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VEGF receptor fusion protein and its use in preparation of medicament for treating eye disease

A fusion protein and receptor technology, applied in sensory diseases, drug combination, drug delivery, etc., can solve problems such as retinal scarring, detachment, and vision loss

Active Publication Date: 2009-12-09
CHENGDU KANGHONG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Abnormal growth of this highly permeable or leaky blood vessel often causes scarring on the retina, which can detach and affect vision

Method used

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  • VEGF receptor fusion protein and its use in preparation of medicament for treating eye disease
  • VEGF receptor fusion protein and its use in preparation of medicament for treating eye disease
  • VEGF receptor fusion protein and its use in preparation of medicament for treating eye disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1, FP 7 build

[0019] The fusion protein FP7 is extracted from HUVEC cells by primers flt-1D2(F), flt-1D2(R), KDR D3(F) and KDRD3-4(R) (see patent application, application number CN200510073595.4) The flt-1 and KDR gene fragments amplified on the cDNA synthesized by mRNA as a template are recombined. The specific conditions are denaturation at 95° for 30 minutes, annealing at 56° for 45 seconds, extension at 72° for 2 minutes, and PCR amplification for 30 cycles to obtain flt-1 and KDR IgG-like domains. PCR product. Using TA cloning kit, clone the PCR product into PCR2.1 plasmid, and transfect E.coli, select white colonies, add LB medium, and culture overnight. Plasmids were extracted with Qiangen plasmid extraction kit, digested and identified by sequencing. The splicing PCR (Sewing PCR) method is used to connect the flt-1 fragment, the KDR fragment and the ribonucleic acid of the partial sequence of the IgG hinge region. Design EcoR1 restriction sites in...

Embodiment 2

[0020] Example 2, FP 8 build

[0021]The fusion protein FP8 was directly amplified by PCR using FP7 as a template, and the primers used in PCR were flt-1D2 (F) and KDR D3-hing (R). The sequence of the latter is: 5'-aggtgctgggcacagtgggcatgtgtgagttttgtctttttcatggaccctgacaaatg. It includes a sequence complementary to the third immunoglobulin-like region of KDR and a partial nucleotide sequence of the human IgG Fc hinge region. The method of PCR amplification and gene cloning is identical with example 1. Finally, the PcDNA3.1 plasmid inserted with FP8 was transfected into CHO cells, and a stable cell line was obtained for protein expression. The amino acid sequence of FP8 is shown in Sequence List 2, and the nucleotide sequence is shown in Sequence List 4.

Embodiment 3

[0022] Example 3 Fusion protein and VEGF binding affinity experiments

[0023] The present invention determines the ability of various fusion proteins to bind VEGF by measuring the amount of VEGF. In this test, a certain amount of VEGF (10PM) was added to the test tube, and then diluted various fusion proteins containing different amounts were added to the test tube containing VEGF, and after mixing, they were placed in a 37 ° incubator. Save for an hour. One hour later, the free VEGF in the test tube was measured by a kit for detecting the amount of VEGF provided by R&D systems——VEGF assay Kit. The measured results are processed by the software and obtained as follows: figure 2 the result of, figure 2 show that FP 1 , FP 3 and FP 7 can effectively bind to VEGF affinity, and its binding affinity can be determined by IC 50 Said, respectively, are 11.2PM, 4.3PM and 4.1PM. This experiment proves that FP 3 and FP 7 Binding ability to VEGF is similar in vitro, and both ...

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Abstract

The present invention relates to two VEGF receptor fusion proteins, their preparation methods and their use in the treatment of eye diseases, including age-related macular degeneration, diabetic retinopathy, diabetic xanthoma glands and tumors caused by neovascular growth Caused failure of treatments such as laser photocoagulation, surgical retinal transplantation.

Description

[0001] This application is a divisional application of a Chinese invention patent application with an application date of March 31, 2006, an application number of 200610066257.2, and an invention title of "Application of VEGF receptor fusion protein in the treatment of eye diseases". Technical field: [0002] The invention relates to the application of VEGF receptor fusion protein in treating various eye diseases caused by the growth of new blood vessels. Background technique: [0003] The retinal vasculature and the choroidal vasculature are important components of the retina. Abnormal changes in the structure or function of blood vessel walls caused by trauma or disease are the main causes of vision loss or loss. For example, diabetic retinopathy (diabetic retinopathy) is the proliferation of retinal blood vessels due to diabetes, which in turn causes retinal detachment. It is the leading cause of vision loss. Growth of new blood vessels in the retina may also occur duri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K38/17A61K9/19A61K9/08A61P27/02
Inventor 俞德超
Owner CHENGDU KANGHONG BIOTECH
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