Construction and application of CHO cell strain suitable for autoantibody detection

Abstract

The invention discloses construction and application of a CHO cell strain suitable for autoantibody detection, and relates to the technical field of biotechnology and autoantibody detection.The construction method includes the steps that a DHFR gene defect type CHO cell strain serves as a receptor, green fluorescent protein (GFP) and dihydrofolate reductase (DHFR) are integrated into the DHFR gene defect type CHO cell strain in a gene transfection mode, and the CHO cell strain suitable for autoantibody detection is obtained. And carrying out label protein GFP fluorescence intensity and nutrition pressure screening and monoclonal culture to obtain a DHFR-CHO cell strain with GFP continuous high-abundance expression, namely GFP-DHFR-CHO. The invention provides construction of a CHO stably transfected cell strain suitable for autoantibody detection, and based on a cell platform, target genes (such as AQP4) are integrated into GFP sites through a Crispr-Cas9 system, so that high-abundance expression of autoantigens is realized, and the sensitivity and specificity of detection are improved; in addition, due to the fact that the uncertainty influence caused by the tedious operation steps of transient rotation is reduced, the stability of production and detection is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology and autoantibody detection technology, in particular to the construction and application of a CHO cell line suitable for autoantibody detection. Background Art

[0002] Autoimmune diseases refer to a type of disease caused by abnormal responses of the human immune system to its own tissues. The characteristics of autoimmune diseases are pathological reactions to self-antigens. Patients show neurological symptoms or inflammatory reactions, that is, the appearance of autoantibodies that bind to self-antigens. Autoimmune diseases affect about 10% of the population and involve almost all tissues and organs. The detection of autoantibodies is an important basis for the diagnosis of autoimmune diseases. It not only helps doctors to clarify the diagnosis and treatment plan, but also has guiding significance for the prognosis of the disease and the prediction of treatment effects.

[0003] Sensitivity and specificity are the key core elements of autoantibody detection. Existing autoantibody detection mainly includes enzyme-linked immunosorbent assay, immunoblot, immunospot or cell-based immunofluorescence assay (CBA). The CBA method is to transfect exogenous target genes into cells to make the cells express autoantigens, and use this as a substrate to detect autoantibodies through cell-based immunofluorescence method. Since it does not involve antigen extraction and homozygosity, it can preserve the protein topological conformation better. It is particularly suitable for the detection of autoantibodies to cell membrane proteins of the nervous system such as central demyelination and autoimmune encephalitis. It has high detection sensitivity and specificity, and its application range and effect are significantly wider than those of enzyme-linked immunosorbent assay and immunoblot.

[0004] However, most existing CBA methods use transient transfection methods with HEK-293 as the cell substrate. The transient transfection method has many operation steps and requires repeated production, and has problems such as low production stability and poor repeatability, which in turn affects the sensitivity and specificity of the test and increases the possibility of misdiagnosis. However, although stable transformation strains can solve the above problems, due to various regulatory mechanisms in the cells themselves, the initial high-expression genes will gradually down-regulate as the number of cell culture generations increases. In addition, each strain of the common stable transformation method needs to undergo long-term screening, and it takes a long time and consumes a lot of energy to obtain the strain. Autoimmune diseases have many types of antigens, and similar long-term screening for each antigen will consume a lot of manpower and material resources. Therefore, it is necessary to develop a solution for constructing a stable cell line for autoantibody detection.

[0005] Due to the hypotriploid chromosomes of HEK-293, the genome is unstable, which increases the difficulty of constructing stable cell lines. Chinese hamster ovary cells (CHO) are commonly used recombinant protein production cells and are more suitable stable transformation target cell lines. The commonly used method for stable transformation of strains is to randomly insert the expression frame into the genome. This method has a strong randomness. Although a better insertion position can be found, the probability is low. In addition, due to the phenomenon that the expression of the target antigen continues to weaken with the increase in the number of culture generations, it takes a long time and a lot of work to screen a relatively stable and highly expressed stable cell line. If only a small amount of target protein is expressed, this scheme can be well applied, but in the face of a large number of self-antigens, the manpower and material resources required for this scheme are too huge. Therefore, it is urgent to find a time-saving and labor-saving method for obtaining stable cell lines with high expression of self-antigens. Summary of the invention

[0006] The present invention provides a construction of a CHO cell line suitable for autoantibody detection and its application, which can effectively solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions: a construction of a CHO cell line suitable for autoantibody detection, comprising taking a DHFR gene-deficient CHO cell line as a receptor, integrating green fluorescent protein GFP and dihydrofolate reductase DHFR into the DHFR gene-deficient CHO cell line by gene transfection, and obtaining a DHFR-CHO cell line GFP-DHFR-CHO with continuous high expression of GFP by measuring the fluorescence intensity of the tag protein GFP, nutritional stress screening and monoclonal culture;

[0008] Provided is a method for site-specific insertion of an autoantigen sequence into a GFP sequence of a GFP-DHFR-CHO stable cell line. The method comprises: using a classic autoantigen water channel protein AQP4, performing double-stranded cutting near the ATG of a GFP gene by using the Crispr-Cas9 technology, inserting the AQP4 gene in front of the GFP gene by homologous recombination repair, and inhibiting non-homologous repair by adding a DNA ligase IV inhibitor Scr7, thereby obtaining a DHFR-CHO cell line AQP4-DHFR-CHO with high expression of AQP4.

[0009] According to the above technical solution, the amino acid sequence of DHFR is shown as SEQ ID NO:1, and the amino acid sequence of AQP4 is shown as SEQ ID NO:2.

[0010] According to the above technical solution, the amino acids of DHFR and AQP4 not only include polypeptides of natural sequences, but also variants of such polypeptides can be used;

[0011] A variant refers to at least a fragment of the full-length sequence mentioned, which is one or more amino acid or nucleic acid sequences truncated at one or both ends relative to the full-length sequence;

[0012] The fragment comprises or encodes a peptide having at least 8 to the full length of the original sequence or a variant thereof;

[0013] The total length of the variant is at least 8 to the full-length amino acid peptide;

[0014] Variants relate to at least a fragment, to a polypeptide or a fragment thereof comprising an amino acid sequence having at least 40% to 99.9% homology to the amino acid sequence or a fragment thereof;

[0015] Variants can also be produced by fusing the polypeptide, its fragment or variant thereof to the N- or / and C-terminus of other known polypeptides or variants thereof and comprise an active portion or domain;

[0016] The active portion or domain has a sequence identity of 70% to 99% when aligned with the active portion of the reference sequence, and the active portion refers to an amino acid sequence shorter than the full-length amino acid sequence or, in the case of a nucleic acid sequence, a nucleic acid sequence encoding less than the full-length amino acid sequence, which is a variant of the natural sequence but retains at least a certain biological activity;

[0017] Specifically, the sequence of the gene encoding DHFR is shown in SEQ ID NO:3, and the sequence of the gene encoding AQP4 is shown in SEQ ID NO:4.

[0018] According to the above technical scheme, DHFR and AQP4 genes are integrated into the primary CHO cell line by gene transfection to obtain a DHFR-CHO stable cell line suitable for overexpression of AQP4;

[0019] It is preferred to transfect DHFR-deficient CHO cell lines with less early differentiation, so DHFR-deficient CHO cell lines with good primary growth conditions are transfected;

[0020] The above construction method includes:

[0021] (1) After trypsin digestion and subculture, DHFR auxotrophic CHO cell lines were inoculated into new culture dishes or culture flasks and cultured in IMDM medium containing hypoxanthine and thymidine;

[0022] (2) cloning GFP and DHRF genes into expression vectors respectively to obtain recombinant overexpression vectors, wherein the sequence of the recombinant overexpression vector is shown in SEQ ID NO:5;

