Recombinant nmda receptor protein and uses thereof

Abstract

The application relates to the technical field of biotechnology, and particularly discloses a recombinant NMDA receptor protein and application thereof, wherein the recombinant NMDA receptor protein comprises an amino acid sequence shown in SEQ ID No. 1. The application further provides a cell stably overexpressing the recombinant NMDA receptor protein and applying the cell to preparation of an anti-NMDA receptor antibody encephalitis detection product. The recombinant NMDA receptor protein is constructed by combining a core sequence of HSP70 protein with a complete sequence of the NMDA receptor protein, so that the expression amount of the recombinant NMDA receptor protein is higher, and the binding capacity with the anti-NMDA receptor antibody is stronger. The cell stably overexpressing the recombinant NMDA receptor has a higher binding efficiency with the anti-NMDA receptor antibody, the sensitivity of a detection reaction is further improved, and the detection result is more accurate.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and more specifically, to a recombinant NMDA receptor protein and its applications. Background Technology

[0002] The NMDA receptor (N-methyl-D-aspartic acid receptor) is a subtype of ionotropic glutamate receptors with a complex molecular structure. NMDA receptors exert their biological functions by forming a complex with their conjugated ion channels and regulatory sites. Functional NMDA receptors must contain an NR1 subunit, with multiple NR2 subunits forming a tetramer (or pentamer) with NR1. NR1 is the basic subunit constituting the ion channel; NR2 is the regulatory subunit. NMDA receptors with different NR2 compositions exhibit different brain distributions and physiological characteristics.

[0003] NMDA receptors are an important class of excitatory amino acid (EAA) receptors in the central nervous system. NMDA receptors not only play important physiological roles in the development of the nervous system, such as regulating neuronal survival, regulating the development of neuronal dendrites and axonal structures, and participating in the formation of synaptic plasticity, but they also play a crucial role in the formation of neuronal circuits. Data suggests that NMDA receptors are a vital class of receptors in learning and memory processes.

[0004] Anti-NMDA receptor antibody encephalitis is an autoimmune encephalitis mediated by anti-NMDA receptor antibodies. It is a neuronal disorder mainly caused by a reversible reduction in NMDA receptors on the surface of neurons. Its main causes include tumors and infectious diseases. It is not contagious and is more common in women and people with tumors. Infection or fatigue are potential triggering factors.

[0005] Typical anti-NMDA receptor antibody encephalitis progresses through five stages: 1. Prodromal stage (flu-like symptoms such as headache, fatigue, and fever); 2. Mental disorder stage: patients present with severe mental disorders, such as bipolar disorder or schizophrenia, and often seek treatment in psychiatric departments; 3. Unresponsive hypoventilation stage: patients exhibit decreased responsiveness to external stimuli and central hypoventilation, requiring endotracheal intubation; 4. Motor and autonomic dysfunction stage: patients present with significant motor impairment (inability to move voluntarily) and various autonomic dysfunctions; 5. Recovery stage or death.

[0006] Currently, purified NMDA receptor protein is mainly used to detect anti-NMDA receptor antibodies in cerebrospinal fluid. However, NMDA receptor protein is a transmembrane protein, and the spatial conformation of the purified protein differs from that in its native state. Furthermore, it is easily affected by the external environment, leading to lower protein expression levels and thus false negative results, resulting in poor accuracy. Summary of the Invention

[0007] To reduce false negatives in anti-NMDA receptor antibody detection, this application provides a recombinant NMDA receptor protein and its application. The recombinant NMDA receptor protein fuses the core sequence of heat shock protein 70 (HSP70) with the complete sequence of the NMDA receptor protein. Cells stably overexpressing the recombinant NMDA receptor protein are further constructed, thereby increasing protein expression while ensuring the correct conformation of the recombinant NMDA receptor protein. This results in more sensitive detection of anti-NMDA receptor antibodies, reduces the probability of false negatives, and provides more accurate results.

[0008] In a first aspect, this application provides a recombinant NMDA receptor protein, employing the following technical solution:

[0009] A recombinant NMDA receptor protein comprising the amino acid sequence shown in SEQ ID No. 1.

[0010] In this application, the core sequence of the HSP70 protein and the complete sequence of the NMDA receptor protein were selected to construct the aforementioned recombinant NMDA receptor protein. Selecting only the core sequence of the HSP70 protein avoids the impact of excessively long sequences on protein expression, thereby improving transcription and translation efficiency and ultimately increasing protein expression levels. In the recombinant NMDA receptor protein, the complete sequence of the NMDA receptor protein retains its function of binding to anti-NMDA receptor antibodies, while the core sequence of the HSP70 protein promotes NMDA receptor protein expression and folding. This allows the recombinant NMDA receptor protein to increase its expression level while maintaining a conformation close to the native conformation, resulting in higher binding efficiency with anti-NMDA receptor antibodies and thus improving the sensitivity and accuracy of the detection reaction.

[0011] Secondly, this application provides a nucleic acid molecule, employing the following technical solution:

[0012] A nucleic acid molecule that encodes the recombinant NMDA receptor protein described in the first aspect.

[0013] Preferably, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID No. 2.

[0014] In this application, the codons of the nucleic acid molecule encoding the above-mentioned recombinant NMDA receptor protein were optimized, which further improved the protein expression level.

[0015] Thirdly, this application provides a carrier, which adopts the following technical solution:

[0016] A vector expressing the recombinant NMDA receptor protein described in the first aspect.

[0017] Preferably, the vector contains at least one copy of the nucleic acid molecule described in the second aspect.

[0018] Preferably, the vector comprises a lentiviral vector.

[0019] Fourthly, this application provides a recombinant lentivirus, employing the following technical solution:

[0020] A recombinant lentivirus containing at least one copy of the vector described in the third aspect.

[0021] Preferably, the genome of the recombinant lentivirus contains at least one copy of the nucleic acid molecule described in the second aspect.

[0022] Fifthly, this application provides a cell that stably overexpresses the recombinant NMDA receptor, employing the following technical solution:

[0023] A cell that stably overexpresses a recombinant NMDA receptor, wherein the cell stably overexpresses the recombinant NMDA receptor protein described in the first aspect.

[0024] Preferably, the cells stably overexpressing the recombinant NMDA receptor contain the recombinant lentivirus described in the fourth aspect.

[0025] Preferably, the cells stably overexpressing the recombinant NMDA receptor contain the vector described in the third aspect.

[0026] Preferably, at least one copy of the nucleic acid molecule described in the second aspect is integrated into the genome of the cell that stably overexpresses the recombinant NMDA receptor.

[0027] In this application, the coding sequence of the recombinant NMDA receptor protein is integrated into the genome of the receptor cell via recombinant lentivirus, thereby achieving sustained and stable protein expression. On the one hand, constructing stable cells that sustainably express the recombinant protein is beneficial for increasing the protein expression level; on the other hand, constructing stable cells that sustainably express the recombinant protein helps the synthesized recombinant protein form a conformation closer to its native state, thereby improving the binding efficiency with anti-NMDA receptor antibodies, increasing sensitivity, and further ensuring the accuracy of the detection results.

[0028] Sixthly, this application provides a method for preparing cells stably overexpressing the recombinant NMDA receptor as described in the fifth aspect, employing the following technical solution:

[0029] A method for preparing cells stably overexpressing recombinant NMDA receptor, the method comprising:

[0030] The recombinant lentivirus described in the fourth aspect was used to infect the recipient cells, and the cells were screened to obtain the cells that stably overexpressed the recombinant NMDA receptor.

[0031] In a seventh aspect, this application provides the use of the recombinant NMDA receptor protein described in the first aspect and / or the cells stably overexpressing the recombinant NMDA receptor described in the fifth aspect in the preparation of an anti-NMDA receptor antibody encephalitis detection product.

[0032] Eighthly, this application provides an anti-NMDA receptor antibody encephalitis detection product, which adopts the following technical solution:

[0033] An anti-NMDA receptor antibody encephalitis detection product, wherein the anti-NMDA receptor antibody encephalitis detection product comprises the recombinant NMDA receptor protein described in the first aspect and / or cells stably overexpressing the recombinant NMDA receptor described in the fifth aspect.

[0034] Ninthly, this application provides a slide for detecting encephalitis using anti-NMDA receptor antibodies, employing the following technical solution:

[0035] An anti-NMDA receptor antibody encephalitis detection slide, wherein the anti-NMDA receptor antibody encephalitis detection slide comprises cells stably overexpressing recombinant NMDA receptors as described in the fifth aspect.

