicity, and the amino acid fragment at the IgE binding site of the protein is generally resistant to digestion in the gastrointestinal tract in vivo, so that it may be absorbed and produce an immune response after reaching the small intestinal mucosa. Therefore, the remaining protein fragments after 60 minutes of simulated digestion in vitro are considered extremely indigestible and have the possibility of sensitization.

[0094] The simulated gastric digestive juice is a digestive juice set to simulate the pH value, salt ion concentration and pepsin concentration in the human gastric juice. It is...

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Abstract

The invention discloses a method for breeding cattle capable of producing hypoallergenic milk and application of the method. The method comprises the following steps: leading a zinc finger nuclease carrier pZFN into fibroblasts of the cattle, so as to obtain donor cells with a gene type which is a biallelic mutation type, wherein the biallelic mutation type is that nucleotide molecules of beta-LGgenes on two homologous chromosomes of the cattle are subjected to frame shift mutation; transferring cell nucleuses of the donor cells into cattle oocytes without the cell nucleuses and developing toform reconstructed embryos; then transferring the reconstructed embryos into uteri of cows and delivering to obtain the cattle capable of producing the hypoallergenic milk. According to the method disclosed by the invention, beta-LG biallelic gene knockout cattle which have no exogenous DNA (Deoxyribonucleic Acid) integration, can produce beta-lactoglobulin-free milk and deliveries mutation to the next generation through normal breeding are successfully bred. By adopting the method disclosed by the invention, an important foundation is laid for allergenic problems of the milk and also provides infinite possibility for preparing humanized milk. The method has important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for cultivating cows producing hypoallergenic milk and an application thereof. Background technique [0002] Milk is an important nutritionally balanced healthy food consumed worldwide, including rich protein, carbohydrates, fats and minerals. Although milk production has been improved through years of breeding strategies, nutritional management and quantitative genetics, the composition of the milk has not changed significantly. With the development of biotechnology and genetic engineering, especially for livestock breed improvement, there is a greater opportunity to produce "designer milk" suitable for human beings. So far, a variety of high-quality transgenic cattle have been reported, such as cattle expressing recombinant human lactoferrin, cattle with increased cow casein content, mastitis-resistant cattle, mad cow disease-resistant cattle, and hornless cat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/873A01K67/027C12Q1/6888
CPCA01K67/0273A01K2227/101A01K2267/02C07K14/4717C12N15/85C12N15/873C12N2800/107C12Q1/6888C12Q2600/124C12Q2600/156
Inventor 戴蕴平孙照霖王明丁方荣王海萍李玲
Owner CHINA AGRI UNIV
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