[0023] (3) The recombinant overexpression vector was transfected into a DHFR-deficient CHO cell line and cultured in IMDM medium without hypoxanthine and thymidine to obtain a GFP-DHFR-CHO stable cell line, and monoclonal cells were selected;

[0024] (4) Obtaining homology arms from the GFP expression frame, splicing through overlap extension PCR and AQP4, and fusing 3×Flag tag proteins to obtain the template required for homologous recombination repair, named Temple, and the sequence of the complete gene is shown in SEQ ID NO:6;

[0025] (5) Use primer asymmetric PCR to amplify Temple to obtain single-stranded DNA, named ssDNA;

[0026] (6) constructing a Crispr-Cas9 vector, the sequence of which is shown in SEQ ID NO:7, and transfecting it together with the ssDNA described in (5) into a GFP-DHFR-CHO cell line, inhibiting non-homologous repair by adding DNA ligase IV inhibitor Scr7, and inserting it into the front end of the GFP gene sequence to obtain a transformed cell line to be screened;

[0027] (7) The cell lines were cultured until the binding degree reached 90%, and then passaged. Before passaged, cells without fluorescence were screened out by flow cytometry, and then the cells were adsorbed and sorted using magnetic beads containing 3×Flag tag proteins to obtain cells expressing AQP4 (fused with 3×Flag tag proteins) but unable to express GFP. These single cells were plated and cultured in 96-well plates. After one to two weeks, a small amount of cells were taken for PCR detection. The detection primers had one end of a primer derived from GFP and the other end of a primer derived from AQP4. The single cell clone that amplified the target band was a positive AQP4-DHFR-CHO stable cell line.

[0028] According to the above technical scheme, the CHO cell line used is a dihydrofolate reductase DHFR nutritional deficiency mutant cell line, which grows in a medium supplemented with hypoxanthine and thymine;

[0029] Alternatively, the introduction of exogenous DHFR gene can also replace the DHFR defect in the body, and DHFR can be used as a screening marker for nutritional deficiency;

[0030] The introduction of exogenous DHFR requires the use of a eukaryotic expression vector, preferably an expression vector selected from pcDNA3.1, pCMV or pENTER, among which pcDNA3.1 can be used for high-level expression and constitutive expression of exogenous genes in a variety of mammalian cells, so pcDNA3.1 is more preferred;

[0031] In step (2) of the above construction method, the expression vector is double-digested with BamhI and XhoI, and the GFP gene is cloned into the double-digestion site to obtain a recombinant expression vector, Escherichia coli is transformed, the bacteria are expanded and the plasmid is extracted to obtain a GFP expression vector, the GFP overexpression vector is double-digested with BsaBI and BstBI, and the DHFR gene is cloned to replace the neomycin resistance gene to obtain a GFP-DHFR overexpression vector, wherein the GFP gene is expressed by the CMV promoter and the DHFR gene is expressed by the SV40 promoter.

[0032] According to the above technical scheme, in step (3) of the above construction method, when GFP-DHFR-CHO cells are obtained by pressure screening culture in IMDM culture medium without hypoxanthine and thymidine, cells expressing GFP fluorescence to a set threshold can be selected by flow cytometry before each transmission culture. This process is continued for 4 months. After 4 months, 40-60 cells with strong GFP expression are selected and transferred to a new 96-well plate for monoclonal culture, and the clone with the highest GFP expression abundance is selected for subsequent experiments.

[0033] According to the above technical solution, in step (3) of the above construction method, the screening time can be 2-6 months;

[0034] In step (3) of the above construction method, methotrexate (MT) can be added to the culture medium to weaken the DHFR enzyme activity, thereby obtaining a CHO cell line with high expression of GFP-DHFR;

[0035] In step (3) of the above construction method, the concentration of methotrexate is 1×10 -9 mol / L-1×10 -6 mol / L, more preferably 1×10 -8 mol / L-2×10 -7 mol / L, specifically, the screening process is as follows:

[0036] (1) Trypsinize GFP-DHFR-CHO cells and subculture them into new T25 or T75 culture flasks. The cell density (confluence) should not exceed 20%.

