CN-1A recombinant protein preparation method and CN-1A antibody imprinting kit

Abstract

The invention discloses a CN-1A recombinant protein preparation method and a CN-1A antibody imprinting kit, histag and Strep-tag II are simultaneously introduced into a C-terminal sequence of a CN-1A sequence to construct lentivirus plasmids and package lentivirus, and then an HEK293F cell strain for stably expressing the recombinant CN-1A protein is constructed in a lentivirus mode. The method comprises the following steps: carrying out serum-free suspension domestication to form stably transfected cells which can be stably expressed and subjected to suspension culture, carrying out large-scale culture and collecting the stably transfected cells, and purifying by using a Strep-tag II tag to obtain CN-1A protein in a lysis solution after collecting the stably transfected cells, after the specificity of the protein is verified by WB, the protein is transferred to an NC membrane to prepare an immunoblotting membrane strip, and the purified CN-1A protein is finally successfully utilized to prepare the anti-CN-1A antibody immunoblotting detection kit through the optimally designed kit composition and detection method. Through parallel comparison of CBA method detection results, it is found that the two detection results are consistent in trend and have a clearer interpretation effect.

Description

Technical Field

[0001] The present invention relates to the field of protein purification and diagnostic reagents in biotechnology, and more specifically to a method for preparing a CN-1A recombinant protein and a CN-1A antibody blotting kit. Background Art

[0002] Sporadic inclusion body myositis (sIBM) is a slowly progressive inflammatory myopathy that occurs more often in patients older than 50 years of age and presents primarily with weakness and atrophy of the quadriceps and flexor digitorum muscles. The search for potential disease biomarkers in sIBM has led to the discovery of antibodies to 50 nucleotidase 1A (CN-1A). CN-1A is a cytoplasmic antigen target and an enzyme that is highly expressed in skeletal muscle and catalyzes the conversion of adenosine monophosphate to adenosine and phosphate. Its accumulation at the vacuolar margins and perinuclear regions of muscle fibers in patients with sIBM suggests that the production of anti-CN-1A antibodies may be secondary to myonuclear degeneration. Anti-CN-1A antibodies were initially considered to be moderately sensitive for sIBM. These antibodies have subsequently been detected in patients with other autoimmune diseases, such as Sjögren's syndrome or systemic erythema, with or without muscle involvement, and in motor neuron disease. Therefore, measurement of anti-CN-1A antibodies continues to be used in clinical practice as a complementary diagnostic test.

[0003] At present, the detection of anti-CN-1A serum or cerebrospinal fluid antibodies mostly adopts immunofluorescence detection (CBA) based on cells expressing antigens. The principle is to make a slide after overexpressing CN-1A protein in cells (mostly 293 cells), permeabilize the cells before detection, add the test sample for incubation, and use fluorescently labeled anti-human IgG secondary antibodies to recognize the autologous anti-CN-1A antibodies bound to the CN-1A protein. This method has high sensitivity and can detect autologous anti-CN-1A antibodies with low titers. However, this method of detecting CN-1A antibodies is relatively complex, time-consuming and expensive, mainly manifested as:

[0004] First, cell immunofluorescence detection usually requires complex sample preparation and processing, including fixation, permeabilization, and blocking steps, which may lead to sample loss or antigen degradation, which also leads to limited yield;

[0005] Second, it requires specialized equipment such as fluorescence microscopes, as well as specific fluorescent dyes and antibodies, which leads to higher experimental costs;

[0006] Third, immunofluorescence experiments require the operator to have certain experimental skills and experience. Beginners may encounter difficulties, and the entire detection process has strict requirements, such as avoiding light operations, not being too vigorous which may cause cell detachment, and non-specific signals caused by complex cell components due to improper operation.

[0007] Immunoblotting, sometimes also called immunoblotting, refers to the detection and analysis of proteins by immunological methods. It usually involves transferring a specific protein to a membrane and then detecting it with the antibody sample to be tested. The main goal is to confirm the presence of antibodies against a specific protein in the sample and detect their relative amount. Compared with CBA detection, immunoblotting does not require special equipment such as fluorescence microscopes, and can be mