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Fusion protein immunosuppressive agent and preparation method and application thereof

A fusion protein and fusion gene technology, applied in the fields of biotechnology and medicine, can solve the problems of low efficiency of immunosuppression

Inactive Publication Date: 2011-06-08
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Soluble HLA-G naturally produced by cells is mainly in the form of monomers, and the dimer form accounts for only 5%, and its immunosuppressive effect is relatively low

Method used

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  • Fusion protein immunosuppressive agent and preparation method and application thereof
  • Fusion protein immunosuppressive agent and preparation method and application thereof
  • Fusion protein immunosuppressive agent and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The construction of embodiment 1 fusion protein sHLA-G / IgG eukaryotic expression vector

[0060] Select the HLA-G5-pcDNA3.0 plasmid and normal human peripheral blood cells to obtain the sHLA-G heavy chain extracellular segment α1-α3 region gene and the human IgG1 heavy chain constant region (IgG-Fc segment) encoding gene, and IgG-Fc Insert the gene fragment of the eukaryotic expression plasmid pcDNA3.1+ to form pcDNA3.1+ / [IgG-Fc], and then recombine the sHLA-G extracellular segment gene into pcDNA3.1+ / [IgG-Fc] to construct a pcDNA3.1+ / [sHLA-G / IgG] plasmid of fusion gene, see the technical route figure 1 .

[0061] 1.1 Obtain the gene fragments of the fusion protein sHLA-G / IgG

[0062] The gene of the recombinant sHLA-G / IgG fusion protein is formed by fusing the extracellular segment (α1-α3 region) of HLA-G1 with the encoding gene of the CH1-CH3 segment of IgG1. The IgG-Fc segment gene amplified by RT-PCR method includes the CH1-CH3 region coding gene of IgG1 and res...

Embodiment 2

[0067] Example 2 Expression, purification and identification of fusion protein sHLA-G / IgG

[0068] 2.1 Establishment of cell lines expressing fusion protein sHLA-G / IgG

[0069] In order to obtain higher protein expression and exclude the interference of MHC molecules and IgG expressed by the cells themselves, we chose LCL 721.221 cells as the expression cells, which are defective in the expression of HLA class I molecule heavy chain and IgG1 heavy chain [30] , but high expression of HLA class I light chain (β 2 M). The plasmid pcDNA3.1+[sHLA-G / IgG] was transfected into 721.221 cells by electroporation, and selected using G418-containing selective medium. After the stable growth of cell clones in G418 was obtained, monoclonalization was carried out, and the expression level of HLA-G fusion protein was further detected, and the clone with efficient and stable expression of the target protein was selected as dimer-721.221, and a large number of cells were expanded and preserved...

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Abstract

In the invention, genes that an alpha 1- alpha 3 area of a code HLA-G (Hepatomaliver Antigen Gene) and a CH1-CH3 area of human IgG (Intravenous Gamma Globulin) are recombined, the obtained infusion protein sHLSA-G / IgG is expressed, and the experiment shows that the infusion protein can significantly inhibit reaction of same reactive T cells at nano-mole level and is more effective than an HLA-G individual in a natural state. The inhibition efficiency of the infusion protein is nearly 10 times of that of the HLA-G individual and the infusion protein not only can inhibit the proliferation of same reactive T cells CD8+T, CD4+T cells, generation of same reactive specific CTL (Cytotoxic T Lymphocyte), but also can regulate the expression of surface ILT2 of same reactive CD8+T so as to further promote the inhibition of the infusion protein. The infusion protein and the acceptor have high affinity, high efficiency for inhibiting same T cell response is realized, and the HLA-G is a multi-state rare human component, therefore, the infusion protein immunosuppressive agent can be used as a safe and efficient immunosuppressive agent.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine, in particular to fusion protein technology and its application in medicine, in particular to fusion protein HLA-G / IgG as a safe and efficient immunosuppressant for the treatment and prevention of transplant rejection and autoimmunity diseases and inflammatory diseases. Background technique [0002] Immunosuppressants are widely used in the treatment of transplant rejection, autoimmune diseases and inflammatory diseases. Commonly used are cyclosporine A, FK506, anti-human CD3 monoclonal antibody, etc., and their common side effects are secondary infection, tumor induction, and drug toxicity. [0003] HLA-G is a non-classic HLA-I molecule. Under physiological conditions, it is only expressed on the maternal-fetal interface and on the surface of some immune-privileged tissue cells, suggesting that it is a class of molecules that induce immune tolerance and its potential application in all...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/85C12P21/02C12N5/10A61K38/17A61P37/06
Inventor 梁智辉吴雄文翁秀芳钟茂华杨敬
Owner HUAZHONG UNIV OF SCI & TECH
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