[0037] (2) Add 1×10 -8 mol / L methotrexate;

[0038] (3) Every 10 days, the methotrexate concentration was increased by multiples to 2.4×10 -7 mol / L;

[0039] (4) After 50 days of screening, observe and evaluate the formation of cell colonies (clones). If necessary, the screening can be extended to 80 days.

[0040] According to the above technical solution, in step (7) of the above construction method, the screening time can be 2-6 months;

[0041] The transfection reagent used in the above construction method is lipo 3000, PEI reagent, more preferably 25kDa PEI reagent;

[0042] The transfection concentration of the recombinant expression vector of the above construction method is 500 μg / mL-4000 μg / mL, such as 700 μg / mL, 1000 μg / mL, 1500 μg / mL, 2000 μg / mL, 2500 μg / mL, 3500 μg / mL or 3800 μg / mL.

[0043] According to the above technical scheme, the AQP4-DHFR-CHO cell line constructed by the construction method of the AQP4-DHFR-CHO cell line suitable for autoantibody detection is an AQP4-DHFR-CHO cell line.

[0044] According to the above technical scheme, an application of a CHO cell line suitable for autoantibody detection, and an application of an AQP4-DHFR-CHO cell line constructed based on a CHO cell line suitable for autoantibody detection in autoantibody detection.

[0045] Compared with the prior art, the present invention has the following beneficial effects: in the technical scheme of the present invention, a CHO stable cell line suitable for autoantibody detection is provided, and the cell can express autoantigens in high abundance, thereby improving the sensitivity and specificity of detection; in addition, the uncertainty caused by the cumbersome operation steps of transient transfection is reduced, thereby improving the stability of production and detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0047] In the attached picture:

[0048] Figure 1 is a schematic diagram of the vector construction of the present invention;

[0049] Figure 2 This is a schematic diagram of the GFP-DHFR-CHO stable cell line expressing GFP green fluorescence of the present invention;

[0050] Figure 3 It is a schematic diagram of immunofluorescence detection of AQP4 autoantibodies in the AQP4-DHFR-CHO cell line of the present invention;

[0051] Figure 4 It is a schematic diagram of negative sample detection results of the present invention;

[0052] Figure 5 It is a schematic diagram of the sample detection results after the QP4-DHFR-CHO stable cell line of the present invention has been continuously cultured for 3 months. DETAILED DESCRIPTION

[0053] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0054] Embodiment: The present invention provides a technical solution, a construction of a CHO stable cell line suitable for autoantibody detection:

[0055] like Figure 1 As shown: the red box indicates the promoter, the green box indicates the gene, the blue box indicates the termination signal, the light green transcribed sgRNA will not express protein, and the yellow line indicates the homology arm; A, recombinant overexpression vector expressing GFP and DHRF genes; B, Temple fragment; C, Crispr-Cas9 vector

[0056] (1) Cultivation of DHFR auxotrophic CHO cells: Primary DHFR auxotrophic CHO cells were cultured in 90% IMDM medium (Thermo Fisher, 12440053) supplemented with 10% fetal bovine serum (Gibco, Catalog No. 10099158), 5 mM hypoxanthine (MCE, H9377), 0.8 mM thymidine (MCE, 50895) and 1% penicillin-streptomycin (Gibco, Catalog No. 15140148). When the cells grew to 95% confluence (i.e., the cells covered more than 95% of the bottom of the flask), they were digested with trypsin at a ratio of 1:6 and transferred to a new culture flask. The cells were cultured for 24 hours and transfected after they were completely attached to the wall.

