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Expression and applications of GluN1/GluN2A tetramer of human N-methyl-D-aspartate receptor

An aspartic acid, tetramer technology, applied in receptor/cell surface antigen/cell surface determinant, application, recombinant DNA technology, etc., can solve the problems of low detection rate and late detection time

Active Publication Date: 2020-06-23
CENT FOR EXCELLENCE IN BRAIN SCI & INTELLIGENCE TECH CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, for the diagnosis of patients with anti-NMDA receptor positive encephalitis, there is still a lack of good reagents and tools, and there are generally problems of low detection rate and late detection time. Therefore, this field needs to be improved on this basis

Method used

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  • Expression and applications of GluN1/GluN2A tetramer of human N-methyl-D-aspartate receptor
  • Expression and applications of GluN1/GluN2A tetramer of human N-methyl-D-aspartate receptor
  • Expression and applications of GluN1/GluN2A tetramer of human N-methyl-D-aspartate receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1. Selection of amino acid coding sequence of human N-methyl-D-aspartate receptor GluN1 / GluN2A tetramer protein and construction of recombinant expression vector

[0067] The amino acid sequence of wild-type GluN1 is shown in SEQ ID NO:1; the amino acid sequence of wild-type GluN2A is shown in SEQ ID NO:2.

[0068] Amino acid sequence of wild-type GluN1 (SEQ ID NO: 1):

[0069]

[0070] Amino acid sequence of wild-type GluN2A (SEQ ID NO:2):

[0071]

[0072] Before recombinant construction and expression, the GluN1 and GluN2A receptor proteins were sequenced to remove their intracellular sequence; at the same time, the glycine at position 612 of the GluN1 subunit was mutated into arginine, and the GluN2A subunit 656, The two glutamic acids at position 657 were mutated to arginines. So far, the amino acid coding sequence of the humanized GluN1 / GluN2A tetramer protein is as follows figure 1 shown.

[0073] The end (3' end) of the cDNA of GluN1 and GluN...

Embodiment 2

[0075] Example 2. Eukaryotic expression and purification of human N-methyl-D-aspartate receptor GluN1 / GluN2A tetramer protein

[0076] The suspended HEK293S GnTI-cells were cultured to a density of 3.0 × 10 in an incubator containing 5% carbon dioxide at 37 °C. 6 cells / ml, add baculovirus. After 12 hours of infection, sodium butyrate with a final concentration of 10 millimolar was added, and the cells were transferred to an incubator at 30°C containing 5% carbon dioxide, and the cells were harvested after continuing to culture for 48 hours.

[0077] The cells were resuspended in TBS buffer (containing NaCl, Tris-Cl pH 8.0), added with protease inhibitors, ultrasonically disrupted, rotated at 4°C for 1.5 hours, then centrifuged at 40,000g for 1 hour, and the supernatant was collected. Mix the supernatant with the Strep-tactin affinity beads, rotate at 4°C for half an hour, wash the beads with TBS buffer, and then use the eluent to elute the protein. The GluN1 / GluN2A tetrameri...

Embodiment 3

[0083] Example 3. The GluN1 / GluN2A tetramer NMDA protein of human N-methyl-D-aspartate receptor is used as an antigen for the detection of anti-NMDA antibody

[0084] Based on the immunological principle of antigen-antibody binding, the full-length membrane protein GluN1 / GluN2A subtype tetramer NMDA protein was used to coat the microtiter plate, and the enzyme-linked immunosorbent assay was used to detect the presence of anti-NMDA receptors in the cerebrospinal fluid of patients with autoimmune encephalitis. Antibodies to the body, as follows:

[0085] (1) GluN1 / GluN2A tetrameric protein is coated on the microtiter plate as an antigen

[0086] Add 100 μL of GluN1 / GluN2A tetrameric protein to each well at a concentration of 1.25 μg / mL or 2.5 μg / mL, and after overnight coating at 4 °C, wash (coating solution, blocking solution and washing solution for GluN1 / GluN2A protein need additional Add detergent (including MNG, the same below); 1% bovine serum albumin BSA 300μL / well at ro...

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Abstract

The invention relates to expression and applications of GluN1 / GluN2A tetramer of a human N-methyl-D-aspartate receptor. It is revealed that some loci at the transmembrane and extracellular domains ofsubunits GluN1 and GluN2A of the N-methyl-D-aspartate receptor are closely related to the stability of recombinant expression of the receptor and the antigen-antibody binding activity. On the basis ofperforming mutation, recombinant expression can be performed by only obtaining the transmembrane and extracellular domains of a mutant, so that an N-methyl-D-aspartate receptor mutant with stable expression and a polymer form of an ideal spatial structure can be obtained. Therefore, the problems of instability of the type of protein and poor antibody binding capacity can be solved.

Description

technical field [0001] The invention belongs to the field of biotechnology, more specifically, the invention relates to the recombinant expression of the GluN1 / GluN2A tetramer of the membrane protein N-methyl-D-aspartate (N-methyl-D-aspartate, NMDA) receptor Vector construction, expression and purification methods, and its application as an antigen are based on the principle of antigen-antibody binding, and are used for the detection of anti-NMDA antibodies. Background technique [0002] N-methyl-D-aspartate (NMDA) receptors are a class of ionotropic glutamate receptors widely present in excitatory synapses in the brain. NMDA receptors are highly permeable to calcium ions, and play an important role in synaptic transmission and synaptic plasticity, and are important molecular switches for learning and memory. Its abnormal function can cause a series of central nervous system diseases, including stroke, depression, chronic pain, schizophrenia and Alzheimer's disease, so NMDA...

Claims

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Application Information

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IPC IPC(8): C07K14/705C12N15/12C12N15/85C12N5/10G01N33/53
CPCC07K14/705G01N33/53
Inventor 竺淑佳谢春张金宝宋楠崔恒祥邓波陈向军
Owner CENT FOR EXCELLENCE IN BRAIN SCI & INTELLIGENCE TECH CHINESE ACAD OF SCI
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