[0036] In this application, recombinant NMDA receptor protein is expressed in cells, and the resulting protein conformation is closer to that in the native state. Then, cells that stably overexpress recombinant NMDA receptor are fixed on a slide, allowing them to bind to anti-NMDA receptor antibodies in the sample. This simulates the binding of NMDA receptor protein to anti-NMDA receptor antibodies in the native state, thereby improving the sensitivity of the detection reaction and making the detection results more accurate.

[0037] In a tenth aspect, this application provides an anti-NMDA receptor antibody encephalitis detection kit, which adopts the following technical solution:

[0038] An anti-NMDA receptor antibody encephalitis detection kit, comprising cells stably overexpressing recombinant NMDA receptors as described in the fifth aspect and / or anti-NMDA receptor antibody encephalitis detection slides as described in the ninth aspect.

[0039] Preferably, the anti-NMDA receptor antibody encephalitis detection kit further includes a FITC-labeled human IgG monoclonal antibody.

[0040] In summary, this application has the following beneficial effects:

[0041] 1. This application constructs a recombinant NMDA receptor protein by fusing the core sequence of the HSP70 protein with the complete sequence of the NMDA receptor protein. The complete sequence of the NMDA receptor protein at its C-terminus retains the function of binding to anti-NMDA receptor antibodies, while the core sequence of the HSP70 protein at its N-terminus promotes protein expression and proper folding. Through the cooperation of the two, the higher-order structure of the above-mentioned recombinant NMDA receptor protein is made as close as possible to the structure of the natural NMDA receptor protein, increasing the binding ability with anti-NMDA receptor antibodies and improving the expression level of the recombinant NMDA receptor protein in cells, thereby improving the sensitivity of the detection reaction.

[0042] 2. This application also constructs cells that stably overexpress recombinant NMDA receptor, immobilizes them on slides, and then detects samples. Compared with directly using purified NMDA receptor protein for detection, the cells stably overexpressing recombinant NMDA receptor significantly increase the protein expression level, which helps reduce the probability of false negatives in the detection reaction. In addition, the conformation of the recombinant NMDA receptor protein in the cells is closer to that of the natural NMDA receptor protein, resulting in higher binding efficiency with anti-NMDA receptor antibodies, further improving the sensitivity of the detection reaction and ensuring the accuracy of the detection results.

[0043] 3. The cells stably overexpressing recombinant NMDA receptor constructed in this application can be stably expressed and passaged. When using the anti-NMDA receptor antibody encephalitis detection product prepared in this application to detect samples, it is only necessary to culture the above-mentioned cells stably overexpressing recombinant NMDA receptor and then fix them on a slide for subsequent detection. When directly using NMDA receptor protein for detection, a complex process of inducing protein expression, collecting protein, and purifying it is required, which is more complicated, has lower detection efficiency, and requires a higher level of technical expertise from the operators. Attached Figure Description

[0044] Figure 1 Image showing the detection results of anti-NMDA receptor antibody in cells transiently expressing recombinant NMDA receptor in Example 5 (magnification = 100x).

[0045] Figure 2 Image showing the detection results of anti-NMDA receptor antibody in cells stably overexpressing the natural NMDA receptor in Example 5 (magnification = 100x).

[0046] Figure 3 Image showing the detection results of anti-NMDA receptor antibody in cells stably overexpressing recombinant NMDA receptor in Example 5 (magnification = 100x).

[0047] Figure 4 Image of the detection results (magnification = 100x) of the anti-NMDA receptor antibody encephalitis detection kit from Example 7 used in Example 8.

[0048] Figure 5 The image shows the detection results (magnification = 100x) obtained using a commercially available anti-glutamate receptor antibody detection kit in Example 8. Detailed Implementation

[0049] This application provides a recombinant NMDA receptor protein, which includes the amino acid sequence shown in SEQ ID No. 1.

[0050] This application also provides a nucleic acid molecule that encodes the aforementioned recombinant NMDA receptor protein.

[0051] Specifically, the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID No. 2.

[0052] This application also provides a vector that expresses the above-described recombinant NMDA receptor protein.

[0053] Specifically, the vector contains at least one copy of the aforementioned nucleic acid molecule.

[0054] This application also provides a recombinant lentivirus containing at least one copy of the aforementioned vector.