[0057] (2) Construction of GFP-DHFR expression vector: Using pcDNA3.1 as a template, double restriction enzymes were used to digest the GFP gene with BamHI and XhoI, and the GFP gene was cloned into the double restriction enzyme site. T4 DNA ligase was used to connect the target gene and the vector site to obtain a recombinant expression vector. Escherichia coli was transformed and the plasmid was extracted by amplification to obtain a GFP expression vector. The GFP overexpression vector was double restricted by BsaBI and BstBI, and the DHFR gene was cloned to replace the neomycin resistance gene to obtain a GFP-DHFR overexpression vector, in which the GFP gene was expressed by the CMV promoter and the DHFR gene was expressed by the SV40 promoter.

[0058] (3) Obtaining GFP-DHFR-CHO cell line: The GFP-DHFR recombinant expression vector was transfected into DHFR auxotrophic CHO cells (transfection concentration was 2000 μg / mL) using Invitrogen's 25 kDa PEI reagent. Six hours after transfection, the culture medium was changed to IMDM medium without hypoxanthine and thymidine and supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. After 2-3 weeks, cell clones visible to the naked eye appeared and green fluorescent cells were observed under a fluorescence microscope, thus obtaining the GFP-DHFR-CHO cell line.

[0059] (4) Selection of GFP-DHFR-CHO monoclonal cell lines: After digestion of the GFP-DHFR-CHO cell line, 40-60 cells with high green fluorescence intensity were selected under a microscope and transferred to a 96-well plate for monoclonal culture. The culture medium was IMDM medium without hypoxanthine and thymidine. Monoclonal cells with good fluorescence intensity were selected for subsequent experiments.

[0060] The results are as follows Figure 2 As shown, a large number of green fluorescent GFP-DHFR-CHO cells can be observed under a fluorescence microscope.

[0061] (5) Acquisition of the Temple fragment: Using the GFP-DHFR overexpression vector as a template, the fragment was linearized by BamHI (NEB, R3136V) and recombined to the front end of GFP using a one step cloning Kit (Novagen, C112). AQP4 and a 700 bp fragment before and after it were amplified using a high-fidelity enzyme (Novagen, P521-d1). The fragment was diluted 100 times and used as a template. Asymmetric PCR was performed with a forward and reverse primer ratio of 1:70. The PCR product was subjected to gel electrophoresis, and the smaller band was cut out and recovered (Tiangen, DP214), which is the Temple fragment.

[0062] (6) Crispr-Cas9 vector:

[0063] The eSpCas9(1.1)_No_FLAG_ATP1A1_G2_Dual_sgRNA vector (vector source addgene, number #86612) was used as a template, and the sticky ends were cut out by BbsI (NEB, R3539S) single enzyme. The primers with sticky ends and sgRNA were gradient annealed to obtain double-stranded DNA designed to express sgRNA. The above-mentioned vector and fragment were connected with T4 ligase (Takara, 2050A) to obtain the Crispr-Cas9 vector containing the target, which was transformed into Escherichia coli, and the plasmid was extracted by bacterial expansion (Tiangen, DP103).

[0064] (7) Obtaining AQP4-DHFR-CHO cell line: The Temple fragment and Crispr-Cas9 vector were co-transfected into the GFP-DHFR-CHO cell line (transfection concentration was 2000 μg / mL) using Invitrogen's 25 kDa PEI reagent for transfection. Six hours after transfection, the medium was replaced with IMDM medium without hypoxanthine and thymidine and supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 50 μM DNA ligase IV inhibitor Scr7 (Merck, SML1546) was used. After one day of culture, the IMDM medium without hypoxanthine and thymidine was replaced, and 10% fetal bovine serum and 1% penicillin-streptomycin were added. After another day of culture, the binding degree reached more than 90% for subculture. The cells were digested with trypsin (Merck, T4674) for 1 minute, and the digested cells were resuspended in IMDM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Then, these cells were added to the sample tube of the flow cytometer, and the parameters were set to separate the cells without GFP fluorescence. The separated cells were collected by centrifugation and fully suspended with a small amount of PBE incubation solution (0.5% BSA, 0.08% EDTA, pH = 7.2 PBS, vacuum filtration sterilization) (about 1×10 in 0.3 mL of liquid). 8 cells), added magnetic beads coated with Flag antibody (Merck, M8823), mixed and incubated at 15°C for 30 minutes, installed the separation column in a magnetic field, added 0.5mL PBE, and naturally drained under gravity to pretreat the separation column, cultured the adsorbed cells in IMDM medium supplemented with 10% fetal bovine serum until the binding degree reached more than 90% again, digested the cells with trypsin for 1 minute, resuspended the digested cells in IMDM medium supplemented with 10% fetal bovine serum, and graded diluted by 10 times until there was only 1 cell in 100μL liquid, cultured single cells in 96-well plates, and amplified the cultured cells with high-fidelity enzyme (Novozyme, P521-d1), used primers with AQP4 at one end and GFP at one end, and the target band was amplified as a positive clone.

