Protein for detecting human p / q-type vgcc autoantibody and application thereof

Abstract

The application discloses a protein for human P / Q type VGCC autoantibody detection and application, relates to the technical field of autoantibody detection, and the protein is a VGCC6 protein, and the amino acid sequence is shown as SEQ ID NO:6. In view of the clinical diagnosis demand of Lambert-Eaton myasthenic syndrome and other diseases related to the antibody, the protein provides strong support for early screening, diagnosis and disease condition monitoring of clinical diseases, fills the short board of the existing detection technology, and promotes the improvement of the diagnosis and treatment level of related autoimmune diseases.

Description

Technical Field

[0001] This invention relates to the field of autoantibody detection technology, and in particular to a protein for detecting human P / Q type VGCC autoantibodies and its application. Background Technology

[0002] Lambert-Eaton Myathenic Syndrome (LEMS) is an autoimmune disease affecting the presynaptic membrane of the neuromuscular junction. Pathogenic autoantibodies bind to voltage-gated calcium channels (VGCCs) at nerve endings, inhibiting their function and leading to a decrease in the release of the neurotransmitter acetylcholine (AChR), resulting in muscle weakness symptoms. Approximately 50-60% of LEMS patients also have tumors, typically small cell lung cancer (SCLC); SCLC-LEMS complications primarily affect men over 50 years of age, and the disease progresses rapidly with shorter survival times. Therefore, VGCC autoantibodies can serve as biomarkers for the diagnosis of LEMS (Zalewski et al., P / Q- and N-type calcium-channel antibodies: oncological, neurological, andserological accompaniments. Muscle Nerve, 2016, 54(2):220-7.), and have very important clinical significance: (1) assisting in the diagnosis of LEMS or other neuroimmunological diseases; (2) providing early attention to the development of complications in small cell lung cancer and guiding prevention; (3) monitoring the treatment effect of LEMS, coordinating with the treatment plan for complications, and prolonging survival.

[0003] VGCC is a large isoform protein, and 10 α1 subtypes of calcium ion channels have been cloned. Among them, the detection rate of Cav2.1 subtype (P / Q-VGCC) antibody in LEMS patients is 90%, and the tumor-associated rate is also higher. Therefore, PQ-VGCC itself can be used as the most effective basis for both auxiliary diagnosis and differential diagnosis. The existing P / Q-VGCC detection method is mainly based on the 125I isotope-labeled VGCC antigen radioimmunoassay (RIA method) (Motomura et al., Animproved diagnostic assay for Lambert-Eaton myasthenic syndrome. Journal of Neuroneursurg Psychiatry, 58(1):85-7.). The RIA method has the advantages of sensitivity, specificity, simplicity and small sample volume. However, the RIA method has problems such as radiation and pollution, and high requirements for instruments and equipment and usage scenarios, which limit its application.

[0004] P / Q-VGCC is a transmembrane protein composed of more than 2250 amino acids, encoding a protein of approximately 257 kDa. P / Q-VGCC can be divided into four domains, each of which transmembrane six times. Research by Parsons et al. showed that the autoantibody epitopes of P / Q-VGCC are mainly distributed between the 5th and 6th transmembrane sequences of domains 2 and 4, with the specific amino acid sequence EDEDSDE (Parsons et al., Linear B-cell epitopes in Lambert-Eatonmyasthenic syndrome defined by cell-free synthetic peptide binding. J Neuroimmunol, 126(1-2):190-5). Earlier research by Takamori showed that the autoantibody epitopes of P / Q-VGCC are mainly distributed between the 5th and 6th transmembrane sequences of the 3rd and 4th domains (Takamori M. An autoimmunechannelopathy asociated with cancer: Lambert-Eaton myasthenic syndrome. Intern Med, 38(2):86-96.). Subsequently, research by Iwasa et al. also showed that the autoantibody epitopes of P / Q-VGCC are distributed in the 3rd domain.

