CN114369154B - N-methyl-D-aspartate receptor recombinant antigen, preparation method thereof and kit containing recombinant antigen - Google Patents

N-methyl-D-aspartate receptor recombinant antigen, preparation method thereof and kit containing recombinant antigen Download PDF

Info

Publication number
CN114369154B
CN114369154B CN202111552136.XA CN202111552136A CN114369154B CN 114369154 B CN114369154 B CN 114369154B CN 202111552136 A CN202111552136 A CN 202111552136A CN 114369154 B CN114369154 B CN 114369154B
Authority
CN
China
Prior art keywords
ser
leu
val
gly
lys
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202111552136.XA
Other languages
Chinese (zh)
Other versions
CN114369154A (en
Inventor
葛霄鹏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dialab Zhangjiagang Biotechnology Co ltd
Original Assignee
Dialab Zhangjiagang Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dialab Zhangjiagang Biotechnology Co ltd filed Critical Dialab Zhangjiagang Biotechnology Co ltd
Priority to CN202111552136.XA priority Critical patent/CN114369154B/en
Publication of CN114369154A publication Critical patent/CN114369154A/en
Application granted granted Critical
Publication of CN114369154B publication Critical patent/CN114369154B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0601—Invertebrate cells or tissues, e.g. insect cells; Culture media therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Urology & Nephrology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention provides an N-methyl-D-aspartate receptor recombinant antigen, a preparation method thereof and a kit containing the recombinant antigen. The recombinant antigen comprises subunit GluN1a recombinant protein, wherein the amino acid sequence of the recombinant protein is that 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-aspartate receptor recombinant antigen also comprises a natural subunit GluN2A recombinant protein and/or a subunit GluN2B recombinant protein, wherein the amino acid sequence is that Asn348 in the sequence shown in SEQ ID NO. 3 is mutated into Asp, and Cys838 and Cys849 are mutated into Ser.

