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Fusion proteins and preparation method thereof, and application of fusion protein to preparation of medicines used for treating ophthalmic diseases and resisting inflammations and tumors

A fusion protein, ophthalmic disease technology, used in anti-tumor drugs, anti-inflammatory agents, metabolic diseases, etc., can solve the problems of single target and short polypeptide half-life, and achieve the effect of reducing the frequency of medication, prolonging the half-life, and enhancing immune function.

Active Publication Date: 2018-10-09
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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  • Fusion proteins and preparation method thereof, and application of fusion protein to preparation of medicines used for treating ophthalmic diseases and resisting inflammations and tumors
  • Fusion proteins and preparation method thereof, and application of fusion protein to preparation of medicines used for treating ophthalmic diseases and resisting inflammations and tumors
  • Fusion proteins and preparation method thereof, and application of fusion protein to preparation of medicines used for treating ophthalmic diseases and resisting inflammations and tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] (1) Fusion protein gene acquisition and expression vector construction

[0045] The sequence of the vascular inhibitory 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 connected through different linking peptides Gly Gly Gly Gly Ser Gly GlyGly Gly Ser Gly Gly Gly Ser Gly Gly Ser flexible (F) linker, AlaGluAlaAlaAlaLysGluAlaAlaAlaLysGluAlaAla AlaLysGluAlaAlaAlaAlaLysAla rigid (R) linker, linked with IL4DM-HM3 protein, designed two new Fc fusion proteins Fc-IL4DM-HM3, with flexible The amino acid sequence of protein 1 constructed with (F) linker is shown in SEQ ID NO.1, and the amino acid sequence of protein 2 constructed with rigid (R) linker is shown in SEQ ID NO.2. According to the codon preference of CHO cells, the The coding sequences of two novel Fc fusion proteins Fc-IL4DM-HM3 were optimized, and NheI restriction site, Kozak sequence, and signal peptide wer...

Embodiment 2

[0097] Inhibitory Effects of Fusion Proteins on the Proliferation of Various Tumor Cells

[0098] MTT method 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 cell B16F10, gastric cancer cell MGC-803, lung cancer cell A549, liver cancer cell Hep-G2, Breast cancer cell MDA-MB-231, colon cancer cell HCT-116, human glioma U87, cervical cancer cell Hela.

[0099] Tumor cells were incubated at 37°C, 5% CO 2 When cultured in an incubator with a density above 90%, it was digested and collected with trypsin, the cells were resuspended in 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 at 37 °C, 5% CO 2 Incubate overnight in the incubator. Dilute fusion protein 1, fusion protein 2, and the positive drug Taxol to respective pre...

Embodiment 3

[0143] Three-dimensional transwell method 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 incubated with endothelial cell culture medium containing 5% fetal bovine serum and 1×ECGS at 37°C, 5% CO 2 When cultured in an incubator to a confluence of more than 90%, the transwell method was used to detect the activity of fusion protein 1 and protein 2 in inhibiting endothelial cell migration. Endothelial cell HUVEC only used the 2nd to 8th passages. The specific operation is as follows:

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

[0146] (2) Digest the HUVEC cells cultivated to the logarithmic growth phase with 0.2% EDTA, collect, wash twice with PBS, resuspend with endothelial cell culture medium containing 0.1% BSA, count under a microscope...

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Abstract

The invention discloses fusion proteins and a preparation method thereof, and application of the fusion proteins to medicines used for treating ophthalmic diseases and resisting inflammations and tumors, belonging to the technical field of biopharmaceuticals. According to the invention, a flexible (F) or rigid (R) linker is used for fusing two polypeptides so as to obtain two bifunctional fusion proteins, respectively; that is, the two angiogenesis-inhibiting polypeptides HM-3 and IL-4 are linked with the Fc fragment of an immunoglobulin in virtue of the amino acid linker so as to form multi-functional fusion protein macromolecules. The fusion proteins can improve drug efficacy, prolong half-life and enhance stability, have the characteristics of strong action, low toxicity and the like, and is applicable to the prevention and treatment of solid tumors, various inflammations and neovascular ophthalmic diseases. The fusion proteins are expressed in eukaryotic cells by using a genetic engineering method and are obtained through affinity chromatographic purification.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and more specifically relates to a fusion protein with functions of anti-inflammation, anti-tumor and treatment of ophthalmic diseases, its preparation method and application. Background technique [0002] Diseases such as arthritis, tumors, inflammation caused by bacteria, and eye diseases such as AMD are all referred to as vascular-related diseases. [0003] Arthritis-like 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. In severe cases, it will 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 Applications(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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