[0055] Specifically, the genome of the recombinant lentivirus contains at least one copy of the aforementioned nucleic acid molecule.

[0056] This application also provides cells that stably overexpress the recombinant NMDA receptor, wherein the cells stably overexpress the recombinant NMDA receptor protein.

[0057] Specifically, the cells that stably overexpress the recombinant NMDA receptor contain the aforementioned recombinant lentivirus.

[0058] Specifically, the cells that stably overexpress the recombinant NMDA receptor contain the aforementioned vector.

[0059] Specifically, the genome of the cells that stably overexpress the recombinant NMDA receptor contains at least one copy of the aforementioned nucleic acid molecule.

[0060] This application also provides a method for preparing cells that stably overexpress the recombinant NMDA receptor, specifically including:

[0061] The recombinant lentivirus described above was used to infect the recipient cells, and the cells were screened to obtain cells that stably overexpressed the recombinant NMDA receptor.

[0062] This application also provides an anti-NMDA receptor antibody encephalitis detection product, wherein the anti-NMDA receptor antibody encephalitis detection product comprises the above-mentioned recombinant NMDA receptor protein and / or cells stably overexpressing the recombinant NMDA receptor.

[0063] This application also provides an anti-NMDA receptor antibody encephalitis detection slide, wherein the anti-NMDA receptor antibody encephalitis detection slide comprises the above-mentioned cells stably overexpressing recombinant NMDA receptor.

[0064] This application also provides an anti-NMDA receptor antibody encephalitis detection kit, which includes the above-mentioned cells stably overexpressing recombinant NMDA receptor and / or anti-NMDA receptor antibody encephalitis detection slides.

[0065] Specifically, the anti-NMDA receptor antibody encephalitis detection kit also includes a FITC-labeled human IgG monoclonal antibody.

[0066] The following is in conjunction with Examples 1-8 and Appendix Figures 1-5 The technical solution of this application will be further explained.

[0067] Example 1

[0068] This embodiment provides a recombinant NMDA receptor protein, the amino acid sequence of which is shown in SEQ ID No. 1.

[0069] The recombinant NMDA receptor protein was constructed based on the core sequence of the HSP70 protein and the complete sequence of the NMDA receptor protein. It has a high expression level in cells, and the higher-order structure formed after folding is very close to the natural structure of the NMDA receptor protein.

[0070] Example 2

[0071] This embodiment provides a lentiviral vector, which is a pHAGE-puro vector with the coding sequence of the recombinant NMDA receptor protein inserted in Example 1.

[0072] The above-mentioned lentiviral vector was prepared by the following method:

[0073] (1) The nucleotide sequence encoding the above recombinant NMDA receptor protein (as shown in SEQ ID No.2) was artificially synthesized. During the synthesis, NotI and XbaI restriction sites were added to both ends of the coding sequence, respectively.

[0074] (2) The pHAGE-puro (Ubisoft Biotechnology Co., Ltd.) vector was digested with NotI and XbaI (ThermoFisher) in a water bath at 37°C for 4 hours. The linearized plasmid fragments after digestion were recovered using a gel extraction kit (ThermoFisher) and homologous recombination was performed with artificially synthesized coding sequences using a homologous recombination kit (Nanjing Novizan Technologies Co., Ltd.). The homologous recombination system is shown in Table 1.

[0075] Table 1 Homologous Recombination System

[0076] Components Volume (μL) Linearized pHAGE-puro vector 2 Artificially synthesized coding sequences 3 2×cloneExpressMix 5 Total volume 10

[0077] The reaction conditions were: incubation at 50°C for 10 minutes, followed by cooling to 4°C and immediately placing on ice.

[0078] The recombinant product was transformed into Escherichia coli DH5α, plated, and incubated overnight at 37°C. Positive colonies were selected the next day, expanded, and sequenced for identification. After correct identification, the plasmid was extracted using an endotoxin-free plasmid extraction kit (ThermoFisher) to obtain the lentiviral vector puro-rNMDA.

[0079] Example 3

[0080] This embodiment provides a recombinant lentivirus, the genome of which integrates a nucleotide sequence encoding the aforementioned recombinant NMDA receptor protein.

[0081] The above-mentioned recombinant lentivirus was prepared by the following method:

[0082] Take 293T cells in good growth condition and seed them in a 10cm culture dish.

