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A kind of uric acid-lowering molecule and its screening method and application
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A technology for lowering uric acid and molecules
Active Publication Date: 2021-06-08
GUANGZHOU JIHENG MEDICAL TECH
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Problems solved by technology
The random polypeptide library technology presented on the surface of phage is a new type of small molecule protein library developed on the basis of phage antibody library. It is based on the same principle as antibody library in terms of construction and screening, so it has similar biological research. However, compared with the antibody library, the random peptide library has many characteristics that the antibody library does not have, which makes up for the deficiencies in the application of the antibody library, making the random peptide library more widely used: 1. It has the ability to recognize natural small molecular organic compounds 2. Small molecular weight, better penetration ability, easy to reach the target site; 3. Strong structural plasticity, small molecular weight, easier to form with ligands Stable chimera structure; 4. Simple structure, composed of a single peptide chain, easier to operate in library construction and molecular transformation, and easier to obtain binding molecules with better specificity; 5. Lack of Fc segment structure of immune effectors, avoiding 6. The polypeptide protein has no glycosylation, does not require post-translational modification, is more suitable for expression in prokaryotic expression systems, and is easy to expand production; the random polypeptide library provides a way for the study of small molecular organics Similar to the biological technology method of antibody library, many human diseases are caused by the metabolic disorder of small molecule organic substances in the body, and it is of great significance to clarify the metabolic mechanism of small molecules for the prevention and treatment of related diseases
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Embodiment 1
[0052] Example 1 Construction of Random Mutation Library
[0053] By self-design, artificially synthesized oligonucleotide fragments encoding random cyclic octapeptides, and amplifying the gene fragments encoding random cyclic octapeptides by extending primers; passing the coding gene fragments and M13KE phage vector through EagI and KpnI restriction endonucleases Ligation was performed after double enzyme digestion, and the recombinant product was electrotransformed into Escherichia coli ER2738 competent cells to obtain a phage display random cyclic octapeptide library, and the capacity and diversity of the library were detected by plaque and sequencing;
[0056] That is, SEQ ID NO.3: GCT TGT NNK NNK NNK NNK NNK NNK NNK NNK NNK TGC GGT GGA GGT
[0057] 3’-CGA ACA(NNM) 8 ACG CCA CCT CCA-5′ negative strand
[0058] That is, SEQ ID NO.4: CGA ACA NNM NNM NNM NNM NNM...
Embodiment 2
[0062] Example 2 Screening of Random Mutation Library
[0063] The uric acid molecule is coupled with BSA, and the coupled uric acid molecule is used as the target molecule. Based on the principle of affinity adsorption, the phage mutant library is subjected to multiple rounds of "adsorption-elution-amplification" with a support ELISA plate. Enrich and amplify phages with specific binding activity to target molecules.
Embodiment 3
[0064] Embodiment 3 identifies the phage obtained by screening
[0065] The phages that can specifically bind uric acid were identified by ELISA and competition ELISA, and the results are shown in Table 1;
[0066] Table 1
[0067]
[0068]
[0069] It can be seen from Table 1 that the screened phages have good binding ability to the coupled uric acid molecules.
[0070] The screened phages were sequenced by sequencing method, and the obtained coding nucleotide sequence and corresponding amino acid sequence were as follows:
[0072] SEQ ID NO.1: ACPSTLIFATCGGGS. (amino acid sequence)
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Abstract
The present invention provides a uric acid-lowering molecule and its screening method and application. The amino acid sequence of the uric acid-lowering molecule is shown in SEQ ID NO.1; the present invention constructs a random mutation library by using phage display technology, and constructs a uric acid target molecule , using the principle of affinity adsorption to screen the library, optimize the construction, screening and identification methods and steps, and obtain the uric acid-lowering molecule, which is used to prepare a uric acid-lowering drug. The molecule has an excellent uric acid-lowering function, and the screening method is simple and efficient. It is worthy of popularization and application, and has broad application prospects and huge market value.
Description
technical field [0001] The invention belongs to the field of biotechnology and relates to a molecule and its screening method and application, in particular to a uric acid-lowering molecule and its screening method and application. Background technique [0002] With the change of people's dietary structure, especially in the coastal Guangdong area, high-nutrition fish, shrimp, shellfish and other seafood, as well as beer and other drinks have become people's main food, and this high-purine eating habit has made my country's high The number of patients with uric acidemia is increasing at a rate of 9.7% per year, and they are showing a younger trend, and the complications such as gout have become the second largest category of metabolic diseases in my country after diabetes, threatening everyone around us. life of one person. The main cause of complications such as hyperuricemia and gout is the excessive concentration of uric acid in the body. Not only gout, uric acid nephropa...
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