Description

N-methyl-D-aspartate receptor recombinant antigen, preparation method thereof and kit containing recombinant antigen
Technical Field
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
The N-methyl-D-aspartate receptor (NMDAR) is one of three ionic glutamate receptors, and is located in the human brain as an ion channel, playing an important role in the development of brain nerves and neuronal changes in the human central system. NMDAR is mainly used for nerve signal conduction induction, when NMDAR is combined with glutamic acid and glycine while the potential difference of brain cell membrane removes the obstruction of magnesium ions, NMDAR is activated and then opened, and positively charged ions pass through the cell membrane to achieve the purpose of transmitting nerve signals. N-methyl-D-aspartate specifically binds to NMDAR, which does not bind to other glutamate receptors.
Unlike other glutamate receptors, NMDAR requires the simultaneous binding of two different agonists, glutamate and glycine, respectively, to open ion channels. After binding magnesium ions, the NMDAR ion channel is closed. NMDAR acts simultaneously before and after the binding of the synapses, influences the release of neural signals and the plasticity of neurons before the binding, and is used for adjusting and relieving the electric signal substances released after the binding of the synapses and adjusting the plasticity of neurons after the binding. Post-translational modification during NMDAR protein expression can also have a tremendous impact on the location of NMDAR expression and channel opening and closing. Due to these unusual modes of action, NMDAR acts as a detector of specific physiological responses by the human brain nerve.
Conventional NMDAR proteins are an isomerised tetrameric protein comprising two glycine binding subunits GluN1 and two glutamate binding subunits GluN2, each of which is also divided into different subtypes, which are expressed at different positions or time points depending on their own characteristics of action, and studies have shown that NMDAR on the nerve synapse is resistant to neurotoxins, whereas extra-synaptic NMDAR leads to apoptosis. Furthermore, the role of NMDAR consisting of different subunits is mainly distinguished from the binding efficiency of agonists, ion passage rate and total probability of channel opening.
Due to the uniqueness of NMDAR action, it is associated with the pathogenesis of a variety of diseases including parkinson's syndrome, alzheimer's disease, huntington's disease, autism, the most predominant of which is anti-NMDAR autoantibody-related encephalitis. anti-NMDAR autoantibody-related encephalitis is most likely to be classified as a paraneoplastic syndrome, which is a sequelae of cross-reaction of the brain with immune responses caused by tumors. Later studies found that specific anti-NMDAR autoantibodies were found in patient samples with associated symptoms, which autoantibodies acted on the hippocampus of the brain, whereby the disease was named anti-NMDAR autoantibody-associated encephalitis. Pathologically, NMDAR-induced penetration of anti-NMDAR antibodies across the cerebral cortex to the hippocampus, whereby NMDAR on the hippocampus is drastically reduced, and several data show that penetration of anti-NMDAR antibodies into the nerve center and attack of NMDAR affect and regulate disease progression. anti-NMDAR autoantibody-related encephalitis is at risk of relapse, which is not necessarily linked to a decrease in antibody concentration, and thus it is inferred that other physiological factors are involved in pathogenesis.
The anti-NMDAR autoantibody-related encephalitis is the most common non-viral encephalitis, and is found in people of all ages, including children and the elderly, mainly in female groups, with the majority of young patients and the average age of the onset being 23 years, and the series of characteristics are consistent with the characteristic that the incidence rate of immune diseases in people with different sexes is greatly different.
Symptoms of NMDAR autoantibody-related encephalitis mainly include psychosis, memory loss, autonomic imbalance, epilepsy. Patients with three to six are afflicted with tumors, especially ovarian tumors. Subsequent studies have found that the following symptoms are also closely linked to anti-NMDAR autoantibodies, including fever of unknown origin, insomnia, anorexia nervosa, childhood unilateral motor dysfunction.
Since NMDAR protein is a single membrane protein and has large molecular weight, prokaryotic cells cannot be used for expressing full-length protein, natural purification is difficult, the preparation yield is extremely low, meanwhile, the interaction of different subunits of NMDAR plays a role in stabilizing the protein structure, recombinant protein which is close to the activity of natural protein is difficult to obtain by singly expressing extracellular regions or single subunits, and the protein stability is poor. Thus, large-scale expression of NMDAR antigen proteins becomes a major difficulty in the development of anti-NMDAR antibody detection reagents.
Disclosure of Invention
The invention aims to provide an N-methyl-D-aspartate receptor recombinant antigen which has high yield, good stability and protein activity close to that of natural NMDAR protein.
It is another object of the present invention to provide a polynucleotide encoding said recombinant antigen of the N-methyl-D-aspartate receptor.
It is another object of the present invention to provide an expression vector containing the polynucleotide.
It is another object of the present invention to provide a cell line containing said polynucleotide or said expression vector.
Another object of the present invention is to provide a method for producing an N-methyl-D-aspartate receptor recombinant antigen having a high expression level.
It is another object of the present invention to provide a kit for detecting an anti-N-methyl-D-aspartate receptor antibody or for detecting a disease positively associated with an anti-N-methyl-D-aspartate receptor antibody.
In order to solve the technical problems, the invention adopts the following technical scheme:
an N-methyl-D-aspartate receptor recombinant antigen comprising a subunit GluN1a recombinant protein having the amino acid sequence: the 22 nd cysteine in the amino acid sequence shown in SEQ ID NO. 1 is mutated to serine, the 61 st asparagine, the 350 th asparagine, the 471 rd asparagine and the 771 th asparagine are mutated to glutamine, the 595 th glutamic acid and the 597 th glutamic acid are mutated to serine, the 598th glutamic acid is mutated to threonine,
the N-methyl-D-aspartate receptor recombinant antigen also comprises subunit GluN2A recombinant protein and/or subunit GluN2B recombinant protein,
the amino acid sequence of the subunit GluN2A recombinant protein is the amino acid sequence shown in SEQ ID NO. 2,
the subunit GluN2B recombinant protein has the amino acid sequence as follows: the amino acid sequence shown in SEQ ID NO. 3, in which the 348 th asparagine is mutated to aspartic acid, the 838 th cysteine and the 849 th cysteine are mutated to serine.
The polynucleotide encoding the recombinant antigen of N-methyl-D-aspartate receptor of claim comprising a nucleotide fragment encoding the subunit GluN1a recombinant protein and a nucleotide fragment encoding the subunit GluN2A recombinant protein,
or, the polynucleotide includes a nucleotide fragment encoding the subunit GluN1a recombinant protein and a nucleotide fragment encoding the subunit GluN2B recombinant protein,
or, the polynucleotide includes a nucleotide fragment encoding the subunit GluN1a recombinant protein, a nucleotide fragment encoding the subunit GluN2A recombinant protein, and a nucleotide fragment encoding the subunit GluN2B recombinant protein.
Preferably, the expression vector containing said polynucleotide is a vector capable of expression in an insect cell.
Further preferably, the expression vector containing said polynucleotide is a baculovirus.
Preferably, the cell line comprising the polynucleotide of claim or the expression vector employs insect cells capable of transfecting baculovirus and expressing the protein.
The preparation method of the N-methyl-D-aspartate receptor recombinant antigen comprises the following steps: simultaneously expressing said subunit GluN1a recombinant protein and said subunit GluN2A recombinant protein in a cell, or simultaneously expressing said subunit GluN1a recombinant protein and said subunit GluN2B recombinant protein in a cell, or simultaneously expressing said subunit GluN1a recombinant protein, said subunit GluN2A recombinant protein and said subunit GluN2B recombinant protein in a cell.
Preferably, the cell strain is cultured to obtain a cell culture solution, and then the N-methyl-D-aspartate receptor recombinant antigen is obtained through separation and purification.
A kit for detecting an anti-N-methyl-D-aspartate receptor antibody or for detecting a disease associated with positive anti-N-methyl-D-aspartate receptor antibodies, said kit comprising said recombinant N-methyl-D-aspartate receptor antigen.
Preferably, the kit is a chemiluminescent detection kit.
Preferably, the test sample type of the kit is one or more of serum, plasma or cerebrospinal fluid.
Preferably, the disease associated with positive anti-N-methyl-D-aspartate receptor antibodies is autoimmune encephalitis.
Compared with the prior art, the invention has the following advantages:
according to the invention, the expression quantity, the protein activity and the stability of the N-methyl-D-aspartate receptor recombinant antigen are improved by changing the amino acid sequence of the NMDAR subunit, the method is suitable for industrial production of the N-methyl-D-aspartate receptor recombinant antigen, the preparation cost of the N-methyl-D-aspartate receptor recombinant antigen is reduced, and when the prepared N-methyl-D-aspartate receptor recombinant antigen is used for detecting the anti-N-methyl-D-aspartate receptor antibody, the detection result is more accurate, the popularization and the utilization of the anti-N-methyl-D-aspartate receptor antibody detection kit are facilitated, and the diagnosis cost of patients with diseases positively related to the anti-N-methyl-D-aspartate receptor antibody is reduced.
Drawings
FIG. 1 is a graph showing the results of NMDAR recombinant antigen protein activity test in examples 1 to 3;
FIG. 2 is a graph showing the results of the NMDAR recombinant antigen stability test in examples 1 to 3;
FIG. 3 is an NMDAR recombinant protein activity validation of comparative example 1;
FIG. 4 is an NMDAR recombinant protein activity validation of comparative example 2.
Detailed Description
The invention is further described below with reference to examples. The present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions which are not noted are conventional conditions in the industry. The technical features of the various embodiments of the present invention may be combined with each other as long as they do not collide with each other.