[0083] When the cell density reached approximately 70%, the lentiviral vector puro-rNMDA, helper plasmid pMD2.G (Ubisoft Biotech Ltd.), and psPAX2 (Ubisoft Biotech Ltd.) were co-transfected into 293T cells. The total volume of the plasmid solution was equal to the transfection reagent Transport5. TM The volume ratio used by ThermoFisher is 1:2.

[0084] After 60 hours, the cell supernatant was collected, centrifuged at 3000g for 15 minutes at 4°C, and the supernatant was filtered through a 0.45μm filter membrane. The filtrate was the viral suspension of the recombinant lentivirus. The viral suspension of the recombinant lentivirus was stored at -80°C.

[0085] Example 4

[0086] This embodiment provides a cell that stably overexpresses the recombinant NMDA receptor, wherein the genome of the cell stably overexpressing the recombinant NMDA receptor integrates the coding sequence of the recombinant NMDA receptor protein from Example 1.

[0087] The cells stably overexpressing the recombinant NMDA receptor were prepared using the following method:

[0088] (1) Determine the optimal screening concentration of puromycin.

[0089] 293T cells in the logarithmic growth phase were seeded into 24-well plates.

[0090] After culturing for 24 hours, a gradient concentration of puromycin was added, with concentrations of 0, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL and 0.7 μg / mL, and the mixture was incubated at 37℃ in a 5% CO2 incubator.

[0091] After 48 hours, cell death was observed in the 24-well plates, and the minimum concentration that killed all cells was taken as the optimal puromycin screening concentration.

[0092] The optimal screening concentration of puromycin was ultimately determined to be 0.3 μg / mL.

[0093] (2) Screening of cells infected with the virus and stably overexpressing the recombinant NMDA receptor.

[0094] Logarithmic growth phase 293T cells were seeded into 6-well plates. When the cell density reached approximately 70%, a viral suspension of recombinant lentivirus prepared in Example 3 and puromycin at a final concentration of 0.3 μg / mL were added at a 1:1 volume ratio of culture medium to viral suspension. After 48 h of infection, the liquid was aspirated, and culture medium was added again, along with puromycin at a final concentration of 0.3 μg / mL for further selection. The same concentration of puromycin was added to untreated 293T cells as a negative control.

[0095] After 48 hours, all negative control cells died, and the cells that survived in the well plates containing the virus suspension were the resistant cells.

[0096] The above process was repeated 3 to 5 times for resistant cells to continue screening, and finally cells that stably overexpressed recombinant NMDA receptor were obtained and cryopreserved.

[0097] Example 5

[0098] This embodiment verifies the binding ability of cells stably overexpressing recombinant NMDA receptor prepared in Example 4 to anti-NMDA receptor antibody, while using cells stably overexpressing natural NMDA receptor and cells transiently expressing recombinant NMDA receptor as controls.

[0099] Cells stably overexpressing the native NMDA receptor were prepared using the following method:

[0100] A nucleotide sequence encoding the natural NMDA receptor protein was artificially synthesized (as shown in SEQ ID No. 3). During synthesis, NotI and XbaI restriction enzyme sites were added to both ends of the coding sequence, respectively. The sequence was then ligated into the pHAGE-puro vector via homologous recombination to obtain the lentiviral vector puro-NMDA. Recombinant lentivirus was then packaged using the puro-NMDA vector, and subsequently used to infect 293T cells. Cells stably overexpressing the natural NMDA receptor were obtained. Specific steps are detailed in Examples 2-4.

[0101] Cells transiently expressing the recombinant NMDA receptor were prepared using the following method:

[0102] (1) The nucleotide sequence encoding the recombinant NMDA receptor protein was artificially synthesized (as shown in SEQ ID No.2). During synthesis, NotI and XbaI restriction sites were added to both ends of the coding sequence, respectively.

[0103] (2) The pCDNA3.1 (U-Bio Biotechnology Co., Ltd.) vector was digested with NotI and XbaI in a water bath at 37°C for 4 hours. The linearized plasmid fragments after digestion were recovered using a gel extraction kit and homologous recombination was performed with an artificially synthesized coding sequence using a homologous recombination kit. The homologous recombination system is shown in Table 1.

[0104] The reaction conditions were: incubation at 50°C for 10 minutes, followed by cooling to 4°C and immediately placing on ice.

