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Insulin-like growth factor binding protein 4 mutant and pharmaceutical application thereof

A growth factor, insulin-like technology, applied in the fields of bioengineering and biomedicine, can solve the problems of high cost, toxic and side effects, poor effect and prognosis

Active Publication Date: 2019-12-17
朱伟东
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the means for treating diabetic ischemic diseases are relatively limited, and the therapy has many disadvantages such as complicated operation, high cost, poor effect and prognosis, and relatively large toxic and side effects.
There is currently no drug therapy for effective prevention and treatment of diabetic ischemic diseases in this field, so there is an urgent need to develop new drugs with excellent effects, no toxic side effects, and capable of replacing existing treatment methods

Method used

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  • Insulin-like growth factor binding protein 4 mutant and pharmaceutical application thereof
  • Insulin-like growth factor binding protein 4 mutant and pharmaceutical application thereof
  • Insulin-like growth factor binding protein 4 mutant and pharmaceutical application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Embodiment 1, IGFBP4-H95P, R147A, the preparation of R149A protein

[0109] 1. Construction of recombinant vectors expressing IGFBP4-H95P, R147A, R149A mutants

[0110] Human mRNA extracted by conventional methods was reverse-transcribed to obtain cDNA. Using the cDNA as a template, use the primer pair 1 corresponding to both sides of the ORF frame of the IGFBP-4 gene (SEQ ID NO: 1), obtain the cDNA sequence by reverse transcription, and perform PCR amplification using DNA polymerase: obtain human IGFBP-4 amplification product. Detected by 1% agarose gel electrophoresis, the length of the amplified product was consistent with the predicted value.

[0111] The obtained coding nucleotide sequence of human IGFBP-4 was cloned into pcDNA3.1V5-6HIS vector (purchased from Agilent Technologies Genomics) to obtain the recombinant vector pcDNA3.1V5-6HIS / IGFBP4. This recombinant vector can be used to transfect HEK293 cells to express wild-type IGFBP-4 protein by the same proced...

Embodiment 2

[0132] Example 2, IGFBP4-H95P, R147A, R149A protein does not bind IGF I or IGF II (ie IGFs)

[0133] Obtain wild IGFBP4 protein and mutated IGFBP4-H95P as in Example 1, R147A, R149A protein, and SDS-PAGE (100mM Tris, pH 6.8, 10% SDS, 0.01% phenol blue) loading buffer containing β-mercaptoethanol Mix and separate by 10% SDS-PAGE gel. The gel protein was transferred to a nitrocellulose membrane for conventional 125I-IGF-I and 125I-IGF-II (purchased from Amersham Pharmacia Biotech, Freibury, Germany) Western ligand blotting ( 125 I-IGF-I and 125 I-IGF-II Western ligand blotting analysis). The amount of natural IGFBP4 protein used and mutated IGFBP4-H95P, R147A, and R149A protein was 105ng each; 125 I-IGF-I and 125 The dosage of I-IGF-II was 250 μCi / μg protein. The analysis results are shown in image 3 . image 3 In , the IGF-binding activity of the protein was quantified by gamma counting the analyzed protein bands, and the background radioactivity of the nitrocellulose m...

Embodiment 3

[0134] Example 3, IGFBP4-H95P, R147A, R149A proteins do not bind to IGF II

[0135] IGF II is known to stimulate DNA synthesis in human osteosarcoma cell MG63, and this stimulation can be neutralized by the binding protein IGFBP4 of IGFs.

[0136] Human osteosarcoma MG63 cells (purchased from American Type Culture Collection; Rockville, MD; CRL 1427) were cultured in vitro until the cells were basically confluent, and fresh DMEM medium (supplemented with 0.1% calf serum) was replaced for 20 hours. , adding natural IGFBP4 and IGFBP4-H95P, R147A, R149A proteins as obtained in Example 1 at different concentrations to the same volume of medium for culturing the same number of cells, respectively. BSA at the same concentration was used as a control. Under normal cell culture conditions, incubate for 48h. Collect the cultured cells, use the CytQUANT cell proliferation kit (purchased from Life Technologies, Beijing), mix the collected cells with lysis buffer and dyeing solution acc...

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Abstract

The invention relates to an insulin-like growth factor binding protein 4 mutant and pharmaceutical application thereof. According to the mutant, part of functions of natural insulin-like growth factorbinding protein are removed, in-vivo degradation caused by specific protease is avoided, and the capacity of the mutant for preventing and treating ischemic diseases by inhibiting a Wnt signal channel is improved. The mutant provided by the invention can prevent, relieve or treat ischemic diseases, and avoids various potential health risks induced by combining IGFs.

Description

technical field [0001] The present invention relates to the fields of bioengineering and biomedicine; specifically, the present invention relates to the insulin-like growth factor binding protein 4 mutant and its pharmaceutical use. Background technique [0002] Diabetes is a group of metabolic diseases characterized by hyperglycemia. During diabetes, long-term high blood sugar causes chronic damage and dysfunction of various tissues, especially the eyes, kidneys, heart, blood vessels, and nerves. According to the statistics released by WHO in 2016, the total number of diabetic patients in China is 110 million, accounting for a quarter of the total number of patients in the world. In the not-too-distant future, it will reach 150 million. Diabetic cardiovascular disease is considered to be the leading cause of death in diabetes, and among cardiovascular diseases, myocardial ischemia is the most common. [0003] Myocardial ischemia refers to a pathological state in which ca...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12C12P21/02A61K38/17A61P9/10
CPCC07K14/4743A61P9/10A61K38/00
Inventor 朱伟东
Owner 朱伟东
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