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Novel application of cysteine dioxygenase, gene and catalysate cysteine sulfinic acid of cysteine dioxygenase

The technology of cysteine ​​dioxygenase and cystine dioxygenase is applied in the field of genetic engineering, and can solve the problems of cysteine ​​dioxygenase cysteine ​​sulfinic acid that have not yet been studied and reported. Achieve the effect of promoting the development of the mammary duct system and improving the lactation ability

Inactive Publication Date: 2017-09-12
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are no studies and reports on the relationship between cysteine ​​dioxygenase and its catalytic product cysteine ​​sulfinic acid and mammary gland development

Method used

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  • Novel application of cysteine dioxygenase, gene and catalysate cysteine sulfinic acid of cysteine dioxygenase
  • Novel application of cysteine dioxygenase, gene and catalysate cysteine sulfinic acid of cysteine dioxygenase
  • Novel application of cysteine dioxygenase, gene and catalysate cysteine sulfinic acid of cysteine dioxygenase

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Experimental program
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Embodiment 1

[0031] Relative quantitative analysis of CDO protein expression in various tissues and organs of 6-week-old female mice was carried out by Western Blot method. The expression level in organs is very low ( figure 1 Middle A panel); consistent with the existing relevant reports. Western Blot quantitative analysis of CDO protein in mammary gland tissue of female mice at different developmental stages found that the expression of CDO protein in mammary gland tissue of each developmental stage showed obvious regular changes. CDO is highly expressed in the mammary gland during puberty to adult development (3 weeks to 12 weeks of age, when the mammary ducts are rapidly extending and massively branched), and during pregnancy and lactation (when the mammary glands are heavily differentiated) The expression level of CDO in the mammary gland decreased significantly, and after the lactation period, the mammary gland regression period (at this time, the mammary acinus degenerated, and the...

Embodiment 2

[0033] In order to reveal the regulatory effect of CDO on mammary gland development, we used CRISPR / Cas9 technology to set two reversely shortened sgRNA targets in the coding region of the first exon of the CDO gene ( figure 2Middle A) Cooperate with Cas9n (D10A) nickase to avoid off-target occurrence. Utilize T7 transcription kit (mMessage Machine T7kit, Life Company) to synthesize Cas9n gene mRNA, utilize T7 efficient transcription kit (Hiscribe TM T7 High yield RNA synthesis Kit, NEB) synthesizes two sgRNAs. CDO gene frameshift mutant mice were obtained by microinjecting Cas9n mRNA (60 ng / μL) and two sgRNAs (20 ng / μL) into fertilized mice and embryo transfer ( figure 2 in panels B and C). Homozygous mutant (CDO gene knockout) individuals were obtained through amplification, and the loss of CDO protein expression in the mammary gland of CDO knockout mice was verified by Western Blot ( figure 2 Middle D panel).

Embodiment 3

[0035] After careful observation, it was found that the offspring of CDO gene knockout female mice were stunted and unable to survive, but their offspring survived normally and developed healthily after being fostered in lactating wild-type (CDO non-knockout) female mice. It shows that the female mice with CDO gene knockout are insufficient in lactation and the offspring are stunted and die. The mammary gland whole tissue staining was further carried out, and the experimental steps included: collecting the fourth pair of mammary glands of the mouse, laying them flat on a glass slide, and fixing them overnight in Carnot's fixative solution (alcohol: chloroform: glacial acetic acid = 6: 3: 1 volume ratio), and then Equilibrate in alcohol with a volume fraction of 70% for 5 minutes, place in acetic acid magenta staining solution for 2 hours, dehydrate with alcohol gradient (80%-90%-95%-100%) (10 minutes per level), xylene transparent 10 Minutes, neutral gum sealing. For staining...

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Abstract

The invention provides novel application of a cysteine dioxygenase gene, cysteine dioxygenase or catalysate cysteine sulfinic acid of the cysteine dioxygenase, and particularly relates to application of cysteine dioxygenase genes, cysteine dioxygenase or catalysate cysteine sulfinic acid to promoting development of mammary glands of mammals. The novel application has the advantages that as shown by research, the cysteine dioxygenase, the gene and the catalysate cysteine sulfinic acid are related to development of mammary ducts, extension and ramification of the mammary ducts can be promoted, and the novel application can be used as a means for promoting the development of the mammary glands, increasing the lactation yields and treating maldevelopment of the mammary glands.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a cysteine ​​dioxygenase, cysteine ​​sulfinic acid, a direct catalyzed product of the enzyme, and a new application of the enzyme gene. Background technique [0002] Breast milk is the most natural, safest and most complete natural food for infant growth, providing the best nutrition for infant growth and development. The survey found that 65.3% of breastfeeding women in China provided formula milk powder for infants under 6 months of age, and the vast majority (86.2%) of them were due to insufficient breast milk (Widespread usage of infant formula in China: a major public health problem, Birth 41 (4): 339-343, 2014). Breast milk insufficiency due to human mammary dysplasia has long-term consequences for infant development and population quality. [0003] According to another study, the growth rate and survival rate of piglets before weaning are closely related to t...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K38/48A61K31/185A61P15/14
Inventor 赵建军韩玉竹刘安芳刘红润马兴宇
Owner SOUTHWEST UNIVERSITY
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