[0065] (8) Detection of AQP4 autoantibodies: AQP4-DHFR-CHO cell line was cultured at 3.5×10 5The cells were densely inoculated on glass slides (Mevid); the glass slides were soaked in 75% ethanol in advance, cleaned with PBS phosphate buffer, and treated with laminin at 37°C for 30 minutes. After 72 hours of cell inoculation, they were fixed with 4% paraformaldehyde for 10 minutes, and 5 AQP4-positive clinical samples with different titers (1:10, 1:32 and 1:100) (Note: the titer was determined based on the results of our company's AQP4 transient transfection test) and 15 negative samples were incubated at 37°C for 60 minutes, and anti-human Alexa 488 secondary antibody (Thermo Fisher, Catalog No. A20000) was incubated at 37°C for 30 minutes. Each step was washed 3 times with PBS phosphate buffer, each time for 5 minutes; observation and photography were carried out under a fluorescence microscope.

[0066] The results are as follows Figure 3 As shown, A1, B1 and C1 are AQP4 transient transfection CHO cell lines. A2, B2 and C2 are AQP4-DHFR-CHO stable transfection cell lines. A1 and A2, B1 and B2, C1 and C2 were all tested with the same sample, and their original titers were 1:10, 1:32 and 1:100, respectively. The test results showed that the green fluorescence intensity of the AQP4-DHFR-CHO stable transfection cell line was significantly higher than that of the AQP4 transient transfection CHO cell line;

[0067] The positive sample detection results showed that the green fluorescence intensity of AQP4 in AQP4-DHFR-CHO stable transfected cells was significantly higher than that in transient transfection, indicating higher sensitivity.

[0068] The results are as follows Figure 4 As shown, the negative sample test results showed that AQP4-DHFR-CHO stable transfected cells rarely had non-specific staining results, indicating better specificity.

[0069] The results are as follows Figure 5 As shown, after continuous culture of AQP4-DHFR-CHO stable cell lines for 3 months, the detection results of positive samples still had high consistency, indicating good stability.

[0070] Finally, it should be noted that the above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Construction of a CHO cell line suitable for autoantibody detection, characterized in that: The method includes using a DHFR gene-deficient CHO cell line as a receptor, integrating green fluorescent protein GFP and dihydrofolate reductase DHFR into the DHFR gene-deficient CHO cell line by gene transfection, and obtaining a DHFR-CHO cell line GFP-DHFR-CHO with continuous high expression of GFP by measuring the fluorescence intensity of the tag protein GFP, nutritional stress screening and monoclonal culture; Provided is a method for site-specific insertion of an autoantigen sequence into a GFP sequence of a GFP-DHFR-CHO stable cell line. The method comprises: using a classic autoantigen water channel protein AQP4, performing double-stranded cutting near the ATG of a GFP gene by using the Crispr-Cas9 technology, inserting the AQP4 gene in front of the GFP gene by homologous recombination repair, and inhibiting non-homologous repair by adding a DNA ligase IV inhibitor Scr7, so as to obtain a DHFR-CHO cell line AQP4-DHFR-CHO with high expression of AQP4.