[0005] These findings clearly provide us with a preliminary understanding of the distribution of P / Q-VGCC autoantibody epitopes. However, since the binding of P / Q-VGCC autoantibodies to epitopes is often conformational, the epitopes revealed by Western blot (WB) and their clinical applications have significant limitations.

[0006] Cell-based assays (CBA) involve transfecting exogenous target genes into cells, causing the cells to express autoantigens. These autoantigens are then used as substrates to detect autoantibodies via immunofluorescence. Because the target protein is expressed in eukaryotic cells, it forms the protein's topological conformation, thus more accurately reflecting the binding of autoantibodies to antigenic epitopes in vivo, significantly improving sample detection rates.

[0007] Therefore, it is essential to develop a protein for the detection of P / Q type VGCC autoantibodies in humans and its application. This method can not only reveal the antigenic epitope distribution of P / Q-VGCC autoantibodies, but also improve the detection sensitivity and specificity of P / Q-VGCC autoantibodies. Summary of the Invention

[0008] This invention aims to address the technical shortcomings of existing human P / Q type VGCC autoantibody detection materials, such as insufficient sensitivity, poor specificity, unstable antigen activity, and cumbersome preparation processes. It provides a protein for detecting human P / Q type VGCC autoantibodies and its application, addressing the clinical diagnostic needs of diseases related to this antibody, such as Lambert-Eaton syndrome. This provides strong support for early screening, diagnosis, and disease monitoring, fills gaps in existing detection technologies, and promotes the improvement of diagnosis and treatment of related autoimmune diseases.

[0009] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: A protein for detecting human P / Q type VGCC autoantibodies, wherein the protein is VGCC6 protein and its amino acid sequence is shown in SEQ ID NO: 6.

[0010] The application of the protein described above for detecting human P / Q type VGCC autoantibodies in the construction of an overexpression vector.

[0011] The construction of the overexpression vector includes the following steps: S1. Linearize the expression vector by XbaI single enzyme digestion; S2. The myc-6×His tag sequence fragment was obtained by amplifying the pcDNA6TR vector using primers MCS-Myc-F / BGH-CX-R. S3. The vector obtained in step S1 and the fragment obtained in step S2 are used to construct a new vector pCDNA3.1NS-mycHis by homologous recombination using a One Step Cloning Kit. S4. The pCDNA3.1NS-mycHis vector was double-digested with NheI and NotI. S5. Amplify the VGCC6 protein using primers 3.1NS-P / Q-VGCC6-F / 3.1NS-P / Q-VGCC6-R to obtain the truncated gene fragment; S6. The vector obtained from the double enzyme digestion in step S4 and the fragment in step S5 are used to construct an overexpression vector by homologous recombination using a One Step Cloning Kit.

[0012] In step S1, the expression vector is pcDNA3.1(+), pCMV, or pENTER.

[0013] Application of one of the proteins described above for detecting human P / Q type VGCC autoantibodies in the construction of cell lines.

[0014] The construction of the cell line includes the following steps: A1. Clean and disinfect the glass slides and treat them with laminar adhesion protein at 37°C for 30 min. A2. Seed HEK-293 cells onto the prepared glass slides and culture them. A3. After culturing for 24 hours, the overexpression vector of VGCC6 protein was transfected into HEK-293 cells using a transfection reagent and cultured for another 24 hours. A4. After culturing for 6 hours, change the medium to HEK-293 cell culture medium and continue culturing for 36-48 hours to obtain transient overexpression cell lines of different P / Q-VGCC antigen fragments.

[0015] In step A3, the transfection concentration of the overexpression vector is 500µg / mL-4000µg / mL.

[0016] In step A3, the transfection reagent is lipo3000 or PEI reagent.

[0017] In step A4, the HEK-293 cell culture medium is DMEM medium containing 10% fetal bovine serum.

[0018] A recombinant polypeptide for detecting human P / Q type VGCC autoantibodies, characterized in that: the recombinant polypeptide is prepared from the above-mentioned cell line.

[0019] The recombinant polypeptide was obtained by disrupting the cell line and purifying the protein to obtain the P / Q-VGCC recombinant polypeptide.

[0020] Application of a recombinant polypeptide, as described above, for the detection of P / Q type VGCC autoantibodies in humans in human P / Q type VGCC autoantibody detection materials.

[0021] The beneficial effects of this invention are: 1. In this invention, by systematically analyzing detection data of different structural domains of P / Q-VGCC, the fragment containing the most important autoantigen epitope mediating the autoimmune response is screened and identified as the specific antigen fragment for detection. This fragment can match the target autoantibody, solving the problems of insufficient specificity and poor binding specificity of traditional antigen fragments from the source, significantly improving detection accuracy, and providing core technical support for the detection of P / Q type VGCC autoantibodies.

[0022] 2. This invention provides a reliable basis for early screening and diagnosis of diseases such as Lambert-Eaton syndrome, provides quantitative support for disease progression assessment, treatment plan adjustment and efficacy judgment, makes up for the shortcomings of traditional testing in disease monitoring, and improves the entire diagnosis and treatment process.

[0023] 3. In this invention, P / Q-VGCC is a transmembrane protein composed of more than 2250 amino acids, encoding a protein of approximately 257 kDa. The large molecular weight of the encoded protein makes in vitro expression of the full-length P / Q-VGCC protein difficult. Furthermore, the P / Q-VGCC antigenic epitope has not been clearly elucidated, and constructing an overexpression plasmid containing the correct antigenic epitope carries some uncertainty. The applicant's research found that existing regions with disclosed antigens did not yield good results (other truncated regions disclosed in this application). This application re-divides the full-length P / Q-VGCC protein with targeted truncated sites. While ensuring the structural integrity of the transmembrane region, six truncated variants covering the entire sequence were designed. Through targeted and exclusive experimental verification, the sequence VGCC6 was finally identified from the novel truncated division. This innovative truncated site design overcomes the limitations of existing technologies, screening for specific sequences, thus avoiding the difficulties in expressing the full-length protein while preserving the structural integrity and antigenic epitope effectiveness of P / Q-VGCC. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the detection results of normal human serum in different P / Q-VGCC antigen fragment overexpressing HEK-293 cell lines in an embodiment of the present invention.

[0025] Figure 2This is a schematic diagram illustrating the detection results of serum (P / Q-VGCC titer 1:32) from Lambert-Eaton myasthenia gravis patients in HEK-293 cell lines overexpressing different P / Q-VGCC antigen fragments, as per an embodiment of the present invention.

[0026] Figure 3 This is a schematic diagram illustrating the detection results of serum (P / Q-VGCC titer 1:100) from Lambert-Eaton myasthenia gravis patients in HEK-293 cell lines overexpressing different P / Q-VGCC antigen fragments, as an embodiment of the present invention.

[0027] Figure 4 This is a schematic diagram of the colorimetric results of autoantibody detection in normal human serum for different P / Q-VGCC recombinant peptides in an application example of the present invention.

[0028] Figure 5 This is a schematic diagram of the colorimetric results of autoantibody detection of different P / Q-VGCC recombinant peptides in the serum of Lambert-Eaton myasthenia gravis patients (P / Q-VGCC titer 1:32) in an application example of the present invention.

[0029] Figure 6 This is a schematic diagram of the colorimetric results of autoantibody detection of different P / Q-VGCC recombinant peptides in the serum of Lambert-Eaton myasthenia gravis patients (P / Q-VGCC titer 1:100) in an application example of the present invention. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0031] The experimental materials used in the following examples included: restriction endonuclease XbaI (NEB, catalog number R0145V); high-fidelity DNA polymerase (Novizan, catalog number P505); One Step Cloning Kit (Novizan, catalog number C117-01); restriction endonuclease KpnI (NEB, catalog number R3142V); restriction endonuclease NotI (NEB, catalog number R3189V); glass slides (Mervide); high-glucose DMEM (Gibco, catalog number 11965092); 10% fetal bovine serum (Gibco, catalog number 10099158); anti-human Alexa 488 secondary antibody (Thermo Fisher Scientific, catalog number A20000); all primers were synthesized by Shanghai Jierui Biotechnology Co., Ltd.; the initial vector pcDNA3.1(+) was a conventional vector provided by Shaoxing University (Shaoxing College of Arts and Sciences).

[0032] Example: The human P / Q type VGCC autoantibody detection material provided in this embodiment is prepared through the following steps: B1. Obtaining the P / Q-VGCC gene fragment: Based on the protein structure of P / Q-VGCC, while ensuring the integrity of the transmembrane region, the P / Q-VGCC protein structure was designed as multiple truncated gene fragments with different transmembrane domains. Multiple truncated gene fragments with different transmembrane domains include P / Q-VGCC1: 1-359aa, P / Q-VGCC2: 360-714aa, P / Q-VGCC3: 715-1008aa, P / Q-VGCC4: 1009-1350aa, P / Q-VGCC5: 1351-1694aa and P / Q-VGCC6: 1695-2262aa; The amino acids of the gene fragments P / Q-VGCC1, P / Q-VGCC2, P / Q-VGCC3, P / Q-VGCC4, P / Q-VGCC5, and P / Q-VGCC6 of this invention not only contain polypeptides with natural sequences, but also can use variants of such polypeptides. The amino acid sequences of P / Q-VGCC1 are shown in SEQ ID NO: 1; the amino acid sequences of P / Q-VGCC2 are shown in SEQ ID NO: 2; the amino acid sequences of P / Q-VGCC3 are shown in SEQ ID NO: 3; the amino acid sequences of P / Q-VGCC4 are shown in SEQ ID NO: 4; the amino acid sequences of P / Q-VGCC5 are shown in SEQ ID NO: 5; and the amino acid sequences of P / Q-VGCC6 are shown in SEQ ID NO: 6. The nucleotide sequence of the gene fragment P / Q-VGCC1 of this invention is shown in SEQ ID NO: 7; the nucleotide sequence of P / Q-VGCC2 is shown in SEQ ID NO: 8; the nucleotide sequence of P / Q-VGCC3 is shown in SEQ ID NO: 9; the nucleotide sequence of P / Q-VGCC4 is shown in SEQ ID NO: 10; the nucleotide sequence of P / Q-VGCC5 is shown in SEQ ID NO: 11; and the nucleotide sequence of P / Q-VGCC6 is shown in SEQ ID NO: 12.

[0033] The overexpression vectors were constructed using the following steps: S1. Linearize the pcDNA3.1(+) vector by XbaI single enzyme digestion; S2. The myc-6×His tag sequence fragment was obtained by amplifying the pcDNA6TR vector using primers MCS-Myc-F / BGH-CX-R. S3. The vector obtained in S1 and the fragment obtained in S2 were used to construct a new vector pCDNA3.1NS-mycHis by homologous recombination using a One Step Cloning Kit. S4. The pCDNA3.1NS-mycHis vector was double-digested with NheI and NotI. S5. Using primers 3.1NS-P / Q-VGCC1-F / 3.1NS-P / Q-VGCC1-R, 3.1NS-P / Q-VGCC2-F / 3.1NS-P / Q-VGCC2-R, 3.1NS-P / Q-VGCC3-F / 3.1NS-P / Q-VGCC3-R, 3.1NS-P / Q-VGCC4-F / 3.1NS-P / Q-VGCC4-R, 3.1NS... -P / Q-VGCC5-F / 3.1NS-P / Q-VGCC5-R, 3.1NS-P / Q-VGCC6-F / 3.1NS-P / Q-VGCC6-R amplified the human P / Q-VGCC gene to obtain P / Q-VGCC1, P / Q-VGCC2, P / Q-VGCC3, P / Q-VGCC4, P / Q-VGCC5, and P / Q-VGCC6 fragments, respectively. S6. The vector obtained from the double enzyme digestion in step S4 and the fragments obtained in step S5 are used to construct new vectors P / Q-VGCC1, P / Q-VGCC2, P / Q-VGCC3, P / Q-VGCC4, P / Q-VGCC5, and P / Q-VGCC6 by homologous recombination using the One Step Cloning Kit.

[0034] In this invention, the primer sequences for constructing the overexpression vector are shown in Table 1.

[0035] Table 1 Primer sequences for constructing overexpression vectors

[0036] The construction of HEK-293 cell lines overexpressing the B2 and P / Q-VGCC fragments includes the following steps: A1. Soak the glass slide in 75% ethanol, clean it with PBS phosphate buffer, and treat it with laminin at 37°C for 30 min. A2. HEK-293 cells were cultured at a rate of 2 × 10⁻⁶. 6 viable cells / 25cm 2 Density seeding is applied to the glass slides prepared in the first step; A3. After culturing for 24 hours, the P / Q-VGCC1, P / Q-VGCC2, P / Q-VGCC3, P / Q-VGCC4, P / Q-VGCC5, and P / Q-VGCC6 vector plasmids (transfection concentration of 2000 µg / mL) were transfected into the cells using 25 kDa PEI reagent. A4. After 6 hours, change the medium to HEK-293 cell culture medium (DMEM medium containing 10% fetal bovine serum) and continue culturing for 36-48 hours before subsequent detection.

[0037] B3. Screening for effective P / Q-VGCC autoantigen fragments includes the following steps: B31. 36-48 hours after transfection, fix with acetone for 10 min; B32. Collect serum from healthy individuals and patients and dilute them at a ratio of 1:10. B33. Mix the diluted serum from step two with the cells treated in step one, and incubate at 37°C for 60 min. B34. After incubation, rinse three times with PBS, each time for 5 minutes. B35. Incubate with the anti-human Alexa488 secondary antibody and the incubation product from step four at 37°C for 30 minutes. B36. After incubation, rinse three times with PBS, 5 minutes each time. B37. Observe and photograph under a fluorescence microscope; In this invention, the detection results of normal human serum in HEK-293 cell lines overexpressing different P / Q-VGCC antigen fragments are as follows: Figure 1 As shown; The detection results of serum (P / Q-VGCC titer 1:32) from patients with Lambert-Eaton myasthenia gravis in HEK-293 cell lines overexpressing different P / Q-VGCC antigen fragments are as follows: Figure 2 As shown; The detection results of serum (P / Q-VGCC titer 1:100) from patients with Lambert-Eaton myasthenia gravis in HEK-293 cell lines overexpressing different P / Q-VGCC antigen fragments are as follows: Figure 3 As shown.

[0038] Results Interpretation: The detection results of normal human plasma in HEK-293 cell lines overexpressing different P / Q-VGCC antigen fragments are as follows: Figure 1 As shown; by Figure 1 It is evident that no obvious green fluorescence was detected in the functional domain truncated forms of VGCC1, VGCC2, VGCC3, VGCC4, VGCC5, and VGCC6 during the detection of normal human serum, indicating that normal human bodies do not contain autoantibodies against P / Q-VGCC.

[0039] The detection results of plasma (P / Q-VGCC titer 1:32) from patients with Lambert-Eaton myasthenia gravis in HEK-293 cell lines overexpressing different P / Q-VGCC antigen fragments are as follows: Figure 2 As shown; the detection results of plasma (P / Q-VGCC titer 1:100) from Lambert-Eaton myasthenia gravis patients in HEK-293 cell lines overexpressing different P / Q-VGCC antigen fragments are as follows. Figure 3 As shown.

[0040] Depend on Figure 2 As can be seen, serum analysis of patients with low-titer (1:32) Lambert-Eaton myasthenia gravis showed that the VGCC6 truncated variant exhibited significant green fluorescence, while the other truncated variants did not. This result indicates that the VGCC6 truncated variant possesses an antigenic epitope.

[0041] Depend on Figure 3 As can be seen, serum samples from patients with high-titer (1:100) Lambert-Eaton myasthenia gravis showed that the VGCC6 truncated fragment exhibited significant green fluorescence, while the other truncated fragments did not. This result indicates that the VGCC6 truncated fragment contains an antigenic epitope and is a suitable fragment for detection.

[0042] Application examples This application example provides the use of human P / Q type VGCC autoantibody detection materials, including the detection of autoantibodies using P / Q-VGCC recombinant peptides via Western blotting. The detection steps are as follows: C1. HEK-293 cell lines with different P / Q-VGCC antigen fragments were subjected to cell disruption, protein separation and purification to obtain recombinant peptides of P / Q-VGCC1-P / Q-VGCC6, which were then prepared into a protein solution (protein concentration of 20 ng / µL). C2. Take 2.5µL of each of the P / QVGCC1-P / QVGCC6 protein solutions and spot them onto the PVDF membrane that has been fully wetted with methanol. After the membrane strip is completely dry, place it into a 96-well plate, add 200µL of 5% skim milk blocking solution, and block at room temperature for 1 hour. C3. Discard the blocking solution and add 200µL of 5% skim milk powder dilution containing 5µL of different serum samples to each well according to the experimental design. Incubate at room temperature for 30 minutes. C4. Discard the liquid in the well, add 250µL of washing solution to each well, let stand or gently shake for 30 seconds and then discard. Repeat washing 5 times. C5. Add 200µL of the secondary antibody working solution diluted 1:1000 to each well and incubate at room temperature for 30 minutes. C6. Discard the secondary antibody diluent and wash 5 times as described in step 3. C7. Add 150µL of chromogenic substrate solution to each well and incubate at room temperature in the dark for 15 minutes. C8. Discard the color developing solution, rinse the membrane strip with deionized water 1-2 times, let it air dry for 1 hour, and observe and record the color development results.

[0043] like Figure 4 The diagram shows the colorimetric results of autoantibody detection of different P / Q-VGCC recombinant peptides in normal human serum in an application example of the present invention.

[0044] like Figure 5 The diagram shows the colorimetric results of autoantibody detection of different P / Q-VGCC recombinant peptides in the serum of Lambert-Eaton myasthenia gravis patients (P / Q-VGCC titer 1:32) in an application example of the present invention.

[0045] like Figure 6 The diagram shows the colorimetric results of autoantibody detection of different P / Q-VGCC recombinant peptides in the serum of Lambert-Eaton myasthenia gravis patients (P / Q-VGCC titer 1:100) in an application example of the present invention.

[0046] from Figures 4-6 It can be seen that neither healthy individuals nor patients with Lambert-Eaton myasthenia gravis showed positive punctate spots with the VGCC1 / VGCC2 / VGCC3 / VGCC4 / VGCC5 recombinant peptide, indicating a negative result. Similarly, healthy individuals also did not show positive punctate spots with the VGCC6 recombinant peptide. However, positive punctate spots were detected in patients with Lambert-Eaton myasthenia gravis using the VGCC6 recombinant peptide, and the spot brightness increased with increasing patient titer. This result indicates that the VGCC6 recombinant peptide can be used for VGCC autoantibody detection using Western blotting.

[0047] Sample positive detection rate detection Immunofluorescence was performed on serum samples from 20 P / Q-VGCC positive patients. The detection rates of truncations of each functional domain are shown in Table 2 below.

[0048] Table 2 Comparison of serum sample detection rates in P / Q-VGCC positive patients

[0049] As shown in Table 2, the detection rate of VGCC6 was 65%, significantly higher than that of other functional domain fragments. This result indicates that VGCC6 has higher detection sensitivity and comprehensive recognition ability. This is because the fragment on VGCC6 is an important site for antigenic epitopes, thus exhibiting higher detection sensitivity.

