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Application of fusion protein of VEGF receptor for treating disease of eye

A fusion protein, eye disease technology, applied in sensory diseases, fusion peptides, cardiovascular system diseases, etc., can solve problems such as affecting vision, detachment, and retinal scarring

Active Publication Date: 2007-02-21
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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  • Application of fusion protein of VEGF receptor for treating disease of eye
  • Application of fusion protein of VEGF receptor for treating disease of eye
  • Application of fusion protein of VEGF receptor for treating disease of eye

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1, FP 7 build

[0018] fusion protein 7 The mRNA extracted from HUVEC cells is used as a template 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 synthetic cDNA were 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

[0019] Example 2, FP 8 build

[0020] fusion protein 8 It is directly amplified by PCR using FP7 as a template, and the primer used in PCR is flt-1D 2(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 stable cell beads were obtained for protein expression. The amino acid sequence of FP8 is shown in Sequence Table 2, and the nucleotide sequence is shown in Sequence Table 3.

Embodiment 3

[0021] Example 3 Experiment of binding affinity between fusion protein and VEGF

[0022] 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, a...

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Abstract

An application of the receptor fusion protein VEGF in treating eye diseases including the age associated macula lutea lesion, retinosis of diabetic, xanthelasma of diabetic, etc is disclosed.

Description

Technical field: [0001] 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: [0002] 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 during recovery from eye injury or surgery. This type of retinal vascular proliferation is also an important cause of vision loss or blindness (Nature 438:932-938, 2005). [0003] Age-related macular degeneration (AMD) is a disease caused by degeneration of cells or tissues and proliferat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395A61K38/18A61P27/02
CPCC07K14/71A61K38/00C07K2319/30A61P9/00A61P27/02A61K38/17C07K19/00C12N15/62
Inventor 俞德超
Owner CHENGDU KANGHONG BIOTECH
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