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Method for preparing humanized immune globulin capable of antagonizing vessel endothelium cell growth factor and combined uses thereof

A technology of immunoglobulin and vascular endothelium, applied in the direction of anti-animal/human immunoglobulin, biochemical equipment and methods, botany equipment and methods, etc., can solve the problems of low expression level and difficult protein extraction

Inactive Publication Date: 2008-03-05
SUZHOU STAINWEI BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression level of the sFlt-1 receptor gene in the body is low, so it is difficult to extract and isolate a sufficient amount of protein for further research; and the soluble KDR receptor has not been reported so far

Method used

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  • Method for preparing humanized immune globulin capable of antagonizing vessel endothelium cell growth factor and combined uses thereof
  • Method for preparing humanized immune globulin capable of antagonizing vessel endothelium cell growth factor and combined uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Construction of expression vectors for fusion immunoglobulin PF1 (Flt1-D123-IgM) and fusion immunoglobulin PF2 (Flt1-D123-IgG)

[0065] The monomer structures of protein PF1 (Flt1-D123-IgM) and protein PF2 (Flt1-D123-IgG) in the present invention are shown in FIG. 1 . The common feature of these two immunoglobulin monomers is that their N-segments contain the extracellular immunoglobulin D1, D2 and D3 regions of the human Flt1 receptor; and their C-segments contain immunoglobulin IgM-Fc or IgG- Fc segment. The cloning of the genes encoding these two fusion immunoglobulins and the construction of their vectors are as follows:

[0066] 1. Cloning of human Flt1-D123 gene fragment.

[0067] The gene fragments encoding the extracellular immunoglobulin regions D1, D2 and D3 of the human Flt1 receptor were cloned from healthy human umbilical vein endothelial cells (HUVEC) by polymerase chain reaction (RT-PCR) . Its specific implementation is as follows: the tota...

Embodiment 2

[0089] Embodiment two: the construction of the expression vector of PK1 (KDR-D123-IgM) and PK2 (KDR-D123-IgG)

[0090] Like the aforementioned protein PF1 (Flt1-D123-IgM) and protein PF2 (Flt1-D123-IgG), PK1 (KDR-D123-IgM) and PK2 (KDR-D123-IgG) in the present invention are also fusion immunoglobulins protein. The monomer structures of these two immunoglobulins are shown in Figure 2, and their common feature is that their N-segment contains the extracellular immunoglobulin D1, D2 and D3 structural regions of the human KDR receptor; and their C-segment contains Immunoglobulin IgM-Fc or IgG-Fc fragment. The cloning of recombinant genes encoding these two fusion immunoglobulins and the construction of their delivery vectors are as follows.

[0091] 1. Cloning of human KDR-D123 gene fragment.

[0092] The gene fragments of extracellular immunoglobulin regions D1, D2 and D3 of human KDR were also cloned by polymerase chain reaction (PCR). The PCR primers used for PCR amplificat...

Embodiment 3

[0104] Example 3: Expression, production, separation and purification of recombinant immunoglobulin.

[0105] In view of the fact that the recombinant immunoglobulin in the present invention has a large molecular weight and contains disulfide bonds S_S and sugar groups, it is better to express it in mammalian cells. In the implementation of the present invention, the expression and production of immunoglobulin KDR-D123-IgM and immunoglobulin KDR-D123-IgG in mammalian cells is taken as an example. The expression and production of fusion immunoglobulin PF1 (Flt1-D123-IgM) and fusion immunoglobulin PF2 (Flt1-D123-IgG) should be roughly the same as this example.

[0106]The expression and production of immunoglobulin KDR-D123-IgM and KDR-D123-IgG in mammalian cells includes three major steps: 1) transfect the expression plasmid containing the fusion immunoglobulin recombinant gene into CHO cells; 2) recombinant gene Identification of expression-positive CHO cells and screening of...

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Abstract

The present invention relates to biotechnology, belongs to the field of recombinant gene engineering, and is six kinds of humanized fused immunoglobulins prepared through recombinant gene engineering and cell culturing process and capable of recognizing and combining with vascular endothelial cell growth factor specifically and their application. These six kinds of fused immunoglobulins feature that each of them has immunoglobulin-like structure regions capable of being combined with VEGF in the N-end and human immunoglobulin constant region in the C-end. These fused immunoglobulins have the bioactivity of high combination with VEGF and the bioactivity of immunoglobulin constant region, and can neutralize the VEGF inside adsorbed body to antagonize and inhibit fission and proliferation of vascular endothelial cell. These fused immunoglobulins are applied as VEGF neutralizing adsorbent alone or in combination.

Description

technical field [0001] The invention belongs to the field of biotechnology-recombinant genetic engineering. More specifically, the present invention reports that six humanized fusion immunoglobulins (three of which are IgM; three of which are IgM; IgG) and its combination applications. The common feature of these six fusion immunoglobulins is that their N-terminals contain several immunoglobulin-like structural regions that can bind VEGF, and their C-terminals contain human immunoglobulin constant regions. This type of fusion immunoglobulin retains the biological activity of highly combining with VEGF and the dual activity of the constant region of immunoglobulin. This type of fusion immunoglobulin can antagonize and inhibit the division and proliferation of vascular endothelial cells by neutralizing the VEGF adsorbed in the body. The six fusion immunoglobulins are used as neutralizing adsorbents for VEGF, and can be used individually or in combination to maximize the drug ...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13C12N15/85G01N33/53G01N33/68A61K39/395
Inventor 周群敏徐一清陶飞龙胡红群
Owner SUZHOU STAINWEI BIOTECH INC
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