CN114957445B - NMDAR NR1 subunit, mutant of NMDAR, construction method and application thereof - Google Patents
NMDAR NR1 subunit, mutant of NMDAR, construction method and application thereof Download PDFInfo
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Abstract
The invention provides an NMDARRR 1 subunit, an NMDAR mutant, and a construction method and application thereof, and relates to the technical field of biological medicine. The NMDARRR 1 subunit mutant does not recognize autoantibodies in patients with NMDAR encephalitis, but shows the same calcium influx function as wild type NR1 when co-transformed with the NR2 subunit of NMDAR, thereby providing important directions for diagnosis and treatment of NMDAR related diseases on a gene level, including related diseases such as replacement of obstacle type NMDAR ion channels, treatment of diseases caused by protruding membrane surface NMDAR reduction caused by pathogenic antibodies, and the like.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to an NMDAR NR1 subunit, an NMDAR mutant, a construction method and application thereof.
Background
NMDAR is an ion channel protein distributed on the surface of the postsynaptic membrane of nerve cells, is one of the subtypes of ionic glutamate receptors, and consists of NR1, NR2 and NR3 subunits. In functionally active NMDARs, at least 1 NR1 and 1 NR2 should be present for 4 subunits, whereas most NMDARs consist of 2 NR1 and 2 NR2, and a small percentage consists of 3 subunits together. NMDA receptors have a complex molecular structure and unique pharmacological properties that are highly permeable to calcium ions, which makes NMDA receptors important in synaptic plasticity and excitotoxicity. When neurotransmitters bind to NMDAR, functional NMDAR ion channels open, ca 2+ Inflow, then triggering a series of biochemical reactions, can promote learning and memory functions; but excessive Ca 2+ The influx causes necrosis of neurons, causing learning and memory impairment. The pathogenesis and pathophysiological processes of various neurodegenerative and neuropsychiatric diseases in humans may be related to NMDAR channel failure, and thus, research on NMDAR channels may provide targets for the development of therapeutic drugs.
anti-NMDAR encephalitis is the most important autoimmune encephalitis, accounting for 70% -80% of patients with autoimmune encephalitis. Mainly present in NMDAR encephalitis patients are specific IgG antibodies directed against the NR1 subunit of NMDAR. These antibodies are pathogenic, induce crosslinking and internalization of NMDA receptors, and block NMDAR-dependent synaptic activity, leading to clinical manifestations of seizures, mental abnormalities, and conscious disturbance. Studies have shown that antibodies against NMDAR encephalitis patients inhibit NMDAR-dependent intracellular Ca 2+ Inflow (Ignacio R A, josep D, teresa S, et al Isolated hemidystonia associated with NMDA receptor antibodies [ J)]Movement disorders: official journal of the Movement Disorder Society,2011,26 (2): 351). Thus, intracellular Ca that maintains NMDAR dependence 2+ Inflow of Ca 2+ The mediated physiological and biochemical reactions are of paramount importance.
Another study showed that, in activating NMDAR channels but not other Ca 2+ Channels (including kainic acid receptor and voltage sensitive Ca) 2+ Channels) calcium readily enters the mitochondria. Ca (Ca) 2+ Direction lineInhibition of granulocyte transport protects neurons from glutamate (Glu) -mediated cell death. Fukudori et al prepared acquired NMDAR channels from Human Embryonic Kidney (HEK) -293 cells, demonstrating that mitochondrial uncoupling protein-2 (UCP 2) may be involved in cellular function and/or dysfunction to some extent, its mechanism and modulation of mitochondrial free Ca following activation of functional NMDAR channels 2+ Level dependent (Fukudori R, takarada T, kambe Y, et al Possible involvement of mitochondrial uncoupling protein-2in cytotoxicity mediated by acquired N-methyl-D-aspartate receptor channels [ J)]Neurochemistry international,2012,61 (4): 498-505). Therefore, the research of the calcium influx function of the NMDAR channel has important significance for the diagnosis and treatment of NMDAR related diseases.
Disclosure of Invention
In view of the above, the present invention aims to provide an NMDAR NR1 subunit, a mutant of NMDAR, which does not recognize autoantibodies in patients with anti-NMDAR encephalitis, but which exhibits the same calcium influx function as wild-type NR1 when co-transformed with the NR2 subunit of NMDAR, thereby providing an important direction for diagnostic treatment of NMDAR-related diseases at the gene level, as well as a method for constructing the same and uses thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a mutant of an NMDAR NR1 subunit that does not recognize autoantibodies against an NMDAR encephalitis patient;
the mutants include the 837-838 and 864-937 positions of the NMDAR NR1 subunit encoding amino acid sequence deleted.
Preferably, the nucleotide sequence of the NMDAR NR1 subunit is shown in SEQ ID NO. 1.
The invention provides a primer pair for constructing the mutant, which comprises 837-838-F, 837-838-R, 864-937-F and 864-937-R, wherein the nucleotide sequence of 837-838-F is shown as SEQ ID NO.4, and the nucleotide sequence of 837-838-R is shown as SEQ ID NO. 5; the nucleotide sequence of 864-937-F is shown as SEQ ID NO.6, and the nucleotide sequence of 864-937-R is shown as SEQ ID NO. 7.
The invention provides a method for constructing the mutant, which comprises the following steps: (1) PCR amplification is carried out by using a nucleotide sequence shown in SEQ ID NO.1 as a template and utilizing 864-937-F and 864-937-R to obtain an NRI subunit mutant with 864-937 amino acid deletion; the nucleotide sequence of 864-937-F is shown as SEQ ID NO.6, and the nucleotide sequence of 864-937-R is shown as SEQ ID NO. 7;
(2) Taking the NRI subunit mutant with the deletion 864-937 amino acid as a template, and carrying out PCR amplification by using 837-838-F and 837-838-R to obtain the mutant of the NMDAR NR1 subunit with the deletion 837-838 amino acid and 864-937 amino acid; the nucleotide sequence of 837-838-F is shown as SEQ ID NO.4, and the nucleotide sequence of 837-838-R is shown as SEQ ID NO. 5.
