Fusion protein of antibody targeted complement regulatory factor for treating myasthenia gravis

A fusion protein and complement technology, applied in the field of medicine and biology, can solve problems such as the reduction of DAF gene expression

Inactive Publication Date: 2011-07-06
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Abstract
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Problems solved by technology

In addition, we detected DAF by real-time quantitative RT-PCR and found that the gene expression of DAF in extraocular muscle was significantly reduced in animal models of myasthenia gravis

Method used

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  • Fusion protein of antibody targeted complement regulatory factor for treating myasthenia gravis
  • Fusion protein of antibody targeted complement regulatory factor for treating myasthenia gravis
  • Fusion protein of antibody targeted complement regulatory factor for treating myasthenia gravis

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Embodiment Construction

[0014] Specifically, the preparation of the fusion protein (scFv-DAF) of the above-mentioned single-chain antibody specifically binding to the acetylcholine receptor and the complement regulatory factor (DAF) can be carried out according to the following steps:

[0015] 1. Cloning of scFv-DAF gene

[0016] Using the plasmid pHEN1 containing anti-human AChR scFv1956# and the plasmid RD37 (NCBI AccessionNo.AB026902) containing DAF cDNA as templates, primers 1, 2 and primers 3 and 4 were used as upstream and downstream primers, respectively, using Primer STAR TM HS DNA polymerase amplifies the structural gene of scFv1956# and the SCR1-4 fragment of DAF. Primers 2 and 3 added a connecting sequence (G 4 S 1 ) 3 , Primers 1 and 4 respectively added two restriction sites NdeI and BamHI.

[0017] Primer 1 (scFv5' primer): The underlined part is the added NdeI restriction site.

[0018] 5' TTT CATATG CAGGTCCA ATTTGTAGAG3';

[0019] Primer 2 (connecting strand 3' primer): The s...

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Abstract

The invention discloses a fusion protein scFV-CD55 of an acetylcholine receptor-resistant single-chain antibody targeted complement regulatory factor CD55. The CD55 is targeted to neuromuscular transmission through single-chain antibody to inhibit combination of the antibody of a pathogenic acetylcholine receptor and the pathogenic acetylcholine receptor, block the cascade of a complement, protect the acetylcholine receptor and eliminate immune injury caused by complement system activation, so that myasthenia gravis is treated in a targeted way. The scFv-CD55 is obtained by coupling acetylcholine receptor-resistant single-chain antibody to the amino terminal of the complement regulatory factor CD55(SCR1-4) through (G4S1)3 connecting peptide. By means of genetic engineering, soluble scFV-CD55 can be obtained through prokaryotic expression and purification. Experiments prove that: the fusion protein scFV-CD55 maintains the affinity of the acetylcholine receptor and the complement inhibition function of the CD55, the complement inhibition function of the scFV-CD55 is obviously improved in cellular experiments, and the complement deposition on immune injury parts is reduced. The invention develops a new biological agent for treating the myasthenia gravis, and the biological agent has wide application prospect.

Description

1. Technical field [0001] The invention belongs to the field of medical biotechnology, and specifically relates to gene cloning, expression of foreign genes in prokaryotic systems, protein purification, inclusion body renaturation, acetylcholine receptor-specific single-chain antibody and complement regulatory factor (DAF) fusion protein ( scFv-DAF) in vitro activity determination and complement attack protection test at the cellular level. The purpose is to develop a new biological agent scFv-DAF for the treatment of myasthenia gravis, which can inhibit the combination of the pathological pathogenic factor-acetylcholine receptor autoantibodies and the acetylcholine receptor and the activation of the complement system in patients with myasthenia gravis, Treatment of myasthenia gravis by protecting acetylcholine receptors and eliminating immune damage caused by activation of the complement system. It has important application prospect and significance. 2. Background technolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/09C12N15/62G01N33/53C12Q1/02
Inventor 李柱一宋琛徐志凯徐江林宏宿长军苗建亭李宏增
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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