Fusion protein used for inhibiting regeneration or growth of blood vessel and medical treatment application thereof

A fusion protein, immunoglobulin technology, applied in cardiovascular system diseases, peptide/protein components, hybrid peptides, etc.

Active Publication Date: 2012-01-11
CHENGDU KANGHONG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current drugs, especially macromolecular antibodies and fusion protein drugs, are all targeting one of them as a therapeutic target. Considering that angiogenesis is a complicated process, in order to improve the defects of current drugs that only target a single target, The present invention provides a fusion protein that simultaneously inhibits the two most important targets of angiogenesis. On the basis of greatly improving biological activity, it ensures that it can more effectively bind or neutralize VEGF and PDGF in vitro and in animals. Block angiogenesis and growth, so as to achieve better therapeutic effect

Method used

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  • Fusion protein used for inhibiting regeneration or growth of blood vessel and medical treatment application thereof
  • Fusion protein used for inhibiting regeneration or growth of blood vessel and medical treatment application thereof
  • Fusion protein used for inhibiting regeneration or growth of blood vessel and medical treatment application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1. Cloning of DNA sequences encoding optimized fusion proteins and construction of recombinant vectors

[0080] The FP2 and FP3 (ZL200510073595.4) constructed by our company were used to amplify FLT-1D2, KDRD3, FLT-1D4 and Fc respectively. The immunoglobulin-like domains encoding PDGFRα and PDGFRβ were obtained from human embryonic liver cells, and then used Different primers use polymerase chain reaction (PCR) to amplify the required fragments, and finally fuse the corresponding sequences to construct the DNA sequences of different fusion proteins. The structures of the 12 fusion proteins constructed in this preferred embodiment are shown in the appendix figure 1 .

[0081] Collect the well-growth extracted human embryonic liver cells (from the American standard cell bank) in the T-175 culture flask, about 1×10 7 For each cell, the total RNA of the cells was extracted using an RNA extraction kit (QIAGEN), and reverse-transcribed into cDNA using an Invitrogen...

Embodiment 2

[0133] Embodiment 2, the stable expression of fusion protein in CHO cell

[0134] The recombinant high-purity plasmids encoding KH-001 to KH-008 were introduced into CHO cells (Invitrogen) by electroporation, and G148 was added after two days in the serum-free culture medium. After the formation of G148 antibody colonies, the limited dilution method was used For clonal culture, after about 2 weeks, monoclonal cells were picked and transferred to culture dishes for expansion.

Embodiment 3

[0135] Purification of embodiment 3 fusion protein

[0136] The cell culture solution containing the fusion protein is collected, the cell fragments are taken out by centrifugation, and purified by staphylococcus protein A affinity chromatography. After protein A-Sepharose chromatography column was washed with PBS buffer to balance, the ultrafilter concentrated culture solution was loaded at a speed of 2 ml / min, and washed with PBS buffer until all unbound proteins were eluted (monitored by A280), then elute the bound protein with 100mM citric acid (pH=3), and immediately neutralize it with enough 2M Tris.

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Abstract

The invention relates to a fusion protein used for inhibiting regeneration or growth of blood vessel, and a medicinal application or pharmaceutical preparation thereof. The fusion protein is capable of inhibiting PDGF (platelet-derived growth factor) and VEGF (vascular endothelial growth factor) at the same time, and effectively treating the different diseases caused by the regeneration and growth of blood vessel, such as tumor, eye disease and the like.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and protein engineering, in particular to a fusion protein capable of effectively inhibiting angiogenesis or growth, as well as its medicinal use or pharmaceutical preparation. Background technique [0002] Under normal physiological conditions, angiogenesis is a process closely regulated by multiple links, which plays an important role in repairing and maintaining the normal function of the body. However, rapid growth of blood vessels in tumors is one of the common clinical phenomena. A large number of animal models and human clinical trials have shown that blocking the formation of new blood vessels in tumors can effectively prevent the growth of tumors and trigger the death of tumor cells, so as to achieve the therapeutic effect on tumors. Therefore, inhibition of angiogenesis is one of the important advances in the research of new anti-tumor drugs. Macromolecular anti-angiogenesi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21C12N1/19C12N5/10A61K38/17A61P35/00A61P9/10
Inventor 柯潇郑强
Owner CHENGDU KANGHONG BIOTECH
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