2. The construction of a CHO cell line suitable for autoantibody detection according to claim 1, characterized in that: The amino acid sequence of DHFR is shown in SEQ ID NO:1, and the amino acid sequence of AQP4 is shown in SEQ ID NO:

2.

3. The construction of a CHO cell line suitable for autoantibody detection according to claim 1, characterized in that: Not only polypeptides containing the amino acids of DHFR and AQP4 in their natural sequences, but also variants of such polypeptides can be used; A variant refers to at least a fragment of the full-length sequence mentioned, which is one or more amino acid or nucleic acid sequences truncated at one or both ends relative to the full-length sequence; The fragment comprises or encodes a peptide having at least 8 to the full length of the original sequence or a variant thereof; The total length of the variant is at least 8 to the full-length amino acid peptide; Variants relate to at least a fragment, to a polypeptide or a fragment thereof comprising an amino acid sequence having at least 40% to 99.9% homology to the amino acid sequence or a fragment thereof; Variants can also be produced by fusing the polypeptide, its fragment or variant thereof to the N- or / and C-terminus of other known polypeptides or variants thereof and comprise an active portion or domain; The active portion or domain has a sequence identity of 70% to 99% when aligned with the active portion of the reference sequence, and the active portion refers to an amino acid sequence shorter than the full-length amino acid sequence or, in the case of a nucleic acid sequence, a nucleic acid sequence encoding less than the full-length amino acid sequence, which is a variant of the natural sequence but retains at least a certain biological activity; Specifically, the sequence of the gene encoding DHFR is shown in SEQ ID NO:3, and the sequence of the gene encoding AQP4 is shown in SEQ ID NO:

4.

4. The construction of a CHO cell line suitable for autoantibody detection according to claim 3, characterized in that: The DHFR and AQP4 genes were integrated into the primary CHO cell line by gene transfection to obtain a DHFR-CHO stable cell line suitable for overexpression of AQP4; It is preferred to transfect DHFR-deficient CHO cell lines with less early differentiation, so DHFR-deficient CHO cell lines with good primary growth conditions are transfected; The above construction method includes: (1) After trypsin digestion and subculture, DHFR auxotrophic CHO cell lines were inoculated into new culture dishes or culture flasks and cultured in IMDM medium containing hypoxanthine and thymidine; (2) cloning GFP and DHRF genes into expression vectors respectively to obtain recombinant overexpression vectors, wherein the sequence of the recombinant overexpression vector is shown in SEQ ID NO:5; (3) The recombinant overexpression vector was transfected into a DHFR-deficient CHO cell line and cultured in IMDM medium without hypoxanthine and thymidine to obtain a GFP-DHFR-CHO stable cell line, and monoclonal cells were selected; (4) Obtaining homology arms from the GFP expression frame, splicing through overlap extension PCR and AQP4, and fusing 3×Flag tag proteins to obtain the template required for homologous recombination repair, named Temple, and the sequence of the complete gene is shown in SEQ ID NO:6; (5) Use primer asymmetric PCR to amplify Temple to obtain single-stranded DNA, named ssDNA; (6) constructing a Crispr-Cas9 vector, the sequence of which is shown in SEQ ID NO:7, and transfecting it together with the ssDNA described in (5) into a GFP-DHFR-CHO cell line, inhibiting non-homologous repair by adding DNA ligase IV inhibitor Scr7, and inserting it into the front end of the GFP gene sequence to obtain a transformed cell line to be screened; (7) The cell lines were cultured until the binding degree reached 90%, and then passaged. Before passaged, cells without fluorescence were screened out by flow cytometry, and then the cells were adsorbed and sorted using magnetic beads containing 3×Flag tag proteins to obtain cells expressing AQP4 (fused with 3×Flag tag proteins) but unable to express GFP. These single cells were plated and cultured in 96-well plates. After one to two weeks, a small amount of cells were taken for PCR detection. The detection primers had one end of a primer derived from GFP and the other end of a primer derived from AQP4. The single cell clone that amplified the target band was a positive AQP4-DHFR-CHO stable cell line.