From a detection and diagnostic standpoint, the epitope against which an anti-NMDAR autoantibody is directed is predominantly located on the surface of the glycine binding subunit GluN 1. The traditional method can express GluN1 on the surface of HEK cells and then test patient samples by indirect immunofluorescence. All variants of GluN1 were recognized by anti-NMDAR antibodies in patient samples. The GluN2 subunits are simultaneously expressed on the cell surface and bind to the GluN1 subunits, and expression of different variants of GluN2 affects epitopes of the GluN1 protein, whereby the interaction of GluN1/GluN2 after binding may alter the spatial configuration of the GluN1 protein and thus the epitope. GluN1 contains multiple glycosylation sites, the most predominant of which is N368, and GluN1 after N368 mutation is not recognized by autoantibodies in the sample.
However, since NMDAR protein is a single membrane protein and has a large molecular weight, prokaryotic cells cannot be used to express full-length protein, natural purification is difficult and the preparation yield is extremely low, recombinant proteins obtained by expressing extracellular regions or single subunits alone are difficult to obtain the activity close to that of natural proteins, and the protein stability is poor, so that the preparation cost of the recombinant antigen of the N-methyl-D-aspartate receptor is high, and the recombinant antigen cannot be popularized and utilized in large scale in the detection of anti-NMDAR antibodies.
Therefore, the inventor provides a novel N-methyl-D-aspartate receptor recombinant antigen with high yield, good stability and protein activity similar to that of natural NMDAR protein and a preparation method thereof through a great deal of researches and experiments.
According to the invention, the N-methyl-D-aspartate receptor recombinant antigen comprises a subunit GluN1a recombinant protein, and the amino acid sequence of the subunit GluN1a recombinant protein is as follows: the 22 nd cysteine in the amino acid sequence shown in SEQ ID NO. 1 is mutated into serine, the 61 st asparagine, the 350 th asparagine, the 471 rd asparagine and the 771 th asparagine are mutated into glutamine, the 595 th glutamic acid and the 597 th glutamic acid are mutated into serine, the 598th glutamic acid is mutated into threonine, the N-methyl-D-aspartate receptor recombinant antigen also comprises subunit GluN2A recombinant protein and/or subunit GluN2B recombinant protein, the amino acid sequence of the subunit GluN2A recombinant protein is the amino acid sequence shown in SEQ ID NO. 2, and the amino acid sequence of the subunit GluN2B recombinant protein is: the amino acid sequence shown in SEQ ID NO. 3, in which the 348 th asparagine is mutated to aspartic acid, the 838 th cysteine and the 849 th cysteine are mutated to serine.
In the present invention, 4 glycosylated amino acids in GluN1a sequence are mutated, in turn Asn61 to gin, asn350 to gin, asn471 to gin, and Asn771 to gin. By mutating the above Asn amino acids, the complexity of the cell in performing post-protein modification steps is reduced, and thus the chance of protein misfolding is reduced.
In the present invention, cys22 in the GluN1a sequence was mutated to serine. Since multiple cysteines may form disulfide bonds with each other, nonspecific or erroneous disulfide bond formation may greatly affect the activity and stability of the protein due to a certain difference from the expression in the natural human body during the expression process. After mutation, the possibility of nonspecific disulfide bond formation in the protein expression process can be effectively reduced, and the protein expression efficiency is improved.
In the present invention, glu595 in GluN1a sequence is mutated to Ser, glu597 is mutated to Ser, and Glu598 is mutated to Thr. Mutation of the positively and negatively charged protein sites to neutral reduces the likelihood of non-specific binding during protein folding. Many charged regions are involved in the binding reaction of the protein to the ligand and removal of these strong charge sites in the absence of ligand can enhance the stability of the expressed protein.
In the invention, one glycosylated amino acid in the GluN2B sequence is mutated and removed, the mutated amino acid is Asn348, and the mutated amino acid is Asp, so that the protein expression quantity and activity are improved.
In the invention, cys838 and Cys849 in GluN2B are mutated into serine, so that the effects of improving the expression efficiency and improving the protein stability can be achieved.
In the invention, 6XHis tag and GFP tag are respectively inserted into C ends of GluN1a, gluN2A and GluN2B, the GFP tag can directly characterize protein through fluorescence, and whether NMDAR is successfully expressed and the expression quantity thereof are judged. The 6xHis tag is mainly used for subsequent protein purification, and the target NMDAR recombinant antigen is obtained after the specific protein is combined with a nickel affinity chromatographic column to remove the hybrid protein.
When GluN1a and GluN2A, gluN a and GluN2B or GluN1a and GluN2B are expressed simultaneously in the same cell, the recombinant protein retains both the transmembrane domain and the extramembranous domain and the GluN2A and/or GluN2B subunits may interact with GluN1a to stabilize the protein structure. The NMDAR antigen thus expressed is a protein that recently approaches the activity of the native antigen.
In the invention, the baculovirus is used for infecting insect sf9 cells, and the insect sf9 cells are passaged after infection, and the concentration of the insect sf9 cells is up to 1x10 after the second passaging 8 The concentration of pfu/ml baculovirus, when used to infect sf9 cells, resulted in a protein yield of about 25 mg/L. Compared with the common membrane protein, the expression yield of 1-2mg/L is obviously improved.
In the present invention, when a cell membrane is lysed and a membrane protein is purified, after the cell is lysed by adding the amphoteric detergent lauryl maltose neopentyl glycol and 1-palmitoyl-2-oleoyl lecithin, the target membrane protein is purified using an anti-6 xHis tag affinity purification column and an anti-GFP tag affinity purification column.
In the invention, the protein product obtained by affinity purification and dissociation is further polished by a reverse chromatography and a size exclusion chromatography, so that the protein product with the purity of more than 90 percent can be obtained
The NMDAR recombinant protein prepared by the invention has good stability, has no change in activity after being stored at 37 ℃ for 2 weeks, can be dissolved in water and various buffer solutions, and can be used for preparing and producing in vitro diagnostic reagents. And after biotinylation of NMDAR antigen, mixing the NMDAR antigen with streptavidin marked magnetic microspheres, adding a sample to be detected, incubating and washing, adding a secondary antibody connected with a luminescent substrate AP, incubating and washing, and detecting a luminescent signal, wherein the sample detection coincidence rate reaches 97%.
The technical scheme and technical effect of the present invention are further illustrated by the following specific examples and comparative examples.
In the following examples, the raw materials, reagents and the like used were all conventional commercial products, as specified.
Example 1
(1) The polynucleotides were synthesized.
Searching genebank obtained the native cDNA sequences of NMDAR subunits GluN1, gluN2A, gluN2B, respectively. All sequences should be searched from a human gene library, the sequence of the natural subunit GluN1 is shown as SEQ ID NO. 1, the sequence of the natural subunit GluN2A is shown as SEQ ID NO. 2, and the sequence of the natural subunit GluN2B is shown as SEQ ID NO. 3.
Analysis of the NMDAR antigen sequence, the glycosylated amino acid positions in the sequence were deduced, and Asn61, asn239, asn350, asn471, asn491 and Asn771 were included in the sequence of GluN1 a. The overlapping mutation method is used for mutating each asparagine into glutamine, and experiments prove that the protein expression and activity after the mutation of other asparagines are improved to different degrees except for Asn239 and Asn 491. Therefore, all glycosylated amino acids except Asn239 and Asn491 in GluN1a were mutated to glutamine.
By sequence analysis, amino acids with strong negative charges in GluN1a sequence were found: glu595, glu597, glu598. Both Glu595 and Glu597 are mutated to serine by single point mutation, glu598 is mutated to threonine.
Cys22 in the GluN1a sequence, which may form disulfide bonds, was mutated to serine by sequence analysis.
Combining the mutations, and synthesizing GluN1a through total genes according to requirements, and sequentially changing the following amino acids on the basis of a natural sequence: asn61Gln, asn350Gln, asn471Gln, asn771Gln, cys22Ser, glu595Ser, glu597Ser, glu598Thr.
The GluN2A native gene was synthesized total gene as desired.
Analysis shows that the glycosylation amino acid locus in GluN2B subunit sequence is obtained, asn348 is mutated into aspartic acid through an overlapping method, and the protein expression quantity after mutation is improved through experimental verification.
Cys838 and Cys849 in the GluN2B sequence, which may form disulfide bonds, were mutated to serine by sequence analysis.
Combining the above mutations, and synthesizing GluN2B through total genes according to requirements, so as to change the following amino acids based on the natural sequence: asn348Asp, cys838Ser, cys849Ser.
At the same time of total gene synthesis, a 6xHis tag was added to the C-terminus of GluN1a, and a GFP tag was added to the C-terminus of GluN2A, gluN B.
During total gene synthesis, notI and XbaI restriction enzyme sites were added to 5 'and 3' of GluN1a nucleic acid sequence, respectively. XhoI and NheI restriction enzyme sites were added to 5 'and 3' of GluN2A, gluN2B nucleic acid sequence, respectively.
(2) pFastBac Dual-GluN1a-GluN2A plasmid was prepared.
1. Mu.g of GluN1a nucleic acid aqueous solution and 2. Mu.g of pFastBac Dual plasmid were respectively added, 100 units of NotI and 100 units of XbaI restriction enzyme were respectively added and reacted at 37℃for 30 minutes, after the completion of the reaction, the excess enzyme was removed by using QIAGEN PCR purification kit, and after the completion of the cleavage, gluN1a and pFastBac Dual plasmid were mixed and 100 units of T4 ligase was added. After incubation for 2 hours at room temperature, the excess T4 enzyme was removed using a QIAGEN PCR purification kit. Will be pureThe pFastBac Dual-GluN1 constructed plasmid obtained by the transformation was transferred into an electroporator, berle Gene Pulser Xcell, into an electrocompetent E.coli DH10Bac TM After 1mL of SOC recovery culture solution was added and incubated at 37℃for 1 hour, the mixture was spread on LB agarose plates containing 100. Mu.g/mL ampicillin and 70. Mu.g/mL gentamicin. After overnight culture, positive clones were selected, inoculated into a culture solution containing 100. Mu.g/mL ampicillin and 70. Mu.g/mL gentamicin SB to culture the bacteria to logarithmic phase, and purified pFastBac Dual-GluN1a plasmid was obtained using a QIAGEN Maxi Prep plasmid megapump kit.