[0105] The recombinant product was transformed into Escherichia coli DH5α, plated, and incubated overnight at 37°C. Positive colonies were selected the next day, expanded, and sequenced for identification. After successful identification, the plasmid was extracted using an endotoxin-free plasmid extraction kit to obtain the expression vector pc3.1-rNMDA.

[0106] (3) Take 293T cells in good growth condition and inoculate them in a 10cm culture dish.

[0107] When the cell density reached approximately 70%, the expression vector pc3.1-rNMDA was transfected into 293T cells. The volume of the plasmid solution was the same as that of the transfection reagent Transport5. TM The volume ratio used is 1:2.

[0108] After successful transfection, the cells were placed in an incubator at 37°C and 5% CO2 concentration and cultured for another 96 hours to obtain cells that transiently expressed the recombinant NMDA receptor.

[0109] The binding affinity of cells stably overexpressing recombinant NMDA receptor, cells stably overexpressing natural NMDA receptor, and cells transiently expressing recombinant NMDA receptor to anti-NMDA receptor antibodies was determined, and the steps are as follows:

[0110] Cell spreaders were placed in 6-well plates, and cells stably overexpressing recombinant NMDA receptor, cells stably overexpressing native NMDA receptor, and cells transiently expressing recombinant NMDA receptor were seeded into each well, approximately 1 × 10-1 cells per well. 6 Cells were collected. After cell adhesion, the cells were gently washed three times with PBS to remove unattached cells, then fixed with 4% paraformaldehyde. The cell slides were then removed and analyzed.

[0111] The testing steps are as follows:

[0112] 1. Remove the cell slide and incubate at room temperature for 10 minutes.

[0113] 2. Sample incubation: Add the sample to be tested to the reaction area of ​​the fixed cells on the cell slide and incubate at room temperature for 1 hour.

[0114] 3. Cleaning: Remove the sample to be tested, add 20 mL of phosphate buffer to the washing box and clean the slide 3 times, 5 min each time.

[0115] 4. Incubation: Add FITC-labeled human IgG monoclonal antibody (purchased from Merck) to each reaction zone and incubate at room temperature for 30 min.

[0116] 5. Washing: Remove the FITC-labeled human IgG monoclonal antibody from the reaction area, and wash the slide three times with 20 mL of phosphate buffer in the washing box, 5 min each time.

[0117] 6. Sealing: Add glycerol to each reaction zone to prevent fluorescence quenching.

[0118] 7. Cover with a coverslip to prevent air bubbles from forming.

[0119] 8. Read the green fluorescence results under a microscope and observe them under a 20x objective lens.

[0120] 9. Results Analysis: Analyze the results.

[0121] The image shows the detection results of anti-NMDA receptor antibodies in cells transiently expressing recombinant NMDA receptor. Figure 1 As shown in the image, the detection results of anti-NMDA receptor antibodies in cells stably overexpressing the native NMDA receptor are as follows. Figure 2 As shown in the image, the detection results of anti-NMDA receptor antibodies in cells stably overexpressing recombinant NMDA receptor are as follows. Figure 3 As shown.

[0122] contrast Figure 1 and Figure 3 As can be seen, compared with transient expression, the constructed cells stably overexpressing recombinant NMDA receptor exhibited stronger fluorescence and a greater number of cells expressing recombinant NMDA receptor protein. This indicates that constructing cells stably overexpressing recombinant NMDA receptor protein can increase protein expression levels and thus improve the sensitivity of the detection reaction compared to transient expression. Furthermore, the constructed cells stably overexpressing recombinant NMDA receptor can be continuously passaged; detection only requires cell culture followed by fixation. Compared to the method of obtaining transiently expressing recombinant NMDA receptor cells, which requires plasmid extraction and cell transfection for each detection, using cells stably overexpressing recombinant NMDA receptor for detection is simpler, more efficient, and less costly.

[0123] contrast Figure 2 and Figure 3 As can be seen, compared with the natural NMDA receptor protein, the recombinant NMDA receptor protein in this application exhibits better binding performance and stronger fluorescence with the anti-NMDA receptor antibody. This application constructs the recombinant protein using only the core sequence of the HSP70 protein and the complete sequence of the NMDA receptor protein, resulting in a short protein sequence and high expression efficiency. HSP70 helps further improve the protein's expression efficiency and also helps the NMDA receptor protein fold correctly, forming a higher-order structure that more closely resembles the natural protein conformation, thereby enhancing its binding ability with the anti-NMDA receptor antibody.

