N-methyl-D-aspartic acid receptor recombinant antigen, preparation method thereof and kit containing recombinant antigen

A technology of aspartic acid and recombinant antigen, applied in the direction of receptor/cell surface antigen/cell surface determinant, botany equipment and method, biochemical equipment and method, etc. It is difficult to obtain recombinant protein and other problems, so as to reduce the cost of preparation, reduce the cost of medical treatment, and achieve the effect of accurate test results

Pending Publication Date: 2022-04-19
迪亚莱博(张家港)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Since NMDAR protein is a membrane protein with a large molecular weight, prokaryotic cells cannot be used to express the full-length protein, and natural purification is also difficult and the production yield is extremely small. At the same time, the interaction of different subunits of NMDAR plays a role in stabilizing the protein structure. Expression alone It is difficult to obtain a recombinant protein with activity close to the natural protein in the extracellular region or a single subunit, and the protein stability is not good

Method used

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  • N-methyl-D-aspartic acid receptor recombinant antigen, preparation method thereof and kit containing recombinant antigen
  • N-methyl-D-aspartic acid receptor recombinant antigen, preparation method thereof and kit containing recombinant antigen
  • N-methyl-D-aspartic acid receptor recombinant antigen, preparation method thereof and kit containing recombinant antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] (1) Polynucleotide synthesis.

[0058] Search the genebank to obtain the native cDNA sequences of NMDAR subunits GluN1, GluN2A, GluN2B, respectively. All sequences should be searched from human gene banks. The natural subunit GluN1 sequence is shown in SEQ ID NO:1, the natural subunit GluN2A sequence is shown in SEQ ID NO:2, and the natural subunit GluN2B sequence is shown in SEQ ID NO:3 shown.

[0059] The NMDAR antigen sequence was analyzed, and the amino acid position of glycosylation in the sequence was deduced, including Asn61, Asn239, Asn350, Asn471, Asn491 and Asn771 in the sequence of GluN1a. Each of the asparagine was mutated into glutamine by the overlapping mutation method. It was verified by experiments that, except for Asn239 and Asn491, the protein expression and activity of the other asparagine mutations were improved to varying degrees. Therefore, all glycosylated amino acids in GluN1a except Asn239 and Asn491 were mutated to glutamine.

[0060] Thr...

Embodiment 2

[0086] Adopt the same method as Example 1 to prepare pFastBac Dual-GluN1a-GluN2B plasmid, then adopt the same method as Example 1 to transfect pFastBac Dual-GluN1a-GluN2B plasmid, express GluN1a and GluN2B simultaneously in sf9 insect cells and separate, The purified NMDAR recombinant protein is labeled as NMDAR recombinant antigen 2, the yield of NMDAR recombinant antigen 2 is 28mg / L, and the protein purity after purification and polishing is 90%.

Embodiment 3

[0088] Using the same method as in Example 1 and Example 2, the pFastBac Dual-GluN1a-GluN2A plasmid and the pFastBac Dual-GluN1a-GluN2B plasmid were simultaneously transfected, GluN1a / GluN2A and GluN1a / GluN2B were simultaneously expressed in sf9 insect cells, separated and purified NMDAR recombinant protein, marked as NMDAR recombinant antigen 3, the yield of NMDAR recombinant antigen 3 is about 23mg / L, and the protein purity after purification and polishing is 90%.

[0089] Protein activity comparison

[0090] Dilute the above-mentioned NMDAR recombinant antigen 1, NMDAR recombinant antigen 2, NMDAR recombinant antigen 3, and NMDAR protein based on the natural cDNA table to 2 μg / mL with PBS, coat each well of the microplate with 0.2 μg protein, and coat at 37 °C. After 2 hours, the coating solution was discarded, and the microwells were washed twice with 100 μL PBS / 0.1% Tween20. After washing, add 100 μL of test samples to the microwells coated with different NMDAR antigen...

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Abstract

The invention provides an N-methyl-D-aspartic acid receptor recombinant antigen, a preparation method thereof and a kit containing the recombinant antigen. The recombinant antigen comprises subunit GluN1a recombinant protein, and the amino acid sequence of the subunit GluN1a recombinant protein is as follows: Asn61, Asn350, Asn471 and Asn771 in the sequence shown in SEQ ID NO: 1 are mutated into Gln, Cys22 is mutated into Ser, Glu595 and Glu597 are mutated into Ser, and Glu598 is mutated into Thr. The N-methyl-D-aspartic acid receptor recombinant antigen further comprises a natural subunit GluN2A recombinant protein and/or a subunit GluN2B recombinant protein, and the amino acid sequence of the N-methyl-D-aspartic acid receptor recombinant antigen is characterized in that Asn348 in the sequence as shown in SEQ ID NO: 3 is mutated into Asp, and Cys838 and Cys849 in the sequence as shown in SEQ ID NO: 3 are mutated into Ser.

Description

technical field [0001] The invention relates to the field of diagnostic raw material reagents, in particular to an N-methyl-D-aspartate receptor recombinant antigen, a preparation method thereof and a kit containing the recombinant antigen. Background technique [0002] N-methyl-D-aspartate receptor (NMDAR) is one of the three ionotropic glutamate receptors, located in the human brain as an ion channel, in the development of cranial nerves and neurons in the human central system played an important role in the process of change. The main function of NMDAR is the induction of nerve signal transduction. When the brain cell membrane potential difference relieves the barrier of magnesium ions and NMDAR combines with glutamic acid and glycine, NMDAR is activated and opened, and positively charged ions pass through the cell membrane to achieve the transmission of nerve signals. Purpose. N-methyl-D-aspartate specifically binds to NMDARs, which do not bind to other glutamate recep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C12N15/12C12N15/866C12N5/10G01N33/68
CPCC07K14/705C12N15/86C12N5/0601G01N33/6893G01N33/6854C12N2710/14043C12N2510/00G01N2800/24G01N2800/28
Inventor 葛霄鹏
Owner 迪亚莱博(张家港)生物科技有限公司
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