Preferably, the PCR amplification system of step (1) and step (2) comprises, in terms of 50. Mu.L: 50ng of template, 2. Mu.L of primer F, 2. Mu.L of primer R, fastPfu DNA Polymerase. Mu.L, 10. Mu.L of 5 XFast Pfukuffer, 4. Mu. L, DMSO 1. Mu.L of 2.5mM dNTP and the balance of nuclease-free water;
the PCR amplification procedures of step (1) and step (2) each comprise: pre-denaturation at 98℃for 2min; denaturation at 98℃for 15s, annealing at 59℃for 15s, extension at 72℃for 4min,33 cycles; and extending at 72 ℃ for 8min.
The invention also provides a method for constructing the NMDAR mutant, which comprises the following steps:
(1) Inserting the mutant into a eukaryotic expression vector to obtain an NR1 recombinant vector;
(2) Inserting NR2 subunit of NMDAR into eukaryotic expression vector to obtain NR2 recombinant vector;
(3) Co-transfecting eukaryotic cells with an NR1 recombinant vector and an NR2 recombinant vector to obtain NMDAR mutants;
step (1) and step (2) do not have a chronological order.
Preferably, the eukaryotic expression vectors of step (1) and step (2) are identical.
Preferably, the NR2 subunit in step (2) comprises NR2a or NR2b, wherein the nucleotide sequence of NR2a is shown as SEQ ID NO.2, and the nucleotide sequence of NR2b is shown as SEQ ID NO. 3.
Preferably, in the co-transfection in the step (3), the mass ratio of the NR1 recombinant vector to the NR2 recombinant vector is (1:1) - (1:4).
The invention also provides application of the mutant or the NMDAR mutant prepared by the method in preparation of a reagent for diagnosis and/or treatment of NMDAR related diseases.
The beneficial effects are that: the invention provides an NMDAR NR1 subunit mutant, which does not recognize autoantibodies in patients with NMDAR encephalitis, but shows the same calcium influx function as wild type NR1 when co-transformed with NR2 subunits of NMDAR, thereby providing important directions for diagnosis and treatment of NMDAR related diseases at the gene level, including related diseases such as diseases caused by replacement of obstacle type NMDAR ion channels and reduction of NMDAR on the membrane surface after protrusion caused by treatment of pathogenic antibodies.
Drawings
FIG. 1 is a graph showing the results of cells after co-transformation of mutants of NR1 subunit with NR2a subunit, respectively;
FIG. 2 is a graph showing the results of cells after co-transformation of mutants of NR1 subunit with NR2b subunit, respectively.
Detailed Description
The invention provides a mutant of an NMDAR NR1 subunit that does not recognize autoantibodies against an NMDAR encephalitis patient;
the mutants include the 837-838 and 864-937 positions of the NMDAR NR1 subunit encoding amino acid sequence deleted.
The nucleotide sequence of the NMDAR NR1 subunit of the invention is preferably shown in SEQ ID NO.1, and in the present invention, the mutant of NMDAR NR1 subunit is referred to as NR1 delta 837-838. In the examples of the present invention, NR1 subunit mutants, namely NR1 delta 837-838, co-transformed with NMDAR NR2a or NR2b subunits, showed substantial cell death under bright field, and displayed an approximation to wild type NR1 subunits, and formed with Ca 2+ Ion channels with internal flow functions; meanwhile, the mutant does not bind with autoantibodies in NMDAR encephalitis patients, and has a good application prospect.
The invention provides a primer pair for constructing the mutant, which comprises 837-838-F, 837-838-R, 864-937-F and 864-937-R, wherein the nucleotide sequence of 837-838-F is shown as SEQ ID NO.4, and the nucleotide sequence of 837-838-R is shown as SEQ ID NO. 5; the nucleotide sequence of 864-937-F is shown as SEQ ID NO.6, and the nucleotide sequence of 864-937-R is shown as SEQ ID NO. 7.
The invention provides a method for constructing the mutant, which comprises the following steps: (1) PCR amplification is carried out by using a nucleotide sequence shown in SEQ ID NO.1 as a template and utilizing 864-937-F and 864-937-R to obtain an NRI subunit mutant with 864-937 amino acid deletion; the nucleotide sequence of 864-937-F is shown as SEQ ID NO.6, and the nucleotide sequence of 864-937-R is shown as SEQ ID NO. 7;
(2) Taking the NRI subunit mutant with the deletion 864-937 amino acid as a template, and carrying out PCR amplification by using 837-838-F and 837-838-R to obtain the mutant of the NMDAR NR1 subunit with the deletion 837-838 amino acid and 864-937 amino acid; the nucleotide sequence of 837-838-F is shown as SEQ ID NO.4, and the nucleotide sequence of 837-838-R is shown as SEQ ID NO. 5.
When the NR1 delta 837-838 is constructed, firstly, a wild NR1 is taken as a template, an NR1 subunit mutant which lacks 864-937 amino acids is constructed, and an NR1 subunit mutant which lacks 864-937 amino acids is taken as a template, so that the NR1 delta 837-838 is constructed.
In the PCR amplification of the two steps, the system and the procedure are the same except that the template and the primer sequences are different. The system for PCR amplification according to the present invention preferably comprises, in 50. Mu.L: 50ng of template, 2. Mu.L of primer F (0.2. Mu.M), 2. Mu.L of primer R (0.2. Mu.M), 1. Mu.L of primer Fast Pfu DNA Polymerase (2.5 units), 10. Mu.L of 5 XFast Pfubuffer, 4. Mu. L, DMSO 1. Mu.L of 2.5mM dNTP and the balance of nuclease-free water. The PCR amplification procedure of the present invention preferably comprises: pre-denaturation at 98℃for 2min; denaturation at 98℃for 15s, annealing at 59℃for 15s, extension at 72℃for 4min,33 cycles; and extending at 72 ℃ for 8min.