[0124] In summary, the results show that cells stably overexpressing recombinant NMDA receptors exhibit better binding and stronger fluorescence with anti-NMDA receptor antibodies, making them suitable for detection in encephalitis related to anti-NMDA receptor antibodies.

[0125] Example 6

[0126] This embodiment provides an anti-NMDA receptor antibody encephalitis detection slide, which is prepared by the following method:

[0127] Place the cell spreaders into 6-well plates, and then seed each well with cells stably overexpressing the recombinant NMDA receptor. The seeding density is approximately 1 × 10⁶ cells per well. 6 Each cell.

[0128] After the cells adhered, they were gently rinsed three times with PBS to remove any unattached cells. Then, they were fixed with 4% paraformaldehyde. The cell slides were then removed, which became the slides for detecting anti-NMDA receptor antibody encephalitis.

[0129] Example 7

[0130] This embodiment provides an anti-NMDA receptor antibody encephalitis detection kit, which includes the anti-NMDA receptor antibody encephalitis detection slide from Example 6, FITC-labeled human IgG monoclonal antibody, and washing solution (i.e., phosphate buffer).

[0131] Example 8

[0132] This embodiment measures the detection limit, specificity, and NMDA receptor protein expression level of the anti-NMDA receptor antibody encephalitis detection kit prepared in Example 7 and the commercially available anti-glutamate receptor antibody detection kit (indirect immunofluorescence method) (Euron Medical Laboratory Diagnostics Co., Ltd., catalog number FA111m-2010-3).

[0133] Limit of detection

[0134] Samples positive for anti-NMDA receptor antibodies were diluted sequentially at ratios of 1:320, 1:640, and 1:1280. The diluted serum was then tested using a commercially available anti-glutamate receptor antibody detection kit and the anti-NMDA receptor antibody encephalitis detection kit from Example 7, and the detection limits of the kits were determined.

[0135] The detection method for commercially available anti-glutamate receptor antibody detection kits is as described in the product's instruction manual. The detection method for the anti-NMDA receptor antibody encephalitis detection kit in Example 7 is as described in Example 5.

[0136] The detection limits of the commercially available anti-glutamate receptor antibody detection kit and the anti-NMDA receptor antibody encephalitis detection kit in Example 7 are shown in Table 2.

[0137] Table 2. Detection Limits and Results of the Anti-Glutamate Receptor Antibody Detection Kit

[0138] Dilution ratio 1:320 1:640 1:1280 Commercially available anti-glutamate receptor antibody detection kits + + - Anti-NMDA receptor antibody encephalitis detection kit + + +

[0139] In Table 2, "+" indicates a positive test result, and "-" indicates a negative test result.

[0140] As shown in Table 2, compared with commercially available kits, the anti-NMDA receptor antibody encephalitis detection kit in this application has a lower limit of detection, indicating that it has higher sensitivity, reduces the probability of false negative results, and improves the accuracy of the detection results.

[0141] Specific detection

[0142] Two hundred clinical samples were tested using both a commercially available anti-glutamate receptor antibody detection kit and the anti-NMDA receptor antibody encephalitis detection kit described in Example 7. Of these, 76 samples were positive for anti-glutamate receptor antibodies, and 124 samples were negative. The specific testing procedure is described in the section on detection limit detection. The results are statistically analyzed and are shown in Table 3.

[0143] Table 3. Detection results of the anti-glutamate receptor antibody detection kit on clinical samples.

[0144]

[0145] As shown in Table 3, the positive and negative concordance rates of both kits were 100%. Compared with the results of a commercially available anti-glutamate receptor antibody detection kit, the anti-NMDA receptor antibody encephalitis detection kit constructed in this application showed a 100% consistency rate in detecting anti-NMDA receptor antibodies in serum samples with the commercially available product, demonstrating its equivalence in clinical use and exhibiting good specificity and high accuracy.

[0146] NMDA receptor protein expression level detection

[0147] Samples known to be positive for NMDA receptor antibodies were tested using both a commercially available anti-glutamate receptor antibody detection kit and the anti-NMDA receptor antibody encephalitis detection kit from Example 7. The specific testing procedures are detailed in Example 5. The test results are as follows: Figure 4 and Figure 5 As shown.