[0050] It is understood that the present invention has been described through some embodiments, and those skilled in the art will recognize that various changes or equivalent substitutions can be made to these features and embodiments without departing from the spirit and scope of the invention. Furthermore, under the teachings of the present invention, these features and embodiments can be modified to adapt to specific situations and materials without departing from the spirit and scope of the invention. Therefore, the present invention is not limited to the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are within the protection scope of the present invention.

Claims

1. A protein for detecting P / Q type VGCC autoantibodies in humans, characterized in that: The protein in question is VGCC6 protein, and its amino acid sequence is shown in SEQ ID NO:

6.

2. The application of the protein as described in claim 1 for the detection of human P / Q type VGCC autoantibodies in constructing an overexpression vector.

3. The application of the protein for detecting human P / Q type VGCC autoantibodies according to claim 2 in the construction of an overexpression vector, characterized in that: The construction of the overexpression vector includes the following steps: S1. Linearize the expression vector by XbaI single enzyme digestion; S2. The myc-6×His tag sequence fragment was obtained by amplifying the pcDNA6TR vector using primers MCS-Myc-F / BGH-CX-R. S3. The vector obtained in step S1 and the fragment obtained in step S2 are used to construct a new vector pCDNA3.1NS-mycHis by homologous recombination using a One Step Cloning Kit. S4. The pCDNA3.1NS-mycHis vector was double-digested with NheI and NotI. S5. Amplify the VGCC6 protein using primers 3.1NS-P / Q-VGCC6-F / 3.1NS-P / Q-VGCC6-R to obtain the truncated gene fragment; S6. The vector obtained from the double enzyme digestion in step S4 and the fragment in step S5 are used to construct an overexpression vector by homologous recombination using a One Step Cloning Kit.

4. The application of the protein for detecting human P / Q type VGCC autoantibodies according to claim 3 in the construction of an overexpression vector, characterized in that: In step S1, the expression vector is pcDNA3.1(+), pCMV, or pENTER.

5. The application of the protein as described in claim 1 for the detection of human P / Q type VGCC autoantibodies in the construction of cell lines.

6. The application of the protein for detecting human P / Q type VGCC autoantibodies according to claim 5 in the construction of cell lines, characterized in that: The construction of the cell line includes the following steps: A1. Clean and disinfect the glass slides and treat them with laminar adhesion protein at 37°C for 30 minutes; A2. Seed HEK-293 cells onto the prepared glass slides and culture them. A3. After culturing for 24 hours, the overexpression vector of VGCC6 protein was transfected into HEK-293 cells using a transfection reagent and cultured for another 24 hours. A4. After culturing for 6 hours, change the medium to HEK-293 cell culture medium and continue culturing for 36-48 hours to obtain cell lines with transient overexpression of different P / Q-VGCC antigen fragments.

7. The application of the protein for detecting human P / Q type VGCC autoantibodies according to claim 6 in the construction of cell lines, characterized in that: In step A3, the transfection concentration of the overexpression vector is 500µg / mL-4000µg / mL.

8. The application of the protein for detecting human P / Q type VGCC autoantibodies according to claim 6 in the construction of cell lines, characterized in that: In step A3, the transfection reagent is lipo 3000 or PEI reagent.

9. The application of the protein for detecting human P / Q type VGCC autoantibodies according to claim 6 in the construction of cell lines, characterized in that: In step A4, the HEK-293 cell culture medium is DMEM medium containing 10% fetal bovine serum.

10. A recombinant polypeptide for detecting human P / Q type VGCC autoantibodies, characterized in that: The recombinant polypeptide was prepared from the cell line described in claim 5.

11. The recombinant polypeptide for detecting human P / Q type VGCC autoantibodies according to claim 10, characterized in that: The recombinant polypeptide is obtained by cell disruption and protein separation and purification of the cell line as described in claim 5.

12. The application of the recombinant polypeptide for detecting human P / Q type VGCC autoantibodies as described in claim 10 in human P / Q type VGCC autoantibody detection materials.

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