The invention also provides a method for constructing the NMDAR mutant, which comprises the following steps:
(1) Inserting the mutant into a eukaryotic expression vector to obtain an NR1 recombinant vector;
(2) Inserting NR2 subunit of NMDAR into eukaryotic expression vector to obtain NR2 recombinant vector;
(3) Co-transfecting eukaryotic cells with an NR1 recombinant vector and an NR2 recombinant vector to obtain NMDAR mutants;
step (1) and step (2) do not have a chronological order.
The mutant is inserted into a eukaryotic expression vector to obtain an NR1 recombinant vector. The eukaryotic expression vectors of the present invention preferably include pCDNA3.1, PIRESpuro3, pCMV-Myc, p3xFLAG-CMV-10 or other eukaryotic vectors which replace part of the vector elements but overexpress proteins, examples are described with pCDNA3.1 and the mutant gene is inserted between the NheI and NotI cleavage sites of pCDNA3.1, but this is not to be construed as a complete scope of the present invention.
According to the invention, NR2 subunits of NMDAR are inserted into eukaryotic expression vectors to obtain NR2 recombinant vectors. The eukaryotic expression vector of the present invention is preferably the same as described above, and will not be described in detail herein. In the present invention, the NR1 mutant and NR2 may be identical or different from each other in the eukaryotic expression vector, and the same insertion site is exemplified in the examples, but it is not intended to be construed as the full scope of the present invention. The NR2 subunit of the invention preferably comprises NR2a or NR2b, the nucleotide sequence of NR2a is preferably as shown in SEQ ID NO.2, and the nucleotide sequence of NR2b is preferably as shown in SEQ ID NO. 3.
After NR1 recombinant vector and NR2 recombinant vector are obtained, the invention transfects eukaryotic cells together with NR1 recombinant vector and NR2 recombinant vector to obtain NMDAR mutant.
The present invention, when carrying out said co-transfection, preferably also comprises the cultivation of eukaryotic cells (cell plating), more preferably comprises: preparing 10% FBS-DMEM high sugar culture medium from DMEM high sugar culture medium and FBS at a ratio of 9:1, and transferring to a new pore plate at a ratio of 1:5-1:6 when eukaryotic cells in the pore plate are fully paved, and placing at 37 ℃ and 5% CO 2 Culturing in a cell culture box, and carrying out the next transfection when the cell density is 30% -40%. The pore plate can be 96 pore plate, 48 pore plate, 24 pore plate, 12 pore plate, 6 pore plate and cell culture dishes with different diameters,an orifice plate containing a suitably sized cell slide is also possible. The eukaryotic cells of the present invention preferably include 293T cells, CHO cells or other cells that overexpress eukaryotic expression vectors, and 293T cells are exemplified in the examples, but are intended to be within the full scope of the present invention.
In the co-transfection of the present invention, the mass ratio of the NR1 recombinant vector to the NR2 recombinant vector is preferably (1:1) to (1:4), and more preferably 1:1. The NR1 recombinant vector and the NR2 recombinant vector are uniformly mixed, and then are co-transfected into the cultured cells by using a transfection reagent PEI.
In the embodiment of the invention, the cells obtained after co-transfection are also verified, wherein the verification comprises calcium ion fluorescent probe staining and immunofluorescence staining, and the method provided by the invention is used for obtaining mutants which have similar calcium influx function as wild type NR1 alpha subunits and do not recognize autoantibodies, thus providing important directions for diagnosis and treatment of NMDAR related diseases, including related diseases such as diseases caused by replacement of obstacle type NMDAR ion channels and reduction of NMDAR on the membrane surface after treatment of pathogenic antibodies.
The invention also provides application of the mutant or the NMDAR mutant prepared by the method in preparation of a reagent for diagnosis and/or treatment of NMDAR related diseases.
The application of the present invention is preferably the same as that described above, and will not be described again here.
The following examples are provided to illustrate a mutant of NMDAR NR1 subunit, NMDAR, and methods of construction and use thereof, but are not to be construed as limiting the scope of the invention.
EXAMPLE 1 construction of NMDAR subunit recombinant vectors
1. Synthesizing an NR1 subunit DNA sequence (SEQ ID NO. 1) of the NMDAR onto pCDNA3.1 by an artificial synthesis method, and inserting restriction enzyme sites into the pCDNA3.1, wherein the restriction enzyme sites are NheI and NotI; synthesizing an NR2a subunit DNA sequence (SEQ ID NO. 2) of NMDAR onto pCDNA3.1, and inserting cleavage sites into the pCDNA3.1, wherein the cleavage sites are NheI and NotI; the NR2b subunit DNA sequence of NMDAR (SEQ ID NO. 3) was synthesized onto pCDNA3.1 with insertion sites NheI and NotI.
2. Primers were designed based on the mutation sites, and specific sequences of the primers are shown in Table 1.
Construction primers for mutant of Table 1
3. And (3) respectively carrying out PCR amplification by using the primers in the step (2), and carrying out agarose gel electrophoresis on the amplified products to identify.
When the primer pair with the number of 1 is used for PCR amplification, the DNA sequence of the NMDAR NR1 subunit with the 864-937 amino acid deleted is used as a template;
when the primer pair with the number of 2-11 is used for PCR amplification, the DNA sequence of NMDAR NR1 subunit is used as a template;
the PCR amplification system is as follows: 50ng of template, 2. Mu.L of primer F (0.2. Mu.M), 2. Mu.L of primer R (0.2. Mu.M), 1. Mu.L of primer Fast Pfu DNA Polymerase (2.5 units), 10. Mu.L of 5 XFast Pfu buffer, 4. Mu. L, DMSO 1. Mu.L of 2.5mM dNTP and finally supplementing the system with 50. Mu.L by using Nuclease-free Water;
the procedure for PCR amplification included: pre-denaturation at 98℃for 2min; denaturation at 98℃for 15s, annealing at 59℃for 15s, extension at 72℃for 4min, denaturation to 33 cycles; and extending at 72 ℃ for 8min.