5. The construction of a CHO cell line suitable for autoantibody detection according to claim 4, characterized in that: The CHO cell line used is a dihydrofolate reductase DHFR auxotrophic mutant cell line, grown in a medium supplemented with hypoxanthine and thymidine; Alternatively, the introduction of exogenous DHFR gene can also replace the DHFR defect in the body, and DHFR can be used as a screening marker for nutritional deficiency; The introduction of exogenous DHFR requires the use of a eukaryotic expression vector, preferably an expression vector selected from pcDNA3.1, pCMV or pENTER, wherein pcDNA3.1 is used for high-level expression and constitutive expression of exogenous genes in a variety of mammalian cells, so pcDNA3.1 is more preferred; In step (2) of the above construction method, the expression vector is double-digested with BamhI and XhoI, and the GFP gene is cloned into the double-digestion site to obtain a recombinant expression vector, Escherichia coli is transformed, the bacteria are expanded and the plasmid is extracted to obtain a GFP expression vector, the GFP overexpression vector is double-digested with BsaBI and BstBI, and the DHFR gene is cloned to replace the neomycin resistance gene to obtain a GFP-DHFR overexpression vector, wherein the GFP gene is expressed by the CMV promoter and the DHFR gene is expressed by the SV40 promoter.

6. The structure of a CHO cell line suitable for autoantibody detection according to claim 5, characterized in that: In step (3) of the above construction method, when GFP-DHFR-CHO cells are obtained by pressure screening culture in IMDM medium without hypoxanthine and thymidine, cells expressing GFP fluorescence to a set threshold can be selected by flow cytometry before each transmission culture. This process is continued for 4 months. After 4 months, 40-60 cells with strong GFP expression are selected and transferred to a new 96-well plate for monoclonal culture. The clone with the highest GFP expression abundance is selected for subsequent experiments.

7. The construction of a CHO cell line suitable for autoantibody detection according to claim 6, characterized in that: In step (3) of the above construction method, the screening time may be 2-6 months; In step (3) of the above construction method, methotrexate (MT) can be added to the culture medium to weaken the DHFR enzyme activity, thereby obtaining a CHO cell line with high expression of GFP-DHFR; In step (3) of the above construction method, the concentration of methotrexate is 1×10 -9 mol / L-1×10 -6 mol / L, more preferably 1×10 -8 mol / L-2×10 -7 mol / L, specifically, the screening process is as follows: (1) Trypsinize and subculture GFP-DHFR-CHO cells into new T25 or T75 culture flasks with a cell density (confluence) not exceeding 20%; (2) Add 1×10 -8 mol / L methotrexate; (3) Every 10 days, the methotrexate concentration was increased by multiples to 2.4×10 -7 mol / L; (4) After 50 days of screening, observe and evaluate the formation of cell colonies (clones). If necessary, the screening can be extended to 80 days.

8. The construction of a CHO cell line suitable for autoantibody detection according to claim 4, characterized in that: In step (7) of the above construction method, the screening time may be 2-6 months; The transfection reagent used in the above construction method is lipo 3000, PEI reagent, more preferably 25kDa PEI reagent; The transfection concentration of the recombinant expression vector of the above construction method is 500 μg / mL-4000 μg / mL, such as 700 μg / mL, 1000 μg / mL, 1500 μg / mL, 2000 μg / mL, 2500 μg / mL, 3500 μg / mL or 3800 μg / mL.

9. The construction of a CHO cell line suitable for autoantibody detection according to claim 6, characterized in that: AQP4-DHFR-CHO cell line constructed by the method for constructing an AQP4-DHFR-CHO cell line suitable for autoantibody detection.

10. An application of a CHO cell line suitable for autoantibody detection, characterized in that: Use of the AQP4-DHFR-CHO cell line constructed according to any one of claims 1 to 9 for the detection of autoantibodies.

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