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A fusion protein and its preparation method and its application in the preparation of ophthalmic diseases, anti-inflammatory and anti-tumor drugs

A fusion protein and ophthalmic disease technology, applied in the direction of anti-tumor drugs, anti-inflammatory agents, metabolic diseases, etc., can solve the problems of single target, short half-life of polypeptides, etc., to reduce the frequency of medication, prolong the half-life, and increase drug compliance Effect

Active Publication Date: 2022-03-25
徐寒梅
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Aiming at the problems of short half-life of existing polypeptides and a single target, the present invention provides a mammalian cell expression method to connect two different active polypeptides and proteins to solve the following problems: 1. Improving the affinity between polypeptide molecules and targets, cell Toxic, enhance the curative effect of polypeptide molecules

Method used

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  • A fusion protein and its preparation method and its application in the preparation of ophthalmic diseases, anti-inflammatory and anti-tumor drugs
  • A fusion protein and its preparation method and its application in the preparation of ophthalmic diseases, anti-inflammatory and anti-tumor drugs
  • A fusion protein and its preparation method and its application in the preparation of ophthalmic diseases, anti-inflammatory and anti-tumor drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] (1) Acquisition of fusion protein gene and construction of expression vector

[0045] The sequence of angiostatic polypeptide HM-3 is shown in SEQ ID NO.5, the sequence of interleukin 4 is shown in SEQ ID NO.6, and the human immunoglobulin IgG1-Fc region (SEQ ID NO.7) is passed through different connecting peptides Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser flexible (F) linker, AlaGluAlaAlaAlaAlaLysGluAlaAlaAlaAlaLysGluAlaAla AlaLysGluAlaAlaAlaLysAla rigid (R) linker, connected with IL4DM-HM3 protein, designed two new Fc fusion proteins Fc-IL4DM-HM3, with flexible (F) The amino acid sequence of protein one constructed by linker is shown in SEQ ID NO.1, and the amino acid sequence of protein two constructed by rigid (R) linker is shown in SEQ ID NO.2. According to CHO cell codon preference, for 2 The coding sequence of a novel Fc fusion protein Fc-IL4DM-HM3 was optimized, and NheI restriction site, Kozak sequence and signal peptide were all introduce...

Embodiment 2

[0097] Inhibitory effect of fusion protein on proliferation of various tumor cells

[0098] MTT assay was used to detect the inhibitory effect of the integrin blocker fusion protein obtained in Example 1 on the proliferation of various tumor cells, including melanoma cells B16F10, gastric cancer cells MGC-803, lung cancer cells A549, liver cancer cells Hep-G2, Breast cancer cells MDA-MB-231, colon cancer cells HCT-116, human glioma U87, cervical cancer cells Hela.

[0099] Tumor cells were incubated at 37°C, 5% CO. 2 When the density was above 90%, the cells were collected by trypsin digestion. The cells were resuspended in the culture medium and counted under a microscope. The cell concentration was adjusted to 3.0×104 cells / mL, and the cell suspension was inoculated into a 96-well plate. medium, 100 μL per well, and incubate at 37 °C, 5% CO 2 Incubate overnight in an incubator. The fusion protein 1, fusion protein 2 and positive drug Taxol were diluted with culture medium...

Embodiment 3

[0143] Three-dimensional transwell assay to detect the activity of fusion protein 1 and protein 2 in inhibiting the migration of human umbilical vein endothelial cells

[0144] Human umbilical vein endothelial cells (HUVEC) were cultured in endothelial cell culture medium containing 5% fetal bovine serum and 1×ECGS at 37°C, 5% CO 2 When the incubator was cultured to a confluency of more than 90%, the transwell method was used to detect the activity of fusion protein 1 and protein 2 to inhibit endothelial cell migration. Only the second to eighth passages of endothelial cell HUVEC were used. The specific operations are as follows:

[0145] (1) Dilute 1:4 with 10 mg / mL Matrigel in DMEM medium, spread on transwell membrane, and air dry at room temperature;

[0146] (2) The HUVEC cells cultured to the logarithmic growth phase were digested with 0.2% EDTA, collected, washed twice with PBS, resuspended in endothelial cell culture medium containing 0.1% BSA, counted under a microscop...

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Abstract

The invention discloses a fusion protein, its preparation method and its application in the treatment of ophthalmic diseases, anti-inflammatory and anti-tumor drugs, and belongs to the technical field of biopharmaceuticals. The present invention uses a flexible (F) or rigid (R) Linker to fuse two polypeptides to obtain two bifunctional fusion proteins, namely two angiogenesis inhibitory polypeptides HM‑3, IL‑4 and immunoglobulin Fc fragments The multifunctional fusion protein macromolecules are connected by amino acid Linker, which can improve drug efficacy, prolong half-life, enhance stability, and have the characteristics of strong effect and low toxicity. It can be used for solid tumors and various inflammatory and neovascular ophthalmic diseases. prevention and treatment. The fusion protein is expressed in eukaryotic cells by genetic engineering and purified by methods such as affinity chromatography.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and more particularly relates to a fusion protein with functions of anti-inflammatory, anti-tumor and treating ophthalmic diseases, and a preparation method and application thereof. Background technique [0002] Diseases such as arthritis, tumors, inflammation caused by bacteria, and ophthalmic diseases such as AMD are all referred to as vascular-related diseases. [0003] Arthritis inflammatory diseases refer to inflammatory diseases that occur in human joints and their surrounding tissues, and can be divided into dozens of types. There are more than 100 million arthritis patients in my country, and the number is increasing. The clinical manifestations are joint redness, swelling, heat, pain, dysfunction and joint deformity, and severe cases lead to joint disability and affect the quality of life of patients. These mainly include rheumatoid arthritis, rheumatoid arthritis, osteoarth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/18A61K38/20A61P29/00A61P35/00A61P27/02A61P19/02A61P19/08A61P19/06A61P37/02A61P17/06A61P3/10A61P31/04A61P31/12A61P33/02
CPCC07K14/475C07K14/5406A61K38/00C07K2319/30A61P35/00A61P27/02A61P19/02A61P29/00
Inventor 徐寒梅
Owner 徐寒梅
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