Method for preparing P2X7R (purinergic ligand-gated ion channel 7 receptor) immunogen

An immunogen and extracellular segment technology, which can be applied in the preparation methods of peptides, chemical instruments and methods, receptors/cell surface antigens/cell surface determinants, etc., and can solve problems such as insignificant effects.

Pending Publication Date: 2019-02-12
WEIFANG MEDICAL UNIV
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Problems solved by technology

[0004] At present, P2X7R antagonists are mainly used to study and treat related inflammatory and autoimmune diseases. For example, P2X7R

Method used

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  • Method for preparing P2X7R (purinergic ligand-gated ion channel 7 receptor) immunogen
  • Method for preparing P2X7R (purinergic ligand-gated ion channel 7 receptor) immunogen
  • Method for preparing P2X7R (purinergic ligand-gated ion channel 7 receptor) immunogen

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Embodiment Construction

[0016] The present invention will be described in detail below in combination with specific embodiments.

[0017] A method for preparing a P2X7R immunogen, carried out according to the following steps:

[0018] Step 1: Cloning of the extracellular region of P2X7R;

[0019] The amino acid sequence of the extracellular segment of P2X7R protein was optimized using codon optimization software, and the gene coding sequence of the extracellular segment of P2X7R was inserted into the expression vector pET30a by whole gene synthesis and restriction enzyme cutting sites NdeI and HindIII, and digested by restriction enzymes. The accuracy of the final expression vector was confirmed by method and sequencing. To facilitate purification, a His-tag was added to the N-terminus of the recombinant protein.

[0020] Step 2: Expression;

[0021] The constructed recombinant plasmid containing the coding sequence of the P2X7R extracellular segment gene for protein expression was transformed int...

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Abstract

The invention discloses a method for preparing a P2X7R (purinergic ligand-gated ion channel 7 receptor) immunogen. The P2X7R protein extracellular section amino acid sequence is optimized; the whole gene synthesis is used; the P2X7R extracellular section gene coding sequence is inserted into an expression carrier pET30a through restrictive enzyme cutting sites NdeI and HindIII; by using a enzyme digestion method and sequencing, the accuracy of the final expression carrier is ascertained; the built protein expression recombinant plasmid containing P2X7R extracellular section gene coding sequence is converted into a BL21 expression strain for IPTG induced expression; P2X7R protein is subjected to affinity chromatography purification in liquid supernatant; detection is performed. The method has the beneficial effects that the immunogenic P2X7R extracellular section can be obtained; the method can be used for P2X7R antibody preparation.

Description

technical field [0001] The invention belongs to the technical field of pharmacy, and relates to the preparation, purification and identification of the extracellular segment immunogen of human purinergic ligand-gated ion channel 7 receptor (P2X7R). Background technique [0002] In the state of cell injury, hypoxia or inflammation, a variety of cells in the body release a large amount of ATP, and the ATP and its decomposition products ADP and AMP that enter the extracellular space act on the P2 receptor. P2 receptors include ion channel P2X receptors (P2X1~7) and metabotropic G-protein coupled P2Y receptors (P2Y1 / 2 / 4 / 6 / 11 / 12 / 13 / 14), in which the ATP / P2X7R signaling pathway Participate in inducing the secretion of inflammatory cytokines, which is closely related to the occurrence and development of inflammation. The expression of P2X7R can be detected on various immune cells such as T lymphocytes, macrophages, dendritic cells, and mast cells. Different cells utilize the ATP / ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K14/705C07K1/22
CPCC07K14/705C12N15/70
Inventor 赵荣兰彭效祥
Owner WEIFANG MEDICAL UNIV
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