1. Mu.g of GluN2A nucleic acid aqueous solution and 2. Mu.g of pFastBac Dual-GluN1a plasmid were taken and reacted at 37℃for 30 minutes with 100 units of XhoI and 100 units of NheI restriction enzyme, respectively, after the completion of the reaction, the excess enzyme was removed by using QIAGEN PCR purification kit, and after the completion of the cleavage, gluN2A/GluN2B and pFastBac Dual-GluN1a plasmids were mixed and 100 units of T4 ligase was added. After incubation for 2 hours at room temperature, the excess T4 enzyme was removed using a QIAGEN PCR purification kit. The purified pFastBac Dual-GluN1-GluN2A/pFastBac Dual-GluN1-GluN2B construction plasmid was transferred into an electrically competent E.coli DH10Bac by means of an electroporator, bere Gene Pulser Xcell TM After 1mL of SOC recovery culture solution was added and incubated at 37℃for 1 hour, the mixture was spread on LB agarose plates containing 100. Mu.g/mL ampicillin and 70. Mu.g/mL gentamicin. After overnight culture, positive clones were selected, inoculated into a culture solution containing 100. Mu.g/mL ampicillin and 70. Mu.g/mL gentamicin SB to grow to a logarithmic phase, and purified pFastBac Dual-GluN1-GluN2A plasmid was obtained using a QIAGEN Bacmid Prep plasmid megapump kit.
(3) NMDAR recombinant antigen expression and purification.
Sf9 insect cells were cultured in Grace insect broth supplemented with 10% calf serum to logarithmic growth phase (1.5-2.5x10) 6 Cells per ml of culture medium and viability was higher than 95%).
2mLGrace insect broth was added to each well of a 6-well plate, and 8X10 was added to each well without additional antibiotics 5 sf9 cells.
mu.L of Cellfectin II was diluted to 100. Mu.L of Grace-free insect culture broth, and 1. Mu.g of pFastBac Dual-GluN1-GluN2A was diluted to 100. Mu.L of Grace-free insect culture broth. The diluted Cellfectin II and pFastBac Dual-GluN1-GluN2A plasmids were mixed and homogenized at room temperature for 15-30 minutes.
The transfection mixture was added dropwise to sf9 cells cultured in 6 well cell culture plates and incubated at 27℃for 3-5 hours. Subsequently, after removing the transfection mixture, 2mL of Grace insect culture solution containing 10% fetal bovine serum was added to each culture well, and 100. Mu.g/mL ampicillin and 70. Mu.g/mL gentamicin were added to the culture solution. Sf9 cells were cultured in culture for 72 hours until virus infection could be observed.
After 72 hours, when the cells enter the late infection stage, 2ml of culture solution supernatant is sucked from each culture hole, the supernatant contains P1 generation virus storage solution, and the supernatant is centrifuged again at 5000xg for 5 minutes to remove the cells and other residues, thus obtaining the P1 generation virus.
Passaging the P1 generation virus, preparing 6-well cell culture plates, and adding 2×10 cells into each well 6 sf9 insect cells were incubated at room temperature for 1 hour to allow the cells to adhere to the culture plates. And selecting a proper amount of P1 generation virus, adding the virus into each culture hole, and controlling the infection copy coefficient (MOI) to be 0.05-0.1 according to the requirement. Cells and viruses were incubated for 48 hours at 27 degrees celsius in humid conditions.
After 48 hours, 2mL of culture solution is sucked from each culture hole, all the culture solutions are mixed and centrifuged at 5000Xg for 5 minutes, the supernatant is taken, the obtained virus is collected as P2 generation, and the virus content needs to reach 1X10 8 pfu/mL can be used to re-infect cells and express the protein.
Add 6X10 to each well in 24 well cell culture plate 5 Individual sf9 insect cells were incubated at room temperature for 30 minutes to allow the cells to attach to the culture plates.
After the incubation was completed, the supernatant was aspirated, and the wells were washed once with fresh sf-900II SFM medium, followed by a further 300. Mu.L of fresh sf-900II SFM medium per well, with 0.1% fetal bovine serum. And continuing adding the P2 generation virus into the cell culture solution, wherein the amount of the virus is enough to enable the MOI coefficient to reach between 5 and 10. Culturing in 27 degree incubator for 24-72 hr, removing supernatant from the culture well, and washing the culture well with culture solution without fetal bovine serum once. The dissociation solution was added to the cell culture wells, all suspended cells and proteins expressed on the cell surface were collected, labeled NMDAR recombinant antigen 1, and the yield of NMDAR recombinant antigen 1 was about 23mg/L.
Amphoteric detergent lauryl maltose neopentyl glycol (MNG-3) and 1-palmitoyl-2-oleoyl lecithin (POPC) were prepared as 0.1% aqueous solutions, and cell solutions were prepared according to 1:10 was diluted in 10mM Tris solution and MNG-3 and POPC were added to the solution to a final concentration of 0.01%. Mixing at room temperature for 2 hours.
After mixing, mixing the protein and nickel particles, mixing for 2 hours at room temperature, transferring the mixed solution into a purification column, discarding the flow channel, cleaning the nickel particles by using PBS with 3-5 particle backlog volume, and finally dissociating by using 100-500mM EDTA solution. The dissociated fraction containing the protein of interest is collected.
Slowly adding the flow channeling in the previous purification process into an anti-GFP label purification column, standing for 30 minutes, discarding the flow channeling, washing the anti-GFP particles by using PBS with the volume of 3-5 particles, and finally dissociating by using glycine solution with the pH value of 4.7-6.7. All NMDAR recombinant antigen 1 obtained by dissociation was collected, with a purity of 95%.
Example 2
pFastBac Dual-GluN1a-GluN2B plasmid was prepared by the same method as in example 1, pFastBac Dual-GluN1a-GluN2B plasmid was transfected by the same method as in example 1, gluN1a and GluN2B were expressed simultaneously in sf9 insect cells and NMDAR recombinant protein was isolated and purified, labeled NMDAR recombinant antigen 2, yield of NMDAR recombinant antigen 2 was 28mg/L, and purity of protein after purification and polishing was 90%.
Example 3
pFastBac Dual-GluN1a-GluN2A plasmid and pFastBac Dual-GluN1a-GluN2B plasmid were transfected simultaneously in the same manner as in example 1 and example 2, gluN1a/GluN2A and GluN1a/GluN2B were expressed simultaneously in sf9 insect cells, NMDAR recombinant protein was isolated and purified, labeled as NMDAR recombinant antigen 3, NMDAR recombinant antigen 3 was produced at a yield of about 23mg/L, and the purified and polished protein had a purity of 90%.
Comparison of protein Activity
The NMDAR recombinant antigen 1, the NMDAR recombinant antigen 2, the NMDAR recombinant antigen 3 and NMDAR proteins based on a natural cDNA table are respectively diluted to 2 mug/mL by PBS, each hole on an ELISA plate is respectively coated by 0.2 mug of protein, the coating liquid is discarded after the coating is carried out for 2 hours at 37 ℃, and the microwells are washed for 2 times by 100 mug of PBS/0.1% Tween 20. After the cleaning is finished, 100 mu L of samples to be tested are added into micropores coated with different NMDAR antigens, the samples to be tested are NMDAR high-value positive samples and 4 1/2 gradient dilution samples thereof, and NMDAR negative samples are tested. Samples were incubated at 37℃for 1 hour and the supernatant was discarded, and each well was blocked with 100. Mu.L PBS/1% BSA after washing was completed, and after blocking for 1 hour, the blocking solution was discarded and the wells were washed 5 times with 200. Mu.L PBS/0.1% Tween 20. mu.L of PBS/0.01% HRP-labeled rabbit anti-human IgG was added, the solution was incubated at 37℃for 1 hour after Fc-specificity, and the microwells were washed 5 times with 200. Mu.L of PBS/0.1% Tween 20. Finally, the color development was observed after adding TMB substrate solution, and after 10 minutes, 50. Mu.L of 2M hydrochloric acid was counted to terminate the reaction, and the reaction was read by an enzyme-labeled instrument. The results show (fig. 1) that compared to the purified recombinant protein expressed after cloning the NMDAR protocdna sequence into the plasmid, the mutated NMDAR sequences expressed and purified proteins NMDAR recombinant antigen 1, NMDAR recombinant antigen 2 and NMDAR recombinant antigen 3 (i.e., NMDAR antigen 1, NMDAR antigen 2 and NMDAR antigen 3 in fig. 1) have about 30% improved reactivity against positive reference samples, at the same level as that of negative serum. Therefore, in the secondary expression system, NMDAR recombinant antigen 1, NMDAR recombinant antigen 2 and NMDAR recombinant antigen 3 have better protein activity, and are more beneficial to the configuration of subsequent detection reagents.
Protein stability test
The NMDAR recombinant antigen 1, the NMDAR recombinant antigen 2 and the NMDAR recombinant antigen 3 are respectively coated according to 0.2 mug/hole, after coating is finished, a coated ELISA plate is respectively taken out for testing by 0,1,3,5,7, 100 mug positive reference is added into each hole for incubation for 1 hour, PBS/1% BSA solution is used for sealing the ELISA plate after incubation is finished, 100 mug PBS/0.01% HRP marked rabbit anti-human IgG is added into each hole after sealing is finished, fc specificity is realized, 100 mug substrate is added into each hole after reaction for 1 hour, and finally 50 mug 2M hydrochloric acid is used for stopping the reaction. During the 7 day 37 ℃ incubation, fig. 2 shows that the reactivity of NMDAR recombinant antigen 1, NMDAR recombinant antigen 2, NMDAR recombinant antigen 3 with positive reference is not significantly changed, and the fluctuation of the reaction OD is within ±10% compared to day 0. Therefore, the NMDAR recombinant antigen 1, the NMDAR recombinant antigen 2 and the NMDAR recombinant antigen 3 have good stability.
anti-NMDAR antibody detection experiments
The purified NMDAR recombinant protein is used to prepare an anti-NMDAR antibody detection reagent. Two portions of the dissociated NMDAR (comprising GluN1a and GluN2A/GluN 2B) recombinant protein were mixed and diluted to 1mg/mL with PBS. 1mL of 1mM magnetic microspheres (200 nm particle size) activated with 50mM EDC was added, and incubated for 2 hours at room temperature. After the labeling was completed, 8000Xg was centrifuged for 10 minutes, the supernatant was removed, and the magnetic microspheres were re-dispersed into 1mL PBS containing 1% BSA. The magnetic microspheres were mixed in solution at room temperature and closed for 1 hour. Centrifugation at 8000Xg for 10 min after completion of the blocking, the supernatant was removed. The magnetic microspheres were dispersed in 20mM Tris 1% BSA solution.
