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Multifunctional fusion polypeptide and its preparation method and application

A fusion polypeptide and multifunctional technology, applied in the field of medicine, can solve the problems of poor vascular specificity and selectivity, unclear target of multifunctional fusion polypeptide, etc., to prevent or treat rheumatoid arthritis, prevent Or the effect of treating retinal neovascular eye disease and reducing neovascular plexus

Active Publication Date: 2016-08-17
NANJING ANJI BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] Aiming at the problems of the existing multifunctional fusion polypeptides with unclear targets and poor specificity and selectivity for blood vessels, the present invention provides a multifunctional fusion polypeptide and its preparation method and application, which can treat various entities Tumors, rheumatoid arthritis and neovascular eye diseases have good preventive and therapeutic effects, increasing the scope of its indications

Method used

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  • Multifunctional fusion polypeptide and its preparation method and application
  • Multifunctional fusion polypeptide and its preparation method and application
  • Multifunctional fusion polypeptide and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1 Preparation and inspection of multifunctional fusion polypeptide polypeptide

[0058] The polypeptide was synthesized by solid-phase synthesis, separated and purified by preparative HPLC, and the purity of the polypeptide was determined by analytical RP-HPLC.

[0059] Polypeptide I-VI solid-phase synthesis method uses Fmoc-wang-resin or Fmoc-CTC-resin (Gill Biochemical Co., Ltd.) as the starting material, and then uses protected amino acids to connect dipeptides to tetrapeptides in sequence, and the peptide connection work is completed After that, fully wash, then cut the peptide, and post-process the crude product of the multifunctional fusion polypeptide. The crude product was dissolved, purified twice by preparative high-performance liquid phase, concentrated and freeze-dried to obtain the pure product, and finally purified for the third time to obtain the refined peptide product. The method can not only ensure the synthesis efficiency, but also improve...

Embodiment 2

[0092] Inhibitory effect of multifunctional fusion polypeptide on the proliferation of human retinal vascular endothelial cells (HRCEC)

[0093] MTT assay was used to detect the activity of the multifunctional fusion polypeptide in inhibiting the proliferation of human retinal vascular endothelial cells. HRCEC cells were incubated at 37°C, 5% CO 2 When cultured in an incubator with a density of more than 90%, it was digested with trypsin and collected, and the cells were resuspended in culture medium and counted under a microscope, and the cell concentration was adjusted to 3.0×10 4 cells / mL, seed the cell suspension into a 96-well plate, 100 μL per well, and inoculate at 37°C, 5% CO 2 Incubate overnight in the incubator. After the cells are completely adhered to the wall, the multifunctional fusion polypeptide polypeptide is used as the administration group, Avastin is used as the positive control group, and the culture solution without any drug is used as the blank control...

Embodiment 3

[0117] Effect of angiogenesis blocking agent polypeptide on corneal neovascularization in BALB / c mice

[0118] (1) Preparation of corneal neovascularization model induced by alkali burn in BALB / c mice:

[0119] Thirty-five healthy BALB / c mice, male, weighing 20-25 g, were examined under a slit-lamp microscope for the anterior segments and appendages of both eyes to exclude eye lesions. 0.3% ofloxacin eye drops were instilled 1 day before the preparation of the alkali burn model, twice a day. After mice were anesthetized by intraperitoneal injection of 1.8% Avertin, a single-layer filter paper sheet with a diameter of 2 mm was clamped with tweezers, immersed in 1mol / L NaOH solution to make it saturated, excess liquid was removed, and the filter paper sheet was placed in BALB / c 40S in the center of the mouse cornea, discard the filter paper, and immediately rinse the burn area and conjunctival sac with 15 mL of PBS for 1 min. Wipe off excess water with a cotton swab, and rota...

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Abstract

The invention discloses a multifunctional fusion polypeptide, a preparation method and application thereof, and belongs to the field of bioengineering and pharmacy. The multifunctional fusion polypeptide is one of six polypeptides or any combination thereof, and can be used for the prevention and treatment of solid tumors, rheumatoid arthritis and neovascular eye diseases. The polypeptide of the present invention is prepared by artificial synthesis, and is characterized in that by inhibiting the proliferation and migration of vascular endothelial cells, inhibiting angiogenesis, and then treating diseases related to angiogenesis, including solid tumors, rheumatoid arthritis, and neovascular eye diseases Wait.

Description

technical field [0001] The invention relates to the field of medicine, in particular to a class of multifunctional fusion polypeptide and its preparation method and its application in the preparation of medicines for treating solid tumors, rheumatoid arthritis and neovascular eye diseases. Background technique [0002] Angiogenesis refers to the process of forming new blood vessels by sprouting on the basis of primitive vascular plexus or existing blood vessels. Angiogenesis plays an important role in wound healing, placenta formation and embryonic development. At the same time, however, angiogenesis also occurs in a variety of pathological conditions, including: tumor growth and metastasis; inflammatory disorders such as rheumatoid arthritis, psoriasis, osteoarthritis, inflammatory bowel disease Crohn's disease, ulcerative colon, and other inflammatory disorders; ocular neovascularization, such as retinopathy of prematurity, ischemic retinopathy, retinal vein or artery occl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K1/06A61K38/16A61P35/00A61P29/00A61P19/02A61P27/02
Inventor 康梦实李梦玮
Owner NANJING ANJI BIOLOGICAL TECH CO LTD
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