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Method for synthesizing inhibitor by screening sialic acid organisms

A sialic acid and inhibitor technology, applied in the field of screening sialic acid biosynthesis inhibitors, can solve the problem of low cost and achieve the effect of less dosage and cost reduction

Inactive Publication Date: 2016-08-24
JIANGNAN UNIV
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Problems solved by technology

Therefore, the development of sialic acid biosynthesis inhibitors is also one of the ways for cancer treatment, but there is no convenient, effective and low-cost method for screening sialic acid biosynthesis inhibitors

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  • Method for synthesizing inhibitor by screening sialic acid organisms
  • Method for synthesizing inhibitor by screening sialic acid organisms
  • Method for synthesizing inhibitor by screening sialic acid organisms

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Embodiment Construction

[0013] Below in conjunction with specific examples, the present invention is further described. It should be understood that the following examples are illustrative of the invention and not intended to limit the scope of the invention.

[0014] Construction of GFP-GAT1 fusion protein expression vector and expression of the fusion protein in various mammalian cells: construction of GFP-GAT1 fusion protein is conducive to rapid detection after protein expression, and the fusion can be detected quickly and conveniently by FACS analysis and fluorescence microscope The protein was highly expressed in CHO, CHOLec3 and Hek293 cells ( figure 1 ). At the same time, it can be measured by GABA uptake experiment that the GAT1 protein activity ( figure 2 ). Through quantitative detection, it can be seen that the activity of GAT1 protein is directly proportional to the content of sialic acid at the end of the sugar chain of the protein. ( image 3 ). Through this association, Hek293 ...

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Abstract

The invention discloses a method for synthesizing inhibitor by screening sialic acid organisms. The method mainly comprises the following steps: constructing an inducible GFP-GAT1 fusion protein expression vector; expressing the GFP-GAT1 fusion protein in Hek293 cells; detecting the GABA magnitude absorbed by a GFP-GAT1 stable cell line Hek293 after a compound is added; and further detecting the sialic acid content by virtue of agglutinin blot blotting if an inhibiting effect on the absorption value exists. According to the method disclosed by the invention, the principle of associating the GAT protein activity with the sialic acid structure at the carbohydrate chain tail end is mainly utilized, the method can be used for screening in the development and application process of synthesizing inhibitors by sialic acid organisms, if the GAT protein activity is not influenced, the possibility of taking the sialic acid organisms as inhibitors can be eliminated, the method is convenient, accurate and small in amount, and the cost can be reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for screening sialic acid biosynthesis inhibitors. Background technique [0002] Sialic acid, also known as N-acetylneuronic acid, is a series of derivatives of 9-carbon monosaccharides. Sialic acid is an essential component of carbohydrate complexes in mammals (Corfield, 1982). In human tissue, there are only 3 different types of sialic acid, the Neu5Ac, 9-O-acetylated Neu5Ac and 9-O-lactosylated Neu5Ac. The sialic acid family plays an important role in the structural diversity of glycocomplexes. Therefore, many biological functions are related to sialic acid. Due to their physical properties, such as negative charge and spatial distribution, sialic acids directly affect the immediate environment of glycocomplexes, so their function can be divided into two categories and explain many phenomena. Sialic acid acts as a biological mask to shield the recognition ...

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Application Information

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IPC IPC(8): C12Q1/02C12N15/85
Inventor 胡静
Owner JIANGNAN UNIV
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