Magnetic microspheres labeled NMDAR at a concentration of 1mg/ml were used as reagent R1, and anti-human IgG antibodies conjugated to AP were diluted 1/20000 in PBS as reagent R2. After mixing 100 mu LR1 with 20 mu L of sample, incubating for 10 minutes at 37 ℃, washing the magnetic microsphere 5 times by using a washing liquid, adding a 100 mu LR2 reagent, incubating for 10 minutes at 37 ℃, washing the magnetic microsphere 5 times by using the washing liquid, and adding a luminescent substrate to detect a luminescent signal value.
Clinical samples were tested using the anti-NMDAR antibody detection reagent prepared, which included 30 positive samples and 50 negative samples. The detection results are shown in Table 1, the detection sensitivity reaches 96.7%, and the detection specificity reaches 100%.
TABLE 1
Comparative example 1
Since 3 cysteines (Cys 588, cys838, cys 849) were included in the GluN2B sequence in total, we examined the effect of mutation using single amino acid mutation, double amino acid cross mutation and a method of simultaneously mutating 3 cysteines. In this comparative example, in which single cysteine was mutated to serine and all cysteines were mutated to serine, the expression rate of the obtained recombinant protein was always low and the total yield was lower than 1mg/L of the culture broth in the other operations as in example 1 or example 2 or example 3. The mutation Cys838 and Cys849 are screened from the combination of two cysteines in mutation to obtain serine which plays an optimal role in promoting protein expression, and the protein expression quantity is improved to 20-50mg/L culture solution, so that Cys588 is kept unchanged. The mutant protein activity was confirmed in FIG. 3, and the results showed that NMDAR mutant protein activity was almost indistinguishable from the native protein activity.
Comparative example 2
The NMDAR recombinant antigen preparation method is the same as that of example 1, example 2 and example 3, except that Asn239 and/or Asn491 in the sequence of GluN1a are mutated into glutamine and co-expressed with GluN2A and/or GluN2B, the protein expression amount is not obviously improved, and the protein activity verification shows that after Asn239 mutation and after Asn491 mutation, the protein activity is not obviously improved compared with the activity of natural protein, so that the Asn239 and Asn491 are unchanged.
The present invention has been described in detail with the purpose of enabling those skilled in the art to understand the contents of the present invention and to implement the same, but not to limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.
Sequence listing
<110> Di ya Lai Bo (Zhang Jiang) Biotech Co., ltd
<120> an N-methyl-D-aspartate receptor recombinant antigen, a method for preparing the same, and a kit containing the same
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 938
<212> PRT
<213> GluN1a(GluN1a)
<400> 1
Met Ser Thr Met Arg Leu Leu Thr Leu Ala Leu Leu Phe Ser Cys Ser
1 5 10 15
Val Ala Arg Ala Ala Cys Asp Pro Lys Ile Val Asn Ile Gly Ala Val
20 25 30
Leu Ser Thr Arg Lys His Glu Gln Met Phe Arg Glu Ala Val Asn Gln
35 40 45
Ala Asn Lys Arg His Gly Ser Trp Lys Ile Gln Leu Asn Ala Thr Ser
50 55 60
Val Thr His Lys Pro Asn Ala Ile Gln Met Ala Leu Ser Val Cys Glu
65 70 75 80
Asp Leu Ile Ser Ser Gln Val Tyr Ala Ile Leu Val Ser His Pro Pro
85 90 95
Thr Pro Asn Asp His Phe Thr Pro Thr Pro Val Ser Tyr Thr Ala Gly
100 105 110
Phe Tyr Arg Ile Pro Val Leu Gly Leu Thr Thr Arg Met Ser Ile Tyr
115 120 125
Ser Asp Lys Ser Ile His Leu Ser Phe Leu Arg Thr Val Pro Pro Tyr
130 135 140
Ser His Gln Ser Ser Val Trp Phe Glu Met Met Arg Val Tyr Ser Trp
145 150 155 160
Asn His Ile Ile Leu Leu Val Ser Asp Asp His Glu Gly Arg Ala Ala
165 170 175
Gln Lys Arg Leu Glu Thr Leu Leu Glu Glu Arg Glu Ser Lys Ala Glu
180 185 190
Lys Val Leu Gln Phe Asp Pro Gly Thr Lys Asn Val Thr Ala Leu Leu
195 200 205
Met Glu Ala Lys Glu Leu Glu Ala Arg Val Ile Ile Leu Ser Ala Ser
210 215 220
Glu Asp Asp Ala Ala Thr Val Tyr Arg Ala Ala Ala Met Leu Asn Met
225 230 235 240
Thr Gly Ser Gly Tyr Val Trp Leu Val Gly Glu Arg Glu Ile Ser Gly
245 250 255
Asn Ala Leu Arg Tyr Ala Pro Asp Gly Ile Leu Gly Leu Gln Leu Ile
260 265 270
Asn Gly Lys Asn Glu Ser Ala His Ile Ser Asp Ala Val Gly Val Val
275 280 285
Ala Gln Ala Val His Glu Leu Leu Glu Lys Glu Asn Ile Thr Asp Pro
290 295 300
Pro Arg Gly Cys Val Gly Asn Thr Asn Ile Trp Lys Thr Gly Pro Leu
305 310 315 320
Phe Lys Arg Val Leu Met Ser Ser Lys Tyr Ala Asp Gly Val Thr Gly
325 330 335
Arg Val Glu Phe Asn Glu Asp Gly Asp Arg Lys Phe Ala Asn Tyr Ser
340 345 350
Ile Met Asn Leu Gln Asn Arg Lys Leu Val Gln Val Gly Ile Tyr Asn
355 360 365
Gly Thr His Val Ile Pro Asn Asp Arg Lys Ile Ile Trp Pro Gly Gly
370 375 380
Glu Thr Glu Lys Pro Arg Gly Tyr Gln Met Ser Thr Arg Leu Lys Ile
385 390 395 400
Val Thr Ile His Gln Glu Pro Phe Val Tyr Val Lys Pro Thr Leu Ser
405 410 415
Asp Gly Thr Cys Lys Glu Glu Phe Thr Val Asn Gly Asp Pro Val Lys
420 425 430
Lys Val Ile Cys Thr Gly Pro Asn Asp Thr Ser Pro Gly Ser Pro Arg
435 440 445
His Thr Val Pro Gln Cys Cys Tyr Gly Phe Cys Ile Asp Leu Leu Ile
450 455 460
Lys Leu Ala Arg Thr Met Asn Phe Thr Tyr Glu Val His Leu Val Ala
465 470 475 480
Asp Gly Lys Phe Gly Thr Gln Glu Arg Val Asn Asn Ser Asn Lys Lys
485 490 495
Glu Trp Asn Gly Met Met Gly Glu Leu Leu Ser Gly Gln Ala Asp Met
500 505 510
Ile Val Ala Pro Leu Thr Ile Asn Asn Glu Arg Ala Gln Tyr Ile Glu
515 520 525
Phe Ser Lys Pro Phe Lys Tyr Gln Gly Leu Thr Ile Leu Val Lys Lys
530 535 540
Glu Ile Pro Arg Ser Thr Leu Asp Ser Phe Met Gln Pro Phe Gln Ser
545 550 555 560
Thr Leu Trp Leu Leu Val Gly Leu Ser Val His Val Val Ala Val Met
565 570 575
Leu Tyr Leu Leu Asp Arg Phe Ser Pro Phe Gly Arg Phe Lys Val Asn
580 585 590
Ser Glu Glu Glu Glu Glu Asp Ala Leu Thr Leu Ser Ser Ala Met Trp
595 600 605
Phe Ser Trp Gly Val Leu Leu Asn Ser Gly Ile Gly Glu Gly Ala Pro
610 615 620
Arg Ser Phe Ser Ala Arg Ile Leu Gly Met Val Trp Ala Gly Phe Ala
625 630 635 640
Met Ile Ile Val Ala Ser Tyr Thr Ala Asn Leu Ala Ala Phe Leu Val
645 650 655
Leu Asp Arg Pro Glu Glu Arg Ile Thr Gly Ile Asn Asp Pro Arg Leu
660 665 670
Arg Asn Pro Ser Asp Lys Phe Ile Tyr Ala Thr Val Lys Gln Ser Ser
675 680 685
Val Asp Ile Tyr Phe Arg Arg Gln Val Glu Leu Ser Thr Met Tyr Arg
690 695 700
His Met Glu Lys His Asn Tyr Glu Ser Ala Ala Glu Ala Ile Gln Ala
705 710 715 720
Val Arg Asp Asn Lys Leu His Ala Phe Ile Trp Asp Ser Ala Val Leu
725 730 735
Glu Phe Glu Ala Ser Gln Lys Cys Asp Leu Val Thr Thr Gly Glu Leu
740 745 750
Phe Phe Arg Ser Gly Phe Gly Ile Gly Met Arg Lys Asp Ser Pro Trp
755 760 765
Lys Gln Asn Val Ser Leu Ser Ile Leu Lys Ser His Glu Asn Gly Phe
770 775 780
Met Glu Asp Leu Asp Lys Thr Trp Val Arg Tyr Gln Glu Cys Asp Ser
785 790 795 800
Arg Ser Asn Ala Pro Ala Thr Leu Thr Phe Glu Asn Met Ala Gly Val
805 810 815
Phe Met Leu Val Ala Gly Gly Ile Val Ala Gly Ile Phe Leu Ile Phe
820 825 830
Ile Glu Ile Ala Tyr Lys Arg His Lys Asp Ala Arg Arg Lys Gln Met
835 840 845
Gln Leu Ala Phe Ala Ala Val Asn Val Trp Arg Lys Asn Leu Gln Asp
850 855 860
Arg Lys Ser Gly Arg Ala Glu Pro Asp Pro Lys Lys Lys Ala Thr Phe
865 870 875 880
Arg Ala Ile Thr Ser Thr Leu Ala Ser Ser Phe Lys Arg Arg Arg Ser
885 890 895
Ser Lys Asp Thr Ser Thr Gly Gly Gly Arg Gly Ala Leu Gln Asn Gln
900 905 910
Lys Asp Thr Val Leu Pro Arg Arg Ala Ile Glu Arg Glu Glu Gly Gln
915 920 925
Leu Gln Leu Cys Ser Arg His Arg Glu Ser
930 935
<210> 2
<211> 1464
<212> PRT
<213> GluN2A(GluN2A)
<400> 2
Met Gly Arg Val Gly Tyr Trp Thr Leu Leu Val Leu Pro Ala Leu Leu
1 5 10 15
Val Trp Arg Gly Pro Ala Pro Ser Ala Ala Ala Glu Lys Gly Pro Pro
20 25 30
Ala Leu Asn Ile Ala Val Met Leu Gly His Ser His Asp Val Thr Glu
35 40 45
Arg Glu Leu Arg Thr Leu Trp Gly Pro Glu Gln Ala Ala Gly Leu Pro
50 55 60
Leu Asp Val Asn Val Val Ala Leu Leu Met Asn Arg Thr Asp Pro Lys
65 70 75 80
Ser Leu Ile Thr His Val Cys Asp Leu Met Ser Gly Ala Arg Ile His
85 90 95
Gly Leu Val Phe Gly Asp Asp Thr Asp Gln Glu Ala Val Ala Gln Met
100 105 110
Leu Asp Phe Ile Ser Ser His Thr Phe Val Pro Ile Leu Gly Ile His
115 120 125
Gly Gly Ala Ser Met Ile Met Ala Asp Lys Asp Pro Thr Ser Thr Phe
130 135 140
Phe Gln Phe Gly Ala Ser Ile Gln Gln Gln Ala Thr Val Met Leu Lys
145 150 155 160
Ile Met Gln Asp Tyr Asp Trp His Val Phe Ser Leu Val Thr Thr Ile
165 170 175
Phe Pro Gly Tyr Arg Glu Phe Ile Ser Phe Val Lys Thr Thr Val Asp
180 185 190
Asn Ser Phe Val Gly Trp Asp Met Gln Asn Val Ile Thr Leu Asp Thr
195 200 205
Ser Phe Glu Asp Ala Lys Thr Gln Val Gln Leu Lys Lys Ile His Ser
210 215 220
Ser Val Ile Leu Leu Tyr Cys Ser Lys Asp Glu Ala Val Leu Ile Leu
225 230 235 240
Ser Glu Ala Arg Ser Leu Gly Leu Thr Gly Tyr Asp Phe Phe Trp Ile
245 250 255
Val Pro Ser Leu Val Ser Gly Asn Thr Glu Leu Ile Pro Lys Glu Phe
260 265 270
Pro Ser Gly Leu Ile Ser Val Ser Tyr Asp Asp Trp Asp Tyr Ser Leu
275 280 285
Glu Ala Arg Val Arg Asp Gly Ile Gly Ile Leu Thr Thr Ala Ala Ser
290 295 300
Ser Met Leu Glu Lys Phe Ser Tyr Ile Pro Glu Ala Lys Ala Ser Cys
305 310 315 320
Tyr Gly Gln Met Glu Arg Pro Glu Val Pro Met His Thr Leu His Pro
325 330 335
Phe Met Val Asn Val Thr Trp Asp Gly Lys Asp Leu Ser Phe Thr Glu
340 345 350
Glu Gly Tyr Gln Val His Pro Arg Leu Val Val Ile Val Leu Asn Lys
355 360 365
Asp Arg Glu Trp Glu Lys Val Gly Lys Trp Glu Asn His Thr Leu Ser
370 375 380
Leu Arg His Ala Val Trp Pro Arg Tyr Lys Ser Phe Ser Asp Cys Glu
385 390 395 400
Pro Asp Asp Asn His Leu Ser Ile Val Thr Leu Glu Glu Ala Pro Phe
405 410 415
Val Ile Val Glu Asp Ile Asp Pro Leu Thr Glu Thr Cys Val Arg Asn
420 425 430
Thr Val Pro Cys Arg Lys Phe Val Lys Ile Asn Asn Ser Thr Asn Glu
435 440 445
Gly Met Asn Val Lys Lys Cys Cys Lys Gly Phe Cys Ile Asp Ile Leu
450 455 460
Lys Lys Leu Ser Arg Thr Val Lys Phe Thr Tyr Asp Leu Tyr Leu Val
465 470 475 480
Thr Asn Gly Lys His Gly Lys Lys Val Asn Asn Val Trp Asn Gly Met
485 490 495
Ile Gly Glu Val Val Tyr Gln Arg Ala Val Met Ala Val Gly Ser Leu
500 505 510
Thr Ile Asn Glu Glu Arg Ser Glu Val Val Asp Phe Ser Val Pro Phe
515 520 525
Val Glu Thr Gly Ile Ser Val Met Val Ser Arg Ser Asn Gly Thr Val
530 535 540
Ser Pro Ser Ala Phe Leu Glu Pro Phe Ser Ala Ser Val Trp Val Met
545 550 555 560
Met Phe Val Met Leu Leu Ile Val Ser Ala Ile Ala Val Phe Val Phe
565 570 575
Glu Tyr Phe Ser Pro Val Gly Tyr Asn Arg Asn Leu Ala Lys Gly Lys
580 585 590
Ala Pro His Gly Pro Ser Phe Thr Ile Gly Lys Ala Ile Trp Leu Leu
595 600 605
Trp Gly Leu Val Phe Asn Asn Ser Val Pro Val Gln Asn Pro Lys Gly
610 615 620
Thr Thr Ser Lys Ile Met Val Ser Val Trp Ala Phe Phe Ala Val Ile
625 630 635 640
Phe Leu Ala Ser Tyr Thr Ala Asn Leu Ala Ala Phe Met Ile Gln Glu
645 650 655
Glu Phe Val Asp Gln Val Thr Gly Leu Ser Asp Lys Lys Phe Gln Arg
660 665 670
Pro His Asp Tyr Ser Pro Pro Phe Arg Phe Gly Thr Val Pro Asn Gly
