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Application of eltas protein as a drug target for the prevention and treatment of insulin resistance-related diseases

An insulin resistance and insulin technology, applied in the field of biomedicine, can solve problems such as limited targets and effective therapeutic drugs, unclear analysis of insulin resistance, and drug side effects.

Active Publication Date: 2019-02-22
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Effective control of insulin resistance is helpful for the clinical prevention and treatment of related diseases including insulin resistance syndrome, type II diabetes, cardiovascular and cerebrovascular diseases, and metabolic syndrome. Due to the lack of effective targets to eliminate the root cause of the disease - insulin resistance, the curative effect of the existing treatment methods is limited, and there are many existing drugs. side effect
[0003] Current studies believe that many factors are involved in the occurrence and development of insulin resistance, including genetic factors, obesity, increased levels of plasma free fatty acids, increased levels of inflammatory factors, etc. Alternative drug targets and effective therapeutic drugs for insulin resistance are very limited, and there is a lack of effective methods to directly antagonize insulin resistance and prevent the diseases caused by it from the source

Method used

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  • Application of eltas protein as a drug target for the prevention and treatment of insulin resistance-related diseases
  • Application of eltas protein as a drug target for the prevention and treatment of insulin resistance-related diseases
  • Application of eltas protein as a drug target for the prevention and treatment of insulin resistance-related diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1, expression and purification of eLtaS protein

[0059] 1. Construction of recombinant plasmid pET-eLtaS

[0060] 1. According to the nucleotide sequence of the whole genome of Staphylococcus aureus NCTC8325 published on the biological information website NCBI (http: / / www.ncbi.nlm.nih.gov) (GI No. 87201381), design and synthesize primer F : 5'-CCG GAATTC TCTGAAGATGACTTAACAAA-3' (the underline is the recognition sequence of restriction endonuclease EcoRI) and primer R: 5'-CCG CTCGAG TTATTTTTTAGAGTTTGCTT-3' (underlined is the restriction endonuclease XhoI restriction endonuclease recognition sequence).

[0061] 2. Extract the genomic DNA of Staphylococcus aureus 8325-4 and use it as a template, use the primer F and primer R synthesized in step 1 to carry out PCR amplification (reaction program: 95°C for 5min; 95°C for 30s, 55°C for 30s, 72°C ℃ 2min, 30 cycles), and then use the PCR product recovery kit to recover the PCR amplification product of about 130...

Embodiment 2

[0075] Specific binding and affinity determination of embodiment 2, eLtaS protein and insulin

[0076] 1. The specific binding between eLtaS protein and insulin

[0077] 1. Take an ELISA plate and coat it with insulin as the coating source (dilute insulin with pH 7.2, 0.01mol / L PBS buffer, set the coating concentration to 10 μg / mL, and add 100 μL to each well).

[0078] 2. After completing step 1, add 200 μL of blocking solution to each well of the microplate, seal the plate with a parafilm, block at 37°C for 90 minutes, discard the supernatant, and wash with washing solution (washing 5 times, adding 300 μL each time) Washing solution, 3min for each wash), and pat dry.

[0079] 3. After completing step 2, add 100 μL of different concentrations of eLtaS protein solution (obtained by diluting eLtaS protein with pH 7.2, 0.01mol / LPBS buffer solution, the concentration is 0M, 5.0×10 -6 M, 1.0×10 -5 M or 1.5×10 -5 M), seal the ELISA plate with a parafilm, and incubate at 37°C fo...

Embodiment 3

[0093] Example 3, eLtaS protein inhibits the binding of insulin to membrane receptor protein

[0094] 1. Competitive ELISA method

[0095] 1. Take an ELISA plate and coat it with insulin as the coating source (dilute insulin with pH 7.2, 0.01mol / L PBS buffer, set the coating concentration to 10 μg / mL, and add 100 μL to each well).

[0096] 2. After completing step 1, add 200 μL of blocking solution to each well of the microplate, seal the plate with a parafilm, block at 37°C for 90 minutes, discard the supernatant, and wash with washing solution (washing 5 times, adding 300 μL each time) Washing solution, 3min for each wash), and pat dry.

[0097] 3. After completing step 2, add 100 μL of membrane receptor protein solution with a concentration of 10 μg / mL and 100 μL of different concentrations of eLtaS protein solution (obtained by diluting eLtaS protein with pH 7.2, 0.01mol / L PBS buffer solution, concentration 0μg / mL, 0.1μg / mL, 0.3μg / mL, 1.0μg / mL, 3.0μg / mL or 5.0μg / mL), sea...

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Abstract

The invention discloses application of eLtaS protein in serving as a drug target for preventing and treating insulin resistance-relevant diseases. An experiment shows that the eLtaS protein can inhibit combination of insulin and membrane receptor protein and combination of the insulin and target cells, reduce phosphorylation of an insulin-mediated receptor, inhibit activation of key molecules of an insulin-mediated cell signaling pathway, inhibit the insulin-mediated sugar transport capacity, inhibit the insulin-mediated glucose uptake capacity, inhibit the hypoglycemic effect of the insulin, increase the concentration of triglyceride in serum, increase the after-meal blood sugar concentration, reduce the sugar tolerance and the like. Therefore, the eLtaS protein can be used as the drug target for preventing and treating the insulin resistance-relevant diseases, and has an important application value.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to the application of eLtaS protein as a drug target for preventing and treating diseases related to insulin resistance. Background technique [0002] Insulin resistance (insulin resistance, IR) refers to the decline in the efficiency of insulin in promoting glucose uptake and utilization. Clinical studies have found that about 25% of normal people have insulin resistance, and about 75% of people with impaired glucose tolerance (IGT) have insulin resistance. The incidence of insulin resistance in patients with type II diabetes is about 85%. Insulin resistance is the initiating factor of many pathological changes, including type II diabetes, obesity, hypertension, hyperlipidemia, high cholesterol, coronary heart disease, insulin resistance syndrome, impaired glucose tolerance, hyperinsulinemia and other metabolic syndromes. The common pathogenesis basis of these symptoms will lead to mult...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N21/552G01N15/14A61K45/00A61P5/50A61P3/10A61P3/00A61P3/06A61P3/04A61P9/10A61P9/12A61P19/06A61P1/16
CPCA61K45/00G01N15/14G01N21/554G01N33/68
Inventor 杨光刘玉高亚萍刘成华
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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