Leg1 protein and application thereof in obesity-related diseases

A protein and obesity technology, applied to Leg1 protein and its application in obesity-related diseases, can solve problems such as few targets

Active Publication Date: 2017-05-31
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, there are still relatively few research results on drugs that effectively treat obesity, and there are relatively few drug targets related to obesity or the regulation of fat synthesis and accumulation in vertebrates

Method used

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  • Leg1 protein and application thereof in obesity-related diseases
  • Leg1 protein and application thereof in obesity-related diseases
  • Leg1 protein and application thereof in obesity-related diseases

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Experimental program
Comparison scheme
Effect test

Embodiment 2

[0077] Since the homolog protein Leg1 of mLeg1 protein in zebrafish is a secreted protein, the above results have shown that mLeg1 gene is mainly expressed in salivary glands, but its mLeg1 protein may also be secreted and transported to other tissues to play a role. Therefore, the inventors of the present invention extracted the total protein from different tissues of mice, and detected the distribution of mLeg1 protein in different tissues by Western Blot.

[0078] Western Blot was used to detect the distribution of mLeg1 protein in different tissues. The experimental method is as follows.

[0079] 2.1 Protein extraction:

[0080] After the mice died suddenly, the target tissues (SG, liver, gut, blood, lung, heat, stomach, kindney, pancrease) were removed, placed in 1.5ml centrifuge tubes, and quickly frozen in liquid nitrogen to prevent degradation. When extracting protein, take out the sample, grind and pulverize by liquid nitrogen, collect the sample powder in a centrif...

Embodiment 3

[0088] Since the salivary gland is a secretory gland, its most important function is to secrete saliva, and mLeg1 is also a secretory protein. Therefore, the inventors of the present invention investigated whether mLeg1 is secreted into saliva.

[0089] The content of mLeg1 in saliva was detected by Western Blot. Specific steps are as follows.

[0090] 3.1 Saliva collection: 0.5 mg / kg of Pilocarpine (Sigma) was injected intraperitoneally into the mice, and a capillary was placed in the mouth of the mice to drain and collect the secreted saliva. Pilocarpine, a drug used to treat dry mouth, stimulates a flood of saliva. The saliva secreted by wild-type mice and mLeg1 systemic knockout mice (how to obtain them are described below) were collected respectively.

[0091] 3.2 Saliva treatment: the collected saliva was boiled at 100° C. for 5 minutes with 5x Laemmli buffer (10% SDS, 250 mM Tris-HCl, 0.1‰ Bromphenol blue, 500 mM DTT, 50% Glycerol) of 1 / 5 saliva volume.

[0092] 3.3...

Embodiment 4

[0095] Whole body knockout mice of mLeg1 gene (mLeg1 Δ / Δ ) of the acquisition

[0096] In order to obtain mleg whole-body knockout mice, the inventors of the present invention used the traditional Cre-loxP system to knock out the mLeg1 gene in mice. The system mainly relies on the Cre enzyme to recognize the loxP sequence and delete the sequences in the two loxP sequences in the same direction, so as to achieve the purpose of gene knockout. When the Cre enzyme is expressed at a specific time and space, mLeg1 can be knocked out at a specific time and space, thereby avoiding the research difficulties caused by embryonic lethality. Here, the inventors of the present invention inserted the loxP sequence into both sides of the third exon of mLeg1, and at the same time added a NEO gene between the third exon and the loxP sequence behind it for forward resistance screening . Through homologous recombination, embryo transfer and genetic screening, the inventors of the present inven...

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Abstract

The invention provides a Leg1 protein and application thereof in obesity-related diseases, relating to the technical field of functions and application of genes. By using an mLeg1-gene knockout mouse as the research object, means of genetics, molecular biology, biochemistry and cytobiology are utilized to perform very comprehensive research on the functions of the mLeg1 gene. The research result indicates that the mLeg1 protein (SEQ ID NO.2) and homologous proteins thereof (SEQ ID NO.1 and SEQ ID NO.5-8) and coding genes thereof can be used as brand-new targets for synthesizing related drugs from fat, can be used in the fields of preparation of drugs for treating obesity or losing weight, preparation of drugs for treating fat deficiency or increasing weight, preparation or screening of drugs for treating diabetes and preparation of drugs for adjusting fat accumulation of vertebrates and other related drugs for adjusting fat synthesis, and provides a brand-new idea for developing and researching drugs for adjusting internal fat synthesis and accumulation of vertebrates or treating fat-related diseases.

Description

technical field [0001] The invention relates to the technical field of gene function and application, in particular to a Leg1 protein and its application in obesity-related diseases. Background technique [0002] Over the past few years, obesity cases have grown rapidly worldwide and are now the fifth leading threat to human death. In developed countries, obesity has emerged as early as the 1980s, and cases have continued to increase since then, but the rate of increase has slowed down in the past 8 years; while in developing countries, obesity is increasing at a very rapid rate every year. speed is increasing. Although obesity generally does not lead directly to death, its complications, especially cardiovascular disease, can be fatal. In 2010, obesity was responsible for an estimated 3.4 million deaths. In addition, studies of obese patients in the United States have shown that obesity is likely to reduce the average life expectancy of humans in the future. According t...

Claims

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Application Information

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IPC IPC(8): C07K14/47A61K38/17A61P3/10A61P3/00A61P3/04
CPCA61K38/00C07K14/47
Inventor 彭金荣胡敏杰
Owner ZHEJIANG UNIV
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