675 680 685
Ser Thr Glu Arg Asn Ile Arg Asn Asn Tyr Pro Tyr Met His Gln Tyr
690 695 700
Met Thr Lys Phe Asn Gln Lys Gly Val Glu Asp Ala Leu Val Ser Leu
705 710 715 720
Lys Thr Gly Lys Leu Asp Ala Phe Ile Tyr Asp Ala Ala Val Leu Asn
725 730 735
Tyr Lys Ala Gly Arg Asp Glu Gly Cys Lys Leu Val Thr Ile Gly Ser
740 745 750
Gly Tyr Ile Phe Ala Thr Thr Gly Tyr Gly Ile Ala Leu Gln Lys Gly
755 760 765
Ser Pro Trp Lys Arg Gln Ile Asp Leu Ala Leu Leu Gln Phe Val Gly
770 775 780
Asp Gly Glu Met Glu Glu Leu Glu Thr Leu Trp Leu Thr Gly Ile Cys
785 790 795 800
His Asn Glu Lys Asn Glu Val Met Ser Ser Gln Leu Asp Ile Asp Asn
805 810 815
Met Ala Gly Val Phe Tyr Met Leu Ala Ala Ala Met Ala Leu Ser Leu
820 825 830
Ile Thr Phe Ile Trp Glu His Leu Phe Tyr Trp Lys Leu Arg Phe Cys
835 840 845
Phe Thr Gly Val Cys Ser Asp Arg Pro Gly Leu Leu Phe Ser Ile Ser
850 855 860
Arg Gly Ile Tyr Ser Cys Ile His Gly Val His Ile Glu Glu Lys Lys
865 870 875 880
Lys Ser Pro Asp Phe Asn Leu Thr Gly Ser Gln Ser Asn Met Leu Lys
885 890 895
Leu Leu Arg Ser Ala Lys Asn Ile Ser Ser Met Ser Asn Met Asn Ser
900 905 910
Ser Arg Met Asp Ser Pro Lys Arg Ala Ala Asp Phe Ile Gln Arg Gly
915 920 925
Ser Leu Ile Met Asp Met Val Ser Asp Lys Gly Asn Leu Met Tyr Ser
930 935 940
Asp Asn Arg Ser Phe Gln Gly Lys Glu Ser Ile Phe Gly Asp Asn Met
945 950 955 960
Asn Glu Leu Gln Thr Phe Val Ala Asn Arg Gln Lys Asp Asn Leu Asn
965 970 975
Asn Tyr Val Phe Gln Gly Gln His Pro Leu Thr Leu Asn Glu Ser Asn
980 985 990
Pro Asn Thr Val Glu Val Ala Val Ser Thr Glu Ser Lys Ala Asn Ser
995 1000 1005
Arg Pro Arg Gln Leu Trp Lys Lys Ser Val Asp Ser Ile Arg Gln Asp
1010 1015 1020
Ser Leu Ser Gln Asn Pro Val Ser Gln Arg Asp Glu Ala Thr Ala Glu
1025 1030 1035 1040
Asn Arg Thr His Ser Leu Lys Ser Pro Arg Tyr Leu Pro Glu Glu Met
1045 1050 1055
Ala His Ser Asp Ile Ser Glu Thr Ser Asn Arg Ala Thr Cys His Arg
1060 1065 1070
Glu Pro Asp Asn Ser Lys Asn His Lys Thr Lys Asp Asn Phe Lys Arg
1075 1080 1085
Ser Val Ala Ser Lys Tyr Pro Lys Asp Cys Ser Glu Val Glu Arg Thr
1090 1095 1100
Tyr Leu Lys Thr Lys Ser Ser Ser Pro Arg Asp Lys Ile Tyr Thr Ile
1105 1110 1115 1120
Asp Gly Glu Lys Glu Pro Gly Phe His Leu Asp Pro Pro Gln Phe Val
1125 1130 1135
Glu Asn Val Thr Leu Pro Glu Asn Val Asp Phe Pro Asp Pro Tyr Gln
1140 1145 1150
Asp Pro Ser Glu Asn Phe Arg Lys Gly Asp Ser Thr Leu Pro Met Asn
1155 1160 1165
Arg Asn Pro Leu His Asn Glu Glu Gly Leu Ser Asn Asn Asp Gln Tyr
1170 1175 1180
Lys Leu Tyr Ser Lys His Phe Thr Leu Lys Asp Lys Gly Ser Pro His
1185 1190 1195 1200
Ser Glu Thr Ser Glu Arg Tyr Arg Gln Asn Ser Thr His Cys Arg Ser
1205 1210 1215
Cys Leu Ser Asn Met Pro Thr Tyr Ser Gly His Phe Thr Met Arg Ser
1220 1225 1230
Pro Phe Lys Cys Asp Ala Cys Leu Arg Met Gly Asn Leu Tyr Asp Ile
1235 1240 1245
Asp Glu Asp Gln Met Leu Gln Glu Thr Gly Asn Pro Ala Thr Gly Glu
1250 1255 1260
Gln Val Tyr Gln Gln Asp Trp Ala Gln Asn Asn Ala Leu Gln Leu Gln
1265 1270 1275 1280
Lys Asn Lys Leu Arg Ile Ser Arg Gln His Ser Tyr Asp Asn Ile Val
1285 1290 1295
Asp Lys Pro Arg Glu Leu Asp Leu Ser Arg Pro Ser Arg Ser Ile Ser
1300 1305 1310
Leu Lys Asp Arg Glu Arg Leu Leu Glu Gly Asn Phe Tyr Gly Ser Leu
1315 1320 1325
Phe Ser Val Pro Ser Ser Lys Leu Ser Gly Lys Lys Ser Ser Leu Phe
1330 1335 1340
Pro Gln Gly Leu Glu Asp Ser Lys Arg Ser Lys Ser Leu Leu Pro Asp
1345 1350 1355 1360
His Thr Ser Asp Asn Pro Phe Leu His Ser His Arg Asp Asp Gln Arg
1365 1370 1375
Leu Val Ile Gly Arg Cys Pro Ser Asp Pro Tyr Lys His Ser Leu Pro
1380 1385 1390
Ser Gln Ala Val Asn Asp Ser Tyr Leu Arg Ser Ser Leu Arg Ser Thr
1395 1400 1405
Ala Ser Tyr Cys Ser Arg Asp Ser Arg Gly His Asn Asp Val Tyr Ile
1410 1415 1420
Ser Glu His Val Met Pro Tyr Ala Ala Asn Lys Asn Asn Met Tyr Ser
1425 1430 1435 1440
Thr Pro Arg Val Leu Asn Ser Cys Ser Asn Arg Arg Val Tyr Lys Lys
1445 1450 1455
Met Pro Ser Ile Glu Ser Asp Val
1460
<210> 3
<211> 1484
<212> PRT
<213> GluN2B(GluN2B)
<400> 3
Met Lys Pro Arg Ala Glu Cys Cys Ser Pro Lys Phe Trp Leu Val Leu
1 5 10 15
Ala Val Leu Ala Val Ser Gly Ser Arg Ala Arg Ser Gln Lys Ser Pro
20 25 30
Pro Ser Ile Gly Ile Ala Val Ile Leu Val Gly Thr Ser Asp Glu Val
35 40 45
Ala Ile Lys Asp Ala His Glu Lys Asp Asp Phe His His Leu Ser Val
50 55 60
Val Pro Arg Val Glu Leu Val Ala Met Asn Glu Thr Asp Pro Lys Ser
65 70 75 80
Ile Ile Thr Arg Ile Cys Asp Leu Met Ser Asp Arg Lys Ile Gln Gly
85 90 95
Val Val Phe Ala Asp Asp Thr Asp Gln Glu Ala Ile Ala Gln Ile Leu
100 105 110
Asp Phe Ile Ser Ala Gln Thr Leu Thr Pro Ile Leu Gly Ile His Gly
115 120 125
Gly Ser Ser Met Ile Met Ala Asp Lys Asp Glu Ser Ser Met Phe Phe
130 135 140
Gln Phe Gly Pro Ser Ile Glu Gln Gln Ala Ser Val Met Leu Asn Ile
145 150 155 160
Met Glu Glu Tyr Asp Trp Tyr Ile Phe Ser Ile Val Thr Thr Tyr Phe
165 170 175
Pro Gly Tyr Gln Asp Phe Val Asn Lys Ile Arg Ser Thr Ile Glu Asn
180 185 190
Ser Phe Val Gly Trp Glu Leu Glu Glu Val Leu Leu Leu Asp Met Ser
195 200 205
Leu Asp Asp Gly Asp Ser Lys Ile Gln Asn Gln Leu Lys Lys Leu Gln
210 215 220
Ser Pro Ile Ile Leu Leu Tyr Cys Thr Lys Glu Glu Ala Thr Tyr Ile
225 230 235 240
Phe Glu Val Ala Asn Ser Val Gly Leu Thr Gly Tyr Gly Tyr Thr Trp
245 250 255
Ile Val Pro Ser Leu Val Ala Gly Asp Thr Asp Thr Val Pro Ala Glu
260 265 270
Phe Pro Thr Gly Leu Ile Ser Val Ser Tyr Asp Glu Trp Asp Tyr Gly
275 280 285
Leu Pro Ala Arg Val Arg Asp Gly Ile Ala Ile Ile Thr Thr Ala Ala
290 295 300
Ser Asp Met Leu Ser Glu His Ser Phe Ile Pro Glu Pro Lys Ser Ser
305 310 315 320
Cys Tyr Asn Thr His Glu Lys Arg Ile Tyr Gln Ser Asn Met Leu Asn
325 330 335
Arg Tyr Leu Ile Asn Val Thr Phe Glu Gly Arg Asn Leu Ser Phe Ser
340 345 350
Glu Asp Gly Tyr Gln Met His Pro Lys Leu Val Ile Ile Leu Leu Asn
355 360 365
Lys Glu Arg Lys Trp Glu Arg Val Gly Lys Trp Lys Asp Lys Ser Leu
370 375 380
Gln Met Lys Tyr Tyr Val Trp Pro Arg Met Cys Pro Glu Thr Glu Glu
385 390 395 400
Gln Glu Asp Asp His Leu Ser Ile Val Thr Leu Glu Glu Ala Pro Phe
405 410 415
Val Ile Val Glu Ser Val Asp Pro Leu Ser Gly Thr Cys Met Arg Asn
420 425 430
Thr Val Pro Cys Gln Lys Arg Ile Val Thr Glu Asn Lys Thr Asp Glu
435 440 445
Glu Pro Gly Tyr Ile Lys Lys Cys Cys Lys Gly Phe Cys Ile Asp Ile
450 455 460
Leu Lys Lys Ile Ser Lys Ser Val Lys Phe Thr Tyr Asp Leu Tyr Leu
465 470 475 480
Val Thr Asn Gly Lys His Gly Lys Lys Ile Asn Gly Thr Trp Asn Gly
485 490 495
Met Ile Gly Glu Val Val Met Lys Arg Ala Tyr Met Ala Val Gly Ser
500 505 510
Leu Thr Ile Asn Glu Glu Arg Ser Glu Val Val Asp Phe Ser Val Pro
515 520 525
Phe Ile Glu Thr Gly Ile Ser Val Met Val Ser Arg Ser Asn Gly Thr
530 535 540
Val Ser Pro Ser Ala Phe Leu Glu Pro Phe Ser Ala Asp Val Trp Val
545 550 555 560
Met Met Phe Val Met Leu Leu Ile Val Ser Ala Val Ala Val Phe Val
565 570 575
Phe Glu Tyr Phe Ser Pro Val Gly Tyr Asn Arg Cys Leu Ala Asp Gly
580 585 590
Arg Glu Pro Gly Gly Pro Ser Phe Thr Ile Gly Lys Ala Ile Trp Leu
595 600 605
Leu Trp Gly Leu Val Phe Asn Asn Ser Val Pro Val Gln Asn Pro Lys
610 615 620
Gly Thr Thr Ser Lys Ile Met Val Ser Val Trp Ala Phe Phe Ala Val
625 630 635 640
Ile Phe Leu Ala Ser Tyr Thr Ala Asn Leu Ala Ala Phe Met Ile Gln
645 650 655
Glu Glu Tyr Val Asp Gln Val Ser Gly Leu Ser Asp Lys Lys Phe Gln
660 665 670
Arg Pro Asn Asp Phe Ser Pro Pro Phe Arg Phe Gly Thr Val Pro Asn
675 680 685
Gly Ser Thr Glu Arg Asn Ile Arg Asn Asn Tyr Ala Glu Met His Ala
690 695 700
Tyr Met Gly Lys Phe Asn Gln Arg Gly Val Asp Asp Ala Leu Leu Ser
705 710 715 720
Leu Lys Thr Gly Lys Leu Asp Ala Phe Ile Tyr Asp Ala Ala Val Leu
725 730 735
Asn Tyr Met Ala Gly Arg Asp Glu Gly Cys Lys Leu Val Thr Ile Gly
740 745 750
Ser Gly Lys Val Phe Ala Ser Thr Gly Tyr Gly Ile Ala Ile Gln Lys
755 760 765
Asp Ser Gly Trp Lys Arg Gln Val Asp Leu Ala Ile Leu Gln Leu Phe
770 775 780
Gly Asp Gly Glu Met Glu Glu Leu Glu Ala Leu Trp Leu Thr Gly Ile
785 790 795 800
Cys His Asn Glu Lys Asn Glu Val Met Ser Ser Gln Leu Asp Ile Asp
805 810 815
Asn Met Ala Gly Val Phe Tyr Met Leu Gly Ala Ala Met Ala Leu Ser
820 825 830
Leu Ile Thr Phe Ile Cys Glu His Leu Phe Tyr Trp Gln Phe Arg His
835 840 845
Cys Phe Met Gly Val Cys Ser Gly Lys Pro Gly Met Val Phe Ser Ile
850 855 860
Ser Arg Gly Ile Tyr Ser Cys Ile His Gly Val Ala Ile Glu Glu Arg
865 870 875 880
Gln Ser Val Met Asn Ser Pro Thr Ala Thr Met Asn Asn Thr His Ser
885 890 895
Asn Ile Leu Arg Leu Leu Arg Thr Ala Lys Asn Met Ala Asn Leu Ser
900 905 910
Gly Val Asn Gly Ser Pro Gln Ser Ala Leu Asp Phe Ile Arg Arg Glu
915 920 925
Ser Ser Val Tyr Asp Ile Ser Glu His Arg Arg Ser Phe Thr His Ser
930 935 940
Asp Cys Lys Ser Tyr Asn Asn Pro Pro Cys Glu Glu Asn Leu Phe Ser
945 950 955 960
Asp Tyr Ile Ser Glu Val Glu Arg Thr Phe Gly Asn Leu Gln Leu Lys
965 970 975
Asp Ser Asn Val Tyr Gln Asp His Tyr His His His His Arg Pro His
980 985 990
Ser Ile Gly Ser Ala Ser Ser Ile Asp Gly Leu Tyr Asp Cys Asp Asn
995 1000 1005
Pro Pro Phe Thr Thr Gln Ser Arg Ser Ile Ser Lys Lys Pro Leu Asp
1010 1015 1020
Ile Gly Leu Pro Ser Ser Lys His Ser Gln Leu Ser Asp Leu Tyr Gly
1025 1030 1035 1040
Lys Phe Ser Phe Lys Ser Asp Arg Tyr Ser Gly His Asp Asp Leu Ile
1045 1050 1055
Arg Ser Asp Val Ser Asp Ile Ser Thr His Thr Val Thr Tyr Gly Asn
1060 1065 1070
Ile Glu Gly Asn Ala Ala Lys Arg Arg Lys Gln Gln Tyr Lys Asp Ser
1075 1080 1085
Leu Lys Lys Arg Pro Ala Ser Ala Lys Ser Arg Arg Glu Phe Asp Glu
1090 1095 1100
Ile Glu Leu Ala Tyr Arg Arg Arg Pro Pro Arg Ser Pro Asp His Lys
1105 1110 1115 1120
Arg Tyr Phe Arg Asp Lys Glu Gly Leu Arg Asp Phe Tyr Leu Asp Gln
1125 1130 1135
Phe Arg Thr Lys Glu Asn Ser Pro His Trp Glu His Val Asp Leu Thr
1140 1145 1150
Asp Ile Tyr Lys Glu Arg Ser Asp Asp Phe Lys Arg Asp Ser Val Ser
1155 1160 1165
Gly Gly Gly Pro Cys Thr Asn Arg Ser His Ile Lys His Gly Thr Gly
1170 1175 1180
Asp Lys His Gly Val Val Ser Gly Val Pro Ala Pro Trp Glu Lys Asn
1185 1190 1195 1200
Leu Thr Asn Val Glu Trp Glu Asp Arg Ser Gly Gly Asn Phe Cys Arg
1205 1210 1215
Ser Cys Pro Ser Lys Leu His Asn Tyr Ser Thr Thr Val Thr Gly Gln
1220 1225 1230
Asn Ser Gly Arg Gln Ala Cys Ile Arg Cys Glu Ala Cys Lys Lys Ala
1235 1240 1245
Gly Asn Leu Tyr Asp Ile Ser Glu Asp Asn Ser Leu Gln Glu Leu Asp
1250 1255 1260
Gln Pro Ala Ala Pro Val Ala Val Thr Ser Asn Ala Ser Thr Thr Lys
1265 1270 1275 1280
Tyr Pro Gln Ser Pro Thr Asn Ser Lys Ala Gln Lys Lys Asn Arg Asn
1285 1290 1295
Lys Leu Arg Arg Gln His Ser Tyr Asp Thr Phe Val Asp Leu Gln Lys
1300 1305 1310
Glu Glu Ala Ala Leu Ala Pro Arg Ser Val Ser Leu Lys Asp Lys Gly
1315 1320 1325
Arg Phe Met Asp Gly Ser Pro Tyr Ala His Met Phe Glu Met Ser Ala
1330 1335 1340
Gly Glu Ser Thr Phe Ala Asn Asn Lys Ser Ser Val Pro Thr Ala Gly
1345 1350 1355 1360
His His His His Asn Asn Pro Gly Gly Gly Tyr Met Leu Ser Lys Ser
1365 1370 1375
Leu Tyr Pro Asp Arg Val Thr Gln Asn Pro Phe Ile Pro Thr Phe Gly
1380 1385 1390
Asp Asp Gln Cys Leu Leu His Gly Ser Lys Ser Tyr Phe Phe Arg Gln
1395 1400 1405
Pro Thr Val Ala Gly Ala Ser Lys Ala Arg Pro Asp Phe Arg Ala Leu
1410 1415 1420
Val Thr Asn Lys Pro Val Val Ser Ala Leu His Gly Ala Val Pro Ala
1425 1430 1435 1440
Arg Phe Gln Lys Asp Ile Cys Ile Gly Asn Gln Ser Asn Pro Cys Val
1445 1450 1455
Pro Asn Asn Lys Asn Pro Arg Ala Phe Asn Gly Ser Ser Asn Gly His
1460 1465 1470
Val Tyr Glu Lys Leu Ser Ser Ile Glu Ser Asp Val
1475 1480