[0148] Figure 4 Images showing the detection results using the anti-NMDA receptor antibody encephalitis detection kit from Example 7. Figure 5 Image showing the test results using a commercially available anti-glutamate receptor antibody detection kit.

[0149] contrast Figure 4 and Figure 5As can be seen, when testing the same sample, the fluorescence intensity of the image obtained using the anti-NMDA receptor antibody encephalitis detection kit in Example 7 was brighter, while the fluorescence intensity of the image obtained using the commercially available anti-glutamate receptor antibody detection kit was weaker. This indicates that the recombinant NMDA receptor protein expression level is higher in the cells stably overexpressing the recombinant NMDA receptor in this application, thus binding to more anti-NMDA receptor antibodies, and consequently binding to more fluorescently labeled secondary antibodies, resulting in more fluorescence. Higher expression levels of recombinant NMDA receptor protein also improve the sensitivity of the detection reaction and increase the accuracy of the results.

[0150] In summary, the cells stably overexpressing recombinant NMDA receptor constructed in this application can be used for the detection of anti-NMDA receptor antibodies. The prepared anti-NMDA receptor antibody encephalitis detection kit shows extremely high consistency with commercially available kits, and can be considered equivalent in clinical use. Compared with commercially available kits, the anti-NMDA receptor antibody encephalitis detection kit prepared in this application has a lower detection limit and a higher expression level of NMDA receptor protein, resulting in better binding to anti-NMDA receptor antibodies, improved detection sensitivity, reduced false negative probability, and increased accuracy of detection results.

[0151] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A recombinant NMDA receptor protein, characterized in that, The amino acid sequence of the recombinant NMDA receptor protein is shown in SEQ ID No.

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant NMDA receptor protein of claim 1; the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No.

2.

3. A carrier, characterized in that, The vector contains at least one copy of the nucleic acid molecule of claim 2.

4. A recombinant lentivirus, characterized in that, The recombinant lentivirus contains at least one copy of the vector of claim 3.

5. The recombinant lentivirus according to claim 4, characterized in that, The genome of the recombinant lentivirus contains at least one copy of the nucleic acid molecule of claim 2.

6. A cell that stably overexpresses recombinant NMDA receptor, characterized in that, The cells stably overexpressing the recombinant NMDA receptor contain the recombinant lentivirus of any one of claims 4-5; or, the cells stably overexpressing the recombinant NMDA receptor contain the vector of claim 3; or, the genome of the cells stably overexpressing the recombinant NMDA receptor is integrated with at least one copy of the nucleic acid molecule of claim 2.

7. A method for preparing cells stably overexpressing recombinant NMDA receptor as described in claim 6, characterized in that, The method for preparing the cells stably overexpressing the recombinant NMDA receptor includes: infecting receptor cells with the recombinant lentivirus as described in any one of claims 4-5, screening, and obtaining the cells stably overexpressing the recombinant NMDA receptor.

8. The use of the recombinant NMDA receptor protein of claim 1 and / or the cells stably overexpressing the recombinant NMDA receptor of claim 6 in the preparation of an anti-NMDA receptor antibody encephalitis detection product.

9. A product for detecting encephalitis using anti-NMDA receptor antibodies, characterized in that, The anti-NMDA receptor antibody encephalitis detection product includes the recombinant NMDA receptor protein of claim 1 and / or the cells stably overexpressing the recombinant NMDA receptor of claim 6.

10. A slide for detecting encephalitis using anti-NMDA receptor antibodies, characterized in that, The anti-NMDA receptor antibody encephalitis detection slide comprises cells stably overexpressing recombinant NMDA receptors as described in claim 6.

11. A kit for detecting encephalitis with anti-NMDA receptor antibodies, characterized in that, The anti-NMDA receptor antibody encephalitis detection kit comprises cells stably overexpressing recombinant NMDA receptors as described in claim 6 and / or anti-NMDA receptor antibody encephalitis detection slides as described in claim 10.

12. The anti-NMDA receptor antibody encephalitis detection kit according to claim 11, characterized in that, The anti-NMDA receptor antibody encephalitis detection kit also includes a FITC-labeled human IgG monoclonal antibody.

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