4. And (3) further processing, converting, sequencing and greatly extracting plasmids with correct sequencing by the PCR product obtained in the step (3).
The PCR products amplified by using the primers numbered 1 and 4 to 11 were treated with DMT enzyme at 37℃for 1 hour, and the template plasmid was removed to recover the gel. The gel recovered product was treated with T4Polynucleotide Kinase (following NEB instructions), the treated product was added to T4 ligase and ligated for 2 hours at room temperature, and then the ligation product was transformed into competent cells (DMT Chemically Competent Cell);
amplifying each PCR product obtained by using primers with the number of 2-3, respectively treating the PCR products for 1h at 37 ℃ by using DMT enzyme, removing template plasmids, and then converting the treated PCR products into competent cells (DMT Chemically Competent Cell);
plating the transformed product, and culturing in an incubator at 37 ℃ overnight; the following day, LB liquid medium was picked up for monoclonal to kana resistance, shake-cultured overnight at 37℃and the plasmid was sent for Jin Weizhi sequencing. The plasmid with correct sequence is greatly saved.
Example 2 preparation of cells overexpressing each mutant and staining with calcium fluorescent probes
(1) Cell plating: the DMEM high sugar culture medium and FBS are prepared into 10% FBS-DMEM high sugar culture medium according to the proportion of 9:1, and the 10% FBS-DMEM high sugar culture medium is passaged into a new 24-well plate according to the proportion of 1:5-1:6 when 293T cells in the 24-well plate are fully paved, and then placed at 37 ℃ and 5% CO 2 Culturing in a cell incubator, and carrying out the next transfection when the cell density is 30% -40%;
(2) PEI transfection: mixing 3 mu gNR subunit mutants with 3 mu g NR2a subunit or 3 mu gNR b subunit respectively according to a ratio of 1:1, and transfecting the mixed plasmid with a transfection reagent PEI according to a ratio of 1:2 to cells obtained by culturing in the step (1);
(3) Staining the cells obtained in step 2 with a calcium ion fluorescent probe
24h-48h after cell transfection, discarding the culture medium, washing 2 times by using PBS, adding 4 mu M rhodo calcium ion fluorescent probe solution for incubation for 10 min, and washing 2 times by using PBS;
(4) Observing the condition of calcium influx in cells under a bright field and a red fluorescent channel under a fluorescent microscope;
(5) Mutants of the NR1 subunit were co-transformed with the NR2a subunit for cell death, calcium fluorescent probe staining, and binding to NMDAR patient autoantibodies, respectively, as shown in Table 2 and FIG. 1.
Table 2 cases of cells after co-transformation of mutants of the NR1 subunit with the NR2a subunit, respectively
(6) The results of the NR1 subunit mutants, co-transferred with the NR2b subunit, under microscopic fields and in the red fluorescence channel are shown in Table 3 and FIG. 2.
Table 3 cases of cells after co-transformation of mutants of the NR1 subunit with the NR2b subunit, respectively
As is clear from the above, NR1 subunits lacking amino acids 837 to 838 and 864 to 937, namely NR 1. DELTA.837 to 838, co-transferred with NMDAR NR2a and NR2b subunits, respectively, and when observed under bright field, cells die in large amounts, showing a similar effect to wild type NR1 subunits, and Ca can be formed 2+ Ion channels with internal flow functions; meanwhile, the mutant does not bind with autoantibodies in NMDAR encephalitis patients, and has a good application prospect.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Shaanxi Maiyuan Biotech Co., ltd
<120> an NMDAR NR1 subunit, mutant of NMDAR, construction method and application thereof
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<170> SIPOSequenceListing 1.0
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atgagcacca tgcgcctgct gacgctcgcc ctgctgttct cctgctccgt cgcccgtgcc 60
gcgtgcgacc ccaagatcgt caacattggc gcggtgctga gcacgcggaa gcacgagcag 120
atgttccgcg aggccgtgaa ccaggccaac aagcggcacg gctcctggaa gattcagctc 180
aatgccacct ccgtcacgca caagcccaac gccatccaga tggctctgtc ggtgtgcgag 240
gacctcatct ccagccaggt ctacgccatc ctagttagcc atccacctac ccccaacgac 300
cacttcactc ccacccctgt ctcctacaca gccggcttct accgcatacc cgtgctgggg 360
ctgaccaccc gcatgtccat ctactcggac aagagcatcc acctgagctt cctgcgcacc 420
gtgccgccct actcccacca gtccagcgtg tggtttgaga tgatgcgtgt ctacagctgg 480
aaccacatca tcctgctggt cagcgacgac cacgagggcc gggcggctca gaaacgcctg 540
gagacgctgc tggaggagcg tgagtccaag gcagagaagg tgctgcagtt tgacccaggg 600
accaagaacg tgacggccct gctgatggag gcgaaagagc tggaggcccg ggtcatcatc 660
ctttctgcca gcgaggacga tgctgccact gtataccgcg cagccgcgat gctgaacatg 720
acgggctccg ggtacgtgtg gctggtcggc gagcgcgaga tctcggggaa cgccctgcgc 780
tacgccccag acggcatcct cgggctgcag ctcatcaacg gcaagaacga gtcggcccac 840
atcagcgacg ccgtgggcgt ggtggcccag gccgtgcacg agctcctcga gaaggagaac 900
atcaccgacc cgccgcgggg ctgcgtgggc aacaccaaca tctggaagac cgggccgctc 960
ttcaagagag tgctgatgtc ttccaagtat gcggatgggg tgactggtcg cgtggagttc 1020
aatgaggatg gggaccggaa gttcgccaac tacagcatca tgaacctgca gaaccgcaag 1080
ctggtgcaag tgggcatcta caatggcacc cacgtcatcc ctaatgacag gaagatcatc 1140
tggccaggcg gagagacaga gaagcctcga gggtaccaga tgtccaccag actgaagatt 1200
gtgacgatcc accaggagcc cttcgtgtac gtcaagccca cgctgagtga tgggacatgc 1260
aaggaggagt tcacagtcaa cggcgaccca gtcaagaagg tgatctgcac cgggcccaac 1320
gacacgtcgc cgggcagccc ccgccacacg gtgcctcagt gttgctacgg cttttgcatc 1380
gacctgctca tcaagctggc acggaccatg aacttcacct acgaggtgca cctggtggca 1440
gatggcaagt tcggcacaca ggagcgggtg aacaacagca acaagaagga gtggaatggg 1500
atgatgggcg agctgctcag cgggcaggca gacatgatcg tggcgccgct aaccataaac 1560
aacgagcgcg cgcagtacat cgagttttcc aagcccttca agtaccaggg cctgactatt 1620
ctggtcaaga aggagattcc ccggagcacg ctggactcgt tcatgcagcc gttccagagc 1680
acactgtggc tgctggtggg gctgtcggtg cacgtggtgg ccgtgatgct gtacctgctg 1740
gaccgcttca gccccttcgg ccggttcaag gtgaacagcg aggaggagga ggaggacgca 1800
ctgaccctgt cctcggccat gtggttctcc tggggcgtcc tgctcaactc cggcatcggg 1860
gaaggcgccc ccagaagctt ctcagcgcgc atcctgggca tggtgtgggc cggctttgcc 1920
atgatcatcg tggcctccta caccgccaac ctggcggcct tcctggtgct ggaccggccg 1980
gaggagcgca tcacgggcat caacgaccct cggctgagga acccctcgga caagtttatc 2040
tacgccacgg tgaagcagag ctccgtggat atctacttcc ggcgccaggt ggagctgagc 2100
accatgtacc ggcatatgga gaagcacaac tacgagagtg cggcggaggc catccaggcc 2160
gtgagagaca acaagctgca tgccttcatc tgggactcgg cggtgctgga gttcgaggcc 2220
tcgcagaagt gcgacctggt gacgactgga gagctgtttt tccgctcggg cttcggcata 2280
ggcatgcgca aagacagccc ctggaagcag aacgtctccc tgtccatcct caagtcccac 2340
gagaatggct tcatggaaga cctggacaag acgtgggttc ggtatcagga atgtgactcg 2400
cgcagcaacg cccctgcgac ccttactttt gagaacatgg ccggggtctt catgctggta 2460
gctgggggca tcgtggccgg gatcttcctg attttcatcg agattgccta caagcggcac 2520
aaggatgctc gccggaagca gatgcagctg gcctttgccg ccgttaacgt gtggcggaag 2580
aacctgcagg atagaaagag tggtagagca gagcctgacc ctaaaaagaa agccacattt 2640
agggctatca cctccaccct ggcttccagc ttcaagaggc gtaggtcctc caaagacacg 2700
agcaccgggg gtggacgcgg cgctttgcaa aaccaaaaag acacagtgct gccgcgacgc 2760
gctattgaga gggaggaggg ccagctgcag ctgtgttccc gtcataggga gagctga 2817
<210> 2
<211> 4395
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atgggcagag tgggctattg gaccctgctg gtgctgccgg cccttctggt ctggcgcggt 60
ccggcgccga gcgcggcggc ggagaagggt ccccccgcgc taaatattgc ggtgatgctg 120
ggtcacagcc acgacgtgac agagcgcgaa cttcgaacac tgtggggccc cgagcaggcg 180
gcggggctgc ccctggacgt gaacgtggta gctctgctga tgaaccgcac cgaccccaag 240
agcctcatca cgcacgtgtg cgacctcatg tccggggcac gcatccacgg cctcgtgttt 300
ggggacgaca cggaccagga ggccgtagcc cagatgctgg attttatctc ctcccacacc 360
ttcgtcccca tcttgggcat tcatgggggc gcatctatga tcatggctga caaggatccg 420
acgtctacct tcttccagtt tggagcgtcc atccagcagc aagccacggt catgctgaag 480
atcatgcagg attatgactg gcatgtcttc tccctggtga ccactatctt ccctggctac 540
agggaattca tcagcttcgt caagaccaca gtggacaaca gctttgtggg ctgggacatg 600
cagaatgtga tcacactgga cacttccttt gaggatgcaa agacacaagt ccagctgaag 660
aagatccact cttctgtcat cttgctctac tgttccaaag acgaggctgt tctcattctg 720
agtgaggccc gctcccttgg cctcaccggg tatgatttct tctggattgt ccccagcttg 780
gtctctggga acacggagct catcccaaaa gagtttccat cgggactcat ttctgtctcc 840
tacgatgact gggactacag cctggaggcg agagtgaggg acggcattgg catcctaacc 900
accgctgcat cttctatgct ggagaagttc tcctacatcc ccgaggccaa ggccagctgc 960
tacgggcaga tggagaggcc agaggtcccg atgcacacct tgcacccatt tatggtcaat 1020
gttacatggg atggcaaaga cttatccttc actgaggaag gctaccaggt gcaccccagg 1080
ctggtggtga ttgtgctgaa caaagaccgg gaatgggaaa aggtgggcaa gtgggagaac 1140
catacgctga gcctgaggca cgccgtgtgg cccaggtaca agtccttctc cgactgtgag 1200
ccggatgaca accatctcag catcgtcacc ctggaggagg ccccattcgt catcgtggaa 1260
gacatagacc ccctgaccga gacgtgtgtg aggaacaccg tgccatgtcg gaagttcgtc 1320
aaaatcaaca attcaaccaa tgaggggatg aatgtgaaga aatgctgcaa ggggttctgc 1380
attgatattc tgaagaagct ttccagaact gtgaagttta cttacgacct ctatctggtg 1440
accaatggga agcatggcaa gaaagttaac aatgtgtgga atggaatgat cggtgaagtg 1500
gtctatcaac gggcagtcat ggcagttggc tcgctcacca tcaatgagga acgttctgaa 1560
gtggtggact tctctgtgcc ctttgtggaa acgggaatca gtgtcatggt ttcaagaagt 1620
aatggcaccg tctcaccttc tgcttttcta gaaccattca gcgcctctgt ctgggtgatg 1680
atgtttgtga tgctgctcat tgtttctgcc atagctgttt ttgtctttga atacttcagc 1740
cctgttggat acaacagaaa cttagccaaa gggaaagcac cccatgggcc ttcttttaca 1800
attggaaaag ctatatggct tctttggggc ctggtgttca ataactccgt gcctgtccag 1860
aatcctaaag ggaccaccag caagatcatg gtatctgtat gggccttctt cgctgtcata 1920
ttcctggcta gctacacagc caatctggct gccttcatga tccaagagga atttgtggac 1980
caagtgaccg gcctcagtga caaaaagttt cagagacctc atgactattc cccacctttt 2040
cgatttggga cagtgcctaa tggaagcacg gagagaaaca ttcggaataa ctatccctac 2100
atgcatcagt acatgaccaa atttaatcag aaaggagtag aggacgcctt ggtcagcctg 2160
aaaacgggga agctggacgc tttcatctac gatgccgcag tcttgaatta caaggctggg 2220
agggatgaag gctgcaagct ggtgaccatc gggagtgggt acatctttgc caccaccggt 2280
tatggaattg cccttcagaa aggctctcct tggaagaggc agatcgacct ggccttgctt 2340
cagtttgtgg gtgatggtga gatggaggag ctggagaccc tgtggctcac tgggatctgc 2400
cacaacgaga agaacgaggt gatgagcagc cagctggaca ttgacaacat ggcgggcgta 2460
ttctacatgc tggctgccgc catggccctt agcctcatca ccttcatctg ggagcacctc 2520
ttctactgga agctgcgctt ctgtttcacg ggcgtgtgct ccgaccggcc tgggttgctc 2580
ttctccatca gcaggggcat ctacagctgc attcatggag tgcacattga agaaaagaag 2640
aagtctccag acttcaatct gacgggatcc cagagcaaca tgttaaaact cctccggtca 2700
gccaaaaaca tttccagcat gtccaacatg aactcctcaa gaatggactc acccaaaaga 2760
gctgctgact tcatccaaag aggttccctc atcatggaca tggtttcaga taaggggaat 2820
ttgatgtact cagacaacag gtcctttcag gggaaagaga gcatttttgg agacaacatg 2880
aacgaactcc aaacatttgt ggccaaccgg cagaaggata acctcaataa ctatgtattc 2940
cagggacaac atcctcttac tctcaatgag tccaacccta acacggtgga ggtggccgtg 3000
agcacagaat ccaaagcgaa ctctagaccc cggcagctgt ggaagaaatc cgtggattcc 3060
atacgccagg attcactatc ccagaatcca gtctcccaga gggatgaggc aacagcagag 3120
aataggaccc actccctaaa gagccctagg tatcttccag aagagatggc ccactctgac 3180
atttcagaaa cgtcaaatcg ggccacgtgc cacagggaac ctgacaacag taagaaccac 3240
aaaaccaagg acaactttaa aaggtcagtg gcctccaaat accccaagga ctgtagtgag 3300
gtcgagcgca cctacctgaa aaccaaatca agctccccta gagacaagat ctacactata 3360
gatggtgaga aggagcctgg tttccactta gatccacccc agtttgttga aaatgtgacc 3420
ctgcccgaga acgtggactt cccggacccc taccaggatc ccagtgaaaa cttccgcaag 3480
ggggactcca cgctgccaat gaaccggaac cccttgcata atgaagaggg gctttccaac 3540
aacgaccagt ataaactcta ctccaagcac ttcaccttga aagacaaggg ttccccgcac 3600
agtgagacca gcgagcgata ccggcagaac tccacgcact gcagaagctg cctttccaac 3660
atgcccacct attcaggcca cttcaccatg aggtccccct tcaagtgcga tgcctgcctg 3720
cggatgggga acctctatga catcgatgaa gaccagatgc ttcaggagac aggtaaccca 3780
gccaccgggg agcaggtcta ccagcaggac tgggcacaga acaatgccct tcaattacaa 3840
aagaacaagc taaggattag ccgtcagcat tcctacgata acattgtcga caaacctagg 3900
gagctagacc ttagcaggcc ctcccggagc ataagcctca aggacaggga acggcttctg 3960
gagggaaatt tttacggcag cctgtttagt gtcccctcaa gcaaactctc ggggaaaaaa 4020
agctcccttt tcccccaagg tctggaggac agcaagagga gcaagtctct cttgccagac 4080
cacacctccg ataacccttt cctccactcc cacagggatg accaacgctt ggttattggg 4140
agatgcccct cggaccctta caaacactcg ttgccatccc aggcggtgaa tgacagctat 4200
cttcggtcgt ccttgaggtc aacggcatcg tactgttcca gggacagtcg gggccacaat 4260
gatgtgtata tttcggagca tgttatgcct tatgctgcaa ataagaataa tatgtactct 4320
acccccaggg ttttaaattc ctgcagcaat agacgcgtgt acaagaaaat gcctagtatc 4380
gaatctgatg tttaa 4395
<210> 3
<211> 4455
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atgaagccca gagcggagtg ctgttctccc aagttctggt tggtgttggc cgtcctggcc 60
gtgtcaggca gcagagctcg ttctcagaag agccccccca gcattggcat tgctgtcatc 120
ctcgtgggca cttccgacga ggtggccatc aaggatgccc acgagaaaga tgatttccac 180
catctctccg tggtaccccg ggtggaactg gtagccatga atgagaccga cccaaagagc 240
atcatcaccc gcatctgtga tctcatgtct gaccggaaga tccagggggt ggtgtttgct 300
gatgacacag accaggaagc catcgcccag atcctcgatt tcatttcagc acagactctc 360
acccccatcc tgggcatcca cgggggctcc tctatgataa tggcagataa ggatgaatcc 420
tccatgttct tccagtttgg cccatcaatt gaacagcaag cttccgtaat gctcaacatc 480
atggaagaat atgactggta catcttttct atcgtcacca cctatttccc tggctaccag 540
gactttgtaa acaagatccg cagcaccatt gagaatagct ttgtgggctg ggagctagag 600
gaggtcctcc tactggacat gtccctggac gatggagatt ctaagatcca gaatcagctc 660
aagaaacttc aaagccccat cattcttctt tactgtacca aggaagaagc cacctacatc 720
tttgaagtgg ccaactcagt agggctgact ggctatggct acacgtggat cgtgcccagt 780
ctggtggcag gggatacaga cacagtgcct gcggagttcc ccactgggct catctctgta 840
tcatatgatg aatgggacta tggcctcccc gccagagtga gagatggaat tgccataatc 900
accactgctg cttctgacat gctgtctgag cacagcttca tccctgagcc caaaagcagt 960
tgttacaaca cccacgagaa gagaatctac cagtccaata tgctaaatag gtatctgatc 1020
aatgtcactt ttgaggggag gaatttgtcc ttcagtgaag atggctacca gatgcacccg 1080
aaactggtga taattcttct gaacaaggag aggaagtggg aaagggtggg gaagtggaaa 1140
gacaagtccc tgcagatgaa gtactatgtg tggccccgaa tgtgtccaga gactgaagag 1200
caggaggatg accatctgag cattgtgacc ctggaggagg caccatttgt cattgtggaa 1260
agtgtggacc ctctgagtgg aacctgcatg aggaacacag tcccctgcca aaaacgcata 1320
gtcactgaga ataaaacaga cgaggagccg ggttacatca aaaaatgctg caaggggttc 1380
tgtattgaca tccttaagaa aatttctaaa tctgtgaagt tcacctatga cctttacctg 1440
gttaccaatg gcaagcatgg gaagaaaatc aatggaacct ggaatggtat gattggagag 1500
gtggtcatga agagggccta catggcagtg ggctcactca ccatcaatga ggaacgatcg 1560
gaggtggtcg acttctctgt gcccttcata gagacaggca tcagtgtcat ggtgtcacgc 1620
agcaatggga ctgtctcacc ttctgccttc ttagagccat tcagcgctga cgtatgggtg 1680
atgatgtttg tgatgctgct catcgtctca gccgtggctg tctttgtctt tgagtacttc 1740
agccctgtgg gttataacag gtgcctcgct gatggcagag agcctggtgg accctctttc 1800
accatcggca aagctatttg gttgctctgg ggtctggtgt ttaacaactc cgtacctgtg 1860
cagaacccaa aggggaccac ctccaagatc atggtgtcag tgtgggcctt ctttgctgtc 1920
atcttcctgg ccagctacac tgccaactta gctgccttca tgatccaaga ggaatatgtg 1980
gaccaggttt ctggcctgag cgacaaaaag ttccagagac ctaatgactt ctcaccccct 2040
ttccgctttg ggaccgtgcc caacggcagc acagagagaa atattcgcaa taactatgca 2100
gaaatgcatg cctacatggg aaagttcaac cagaggggtg tagatgatgc attgctctcc 2160
ctgaaaacag ggaaactgga tgccttcatc tatgatgcag cagtgctgaa ctatatggca 2220
ggcagagatg aaggctgcaa gctggtgacc attggcagtg ggaaggtctt tgcttccact 2280
ggctatggca ttgccatcca aaaagattct gggtggaagc gccaggtgga ccttgctatc 2340
ctgcagctct ttggagatgg ggagatggaa gaactggaag ctctctggct cactggcatt 2400
tgtcacaatg agaagaatga ggtcatgagc agccagctgg acattgacaa catggcaggg 2460
gtcttctaca tgttgggggc ggccatggct ctcagcctca tcaccttcat ctgcgaacac 2520
cttttctatt ggcagttccg acattgcttt atgggtgtct gttctggcaa gcctggcatg 2580
gtcttctcca tcagcagagg tatctacagc tgcatccatg gggtggcgat cgaggagcgc 2640
cagtctgtaa tgaactcccc caccgcaacc atgaacaaca cacactccaa catcctgcgc 2700
ctgctgcgca cggccaagaa catggctaac ctgtctggtg tgaatggctc accgcagagc 2760
gccctggact tcatccgacg ggagtcatcc gtctatgaca tctcagagca ccgccgcagc 2820
ttcacgcatt ctgactgcaa atcctacaac aacccgccct gtgaggagaa cctcttcagt 2880
gactacatca gtgaggtaga gagaacgttc gggaacctgc agctgaagga cagcaacgtg 2940
taccaagatc actaccacca tcaccaccgg ccccatagta ttggcagtgc cagctccatc 3000
gatgggctct acgactgtga caacccaccc ttcaccaccc agtccaggtc catcagcaag 3060
aagcccctgg acatcggcct cccctcctcc aagcacagcc agctcagtga cctgtacggc 3120
aaattctcct tcaagagcga ccgctacagt ggccacgacg acttgatccg ctccgatgtc 3180
tctgacatct caacccacac cgtcacctat gggaacatcg agggcaatgc cgccaagagg 3240
cgtaagcagc aatataagga cagcctgaag aagcggcctg cctcggccaa gtcccgcagg 3300
gagtttgacg agatcgagct ggcctaccgt cgccgaccgc cccgctcccc tgaccacaag 3360
cgctacttca gggacaagga agggctacgg gacttctacc tggaccagtt ccgaacaaag 3420
gagaactcac cccactggga gcacgtagac ctgaccgaca tctacaagga gcggagtgat 3480
gactttaagc gcgactccgt cagcggagga gggccctgta ccaacaggtc tcacatcaag 3540
cacgggacgg gcgacaaaca cggcgtggtc agcggggtac ctgcaccttg ggagaagaac 3600
ctgaccaacg tggagtggga ggaccggtcc gggggcaact tctgccgcag ctgtccctcc 3660
aagctgcaca actactccac gacggtgacg ggtcagaact cgggcaggca ggcgtgcatc 3720
cggtgtgagg cttgcaagaa agcaggcaac ctgtatgaca tcagtgagga caactccctg 3780
caggaactgg accagccggc tgccccagtg gcggtgacgt caaacgcctc caccactaag 3840
taccctcaga gcccgactaa ttccaaggcc cagaagaaga accggaacaa actgcgccgg 3900
cagcactcct acgacacctt cgtggacctg cagaaggaag aagccgccct ggccccgcgc 3960
agcgtaagcc tgaaagacaa gggccgattc atggatggga gcccctacgc ccacatgttt 4020
gagatgtcag ctggcgagag cacctttgcc aacaacaagt cctcagtgcc cactgccgga 4080
catcaccacc acaacaaccc cggcggcggg tacatgctca gcaagtcgct ctaccctgac 4140
cgggtcacgc aaaacccttt catccccact tttggggacg accagtgctt gctccatggc 4200
agcaaatcct acttcttcag gcagcccacg gtggcggggg cgtcgaaagc caggccggac 4260
ttccgggccc ttgtcaccaa caagccggtg gtctcggccc ttcatggggc cgtgccagcc 4320
cgtttccaga aggacatctg tatagggaac cagtccaacc cctgtgtgcc taacaacaaa 4380
aaccccaggg ctttcaatgg ctccagcaat gggcatgttt atgagaaact ttctagtatt 4440
gagtctgatg tctga 4455
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
cggcacaagg atgctcgccg 20
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
ggcaatctcg atgaaaatca 20
<210> 6
<211> 56
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
aacctctcag atccctcggt cagcaccgtg gtgtgagatt acaaggatga cgacga 56
<210> 7
<211> 53
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
gagcgggccc gtgatatcag tgggatggta ctgctgcagg ttcttccgcc aca 53
<210> 8
<211> 35
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
gtacctgctg agaagcttct cagcgcgcat cctgg 35
<210> 9
<211> 34
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
gaagcttctc agcaggtaca gcatcacggc cacc 34
<210> 10
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
gcatcgggga aggcgccccc agaagcatcc tgggcatggt gtgggc 46
<210> 11
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
tctgggggcg ccttccccga tgc 23
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
agcacactgt ggctgctggt 20
<210> 13
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
cacgaagggc tcctggtgga 20
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
gaggcgaaag agctggaggc 20
<210> 15
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
gtaggagaca ggggtgggag 20
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
ttctcagcgc gcatcctggg 20
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
gggggcgcct tccccgatgc 20
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
atcctgggca tggtgtgggc 20
<210> 19
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
tgagaagctt ctgggggcgc 20
<210> 20
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
ggcaacacca acatctggaa 20
<210> 21
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
catcagcagg gccgtcacgt 20
<210> 22
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
gcgcgcatcc tgggcatggt 20
<210> 23
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
gcttctgggg gcgccttccc 20
<210> 24
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
acagccggct tctaccgcat 20
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
ggcgacggag caggagaaca 20
Claims (7)
1. A mutant of an NMDARNR1 subunit, wherein the mutant does not recognize an autoantibody against an NMDAR encephalitis patient;
the mutant lacks the 837-838 and 864-937 positions of the NMDANR 1 subunit coding amino acid sequence;
the nucleotide sequence of the NMDANR 1 subunit is shown as SEQ ID NO. 1.
2. A method of constructing the mutant of claim 1, comprising the steps of:
(1) PCR amplification is carried out by using a nucleotide sequence shown in SEQ ID NO.1 as a template and utilizing 864-937-F and 864-937-R to obtain an NRI subunit mutant with 864-937 amino acid deletion; the nucleotide sequence of 864-937-F is shown as SEQ ID NO.6, and the nucleotide sequence of 864-937-R is shown as SEQ ID NO. 7;
(2) Taking the NRI subunit mutant with the deletion 864-937 amino acid as a template, and carrying out PCR amplification by using 837-838-F and 837-838-R to obtain the NMDANR 1 subunit mutant with the deletion 837-838 amino acid and 864-937 amino acid; the nucleotide sequence of 837-838-F is shown as SEQ ID NO.4, and the nucleotide sequence of 837-838-R is shown as SEQ ID NO. 5.
3. The method of claim 2, wherein the PCR amplification system of step (1) and step (2) is each 50 μl, comprising: 50ng of template, 2. Mu.L of primer F, 2. Mu. L, fastPfuDNAPolymerase 1. Mu.L of primer R, 10. Mu.L of 5 XFastPfuubufer, 4. Mu. L, DMSO 1. Mu.L of 2.5mM dNTP and the balance of nuclease-free water;
the PCR amplification procedures of step (1) and step (2) each comprise: pre-denaturation at 98℃for 2min; denaturation at 98℃for 15s, annealing at 59℃for 15s, extension at 72℃for 4min,33 cycles; and extending at 72 ℃ for 8min.
4. A method of constructing an NMDAR mutant comprising the steps of:
(1) Inserting the mutant of claim 1 into a eukaryotic expression vector to obtain an NR1 recombinant vector;
(2) Inserting NR2 subunit of NMDAR into eukaryotic expression vector to obtain NR2 recombinant vector;
(3) Co-transfecting eukaryotic cells with an NR1 recombinant vector and an NR2 recombinant vector to obtain NMDAR mutants;
step (1) and step (2) do not have a chronological order.
5. The method of claim 4, wherein the eukaryotic expression vectors of step (1) and step (2) are the same.
6. The method of claim 4, wherein said NR2 subunit of step (2) comprises NR2a or NR2b, said NR2a having a nucleotide sequence as set forth in SEQ ID No.2 and said NR2b having a nucleotide sequence as set forth in SEQ ID No. 3.
7. The method of claim 4, wherein the mass ratio of said NR1 recombinant vector to NR2 recombinant vector at the time of said co-transfection of step (3) is from (1:1) to (1:4).
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