Claims (11)

1. The N-methyl-D-aspartate receptor recombinant antigen is characterized by comprising subunit GluN1a recombinant protein and subunit GluN2A recombinant protein and/or subunit GluN2B recombinant protein, wherein the amino acid sequence of the subunit GluN1a recombinant protein is as follows: the 22 nd cysteine in the amino acid sequence shown in SEQ ID NO. 1 is mutated to serine, the 61 st asparagine, the 350 th asparagine, the 471 rd asparagine and the 771 th asparagine are mutated to glutamine, the 595 th glutamic acid and the 597 th glutamic acid are mutated to serine, the 598th glutamic acid is mutated to threonine,
the amino acid sequence of the subunit GluN2A recombinant protein is the amino acid sequence shown in SEQ ID NO. 2,
the subunit GluN2B recombinant protein has the amino acid sequence as follows: the amino acid sequence shown in SEQ ID NO. 3, in which the 348 th asparagine is mutated to aspartic acid, the 838 th cysteine and the 849 th cysteine are mutated to serine.
2. A polynucleotide encoding a recombinant N-methyl-D-aspartate receptor antigen according to claim 1, wherein said polynucleotide comprises a nucleotide fragment encoding said subunit GluN1a recombinant protein and a nucleotide fragment encoding said subunit GluN2A recombinant protein,
or, the polynucleotide includes a nucleotide fragment encoding the subunit GluN1a recombinant protein and a nucleotide fragment encoding the subunit GluN2B recombinant protein,
or, the polynucleotide includes a nucleotide fragment encoding the subunit GluN1a recombinant protein, a nucleotide fragment encoding the subunit GluN2A recombinant protein, and a nucleotide fragment encoding the subunit GluN2B recombinant protein.
3. An expression vector comprising the polynucleotide of claim 2.
4. The expression vector of claim 3, wherein the expression vector is a vector capable of expression in an insect cell.
5. The expression vector of claim 4, wherein the expression vector is a baculovirus.
6. A cell line comprising the polynucleotide of claim 2 or the expression vector of any one of claims 3 to 5.
7. The cell line of claim 6, wherein said cell line is an insect cell transfected with baculovirus and expressing a protein.
8. A method of preparing the recombinant N-methyl-D-aspartate receptor antigen according to claim 1, wherein the method comprises: simultaneously expressing said subunit GluN1a recombinant protein and said subunit GluN2A recombinant protein in a cell, or simultaneously expressing said subunit GluN1a recombinant protein and said subunit GluN2B recombinant protein in a cell, or simultaneously expressing said subunit GluN1a recombinant protein, said subunit GluN2A recombinant protein and said subunit GluN2B recombinant protein in a cell.
9. The method according to claim 8, wherein the cell strain of claim 6 is cultured to obtain a cell culture solution, and then the recombinant antigen of N-methyl-D-aspartate receptor is obtained by separation and purification.
10. A kit for detecting an anti-N-methyl-D-aspartate receptor antibody, comprising the recombinant N-methyl-D-aspartate receptor antigen of claim 1.
11. The kit of claim 10, wherein the kit is a chemiluminescent assay kit;
and/or the detection sample type of the kit is one or more of serum, plasma or cerebrospinal fluid.
CN202111552136.XA 2021-12-17 2021-12-17 N-methyl-D-aspartate receptor recombinant antigen, preparation method thereof and kit containing recombinant antigen Active CN114369154B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111552136.XA CN114369154B (en) 2021-12-17 2021-12-17 N-methyl-D-aspartate receptor recombinant antigen, preparation method thereof and kit containing recombinant antigen

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111552136.XA CN114369154B (en) 2021-12-17 2021-12-17 N-methyl-D-aspartate receptor recombinant antigen, preparation method thereof and kit containing recombinant antigen

Publications (2)

Publication Number Publication Date
CN114369154A CN114369154A (en) 2022-04-19
CN114369154B true CN114369154B (en) 2024-03-08

Family

ID=81140797

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111552136.XA Active CN114369154B (en) 2021-12-17 2021-12-17 N-methyl-D-aspartate receptor recombinant antigen, preparation method thereof and kit containing recombinant antigen

Country Status (1)

Country Link
CN (1) CN114369154B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116253790A (en) * 2022-12-13 2023-06-13 迪亚莱博（张家港）生物科技有限公司 NMDAR recombinant protein for anti-NMDAR antibody detection and preparation method and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5502166A (en) * 1992-02-26 1996-03-26 Mitsubishi Chemical Corporation NMDH receptor proteins and genes encoding the same
CN111320684A (en) * 2018-12-13 2020-06-23 中国科学院脑科学与智能技术卓越创新中心 Expression of GluN1/GluN2A tetramer of human N-methyl-D-aspartate receptor and application thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IN178305B (en) * 1992-10-23 1997-03-22 Hoechst Ag

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5502166A (en) * 1992-02-26 1996-03-26 Mitsubishi Chemical Corporation NMDH receptor proteins and genes encoding the same
CN111320684A (en) * 2018-12-13 2020-06-23 中国科学院脑科学与智能技术卓越创新中心 Expression of GluN1/GluN2A tetramer of human N-methyl-D-aspartate receptor and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
海马神经元的瞬间外向钾电流;龚波;黄美燕;马义才;戚智;;生物化学与生物物理进展(04);第6-11页 *

Also Published As

Publication number Publication date
CN114369154A (en) 2022-04-19

Similar Documents

Publication Publication Date Title
CN111499746B (en) High-affinity rabbit monoclonal antibody for human interleukin-2 and application thereof
CN107643405B (en) Lung cancer diagnostic kit based on autoantibody detection and application thereof
CN111320684B (en) Expression of GluN1/GluN2A tetramer of human N-methyl-D-aspartate receptor and application thereof
CN108490174A (en) Detect the method and its application of the cell of CAR
CN114369154B (en) N-methyl-D-aspartate receptor recombinant antigen, preparation method thereof and kit containing recombinant antigen
KR100771402B1 (en) Srsv detection kit
CN114736292B (en) Nano antibody targeting norovirus protein and application thereof
WO2008001944A1 (en) Reagent for detection of autoantibody and kit for diagnosis of autoimmune disease
CN117700534A (en) Antibody for resisting monkey poxvirus A35R protein and application thereof
CN114891091B (en) NMDAR encephalitis resistant recombinant antigen and application thereof
CN116102643B (en) Monoclonal antibody for monkey poxvirus A35 protein and application thereof
CN111548423A (en) Mycoplasma pneumoniae fusion antigen and preparation method and application thereof
CN113308491B (en) Recombinant plasmid and recombinant cell for co-expressing NFAT and human DNAM-1 protein, and construction method and application thereof
CN109765370B (en) Prostate cancer diagnostic kit, preparation method and application
CN107163131B (en) Antigenic polypeptide of tumor suppressor factor p16 and application thereof
CN107828740B (en) Homer3 monoclonal antibody and application thereof
CN111778257A (en) Genetically modified CD96 protein, monoclonal antibody thereof and application
CN114487448B (en) Composition and kit for detecting myasthenia gravis-related antibody and application of composition and kit
KR102466369B1 (en) Diagnosis method for autoimmune encephalitis
JP7517659B2 (en) Polypeptides encoded by the Epstein-Barr virus BNLF2b gene and their use in detection - Patents.com
CN114957444B (en) NR1 subunit deletion mutant of NMDAR, mutant cell, construction method and application
CN110981947B (en) Preparation and application of treponema pallidum TP47 recombinant antigen
KR102650819B1 (en) Diagnostic kit for skin comprising antibody specific for LDH1 of Lactobacillus plantarum
CN110240644B (en) Human growth hormone receptor mutant, human growth hormone immunogen, polyclonal antibody and detection kit
CN116253790A (en) NMDAR recombinant protein for anti-NMDAR antibody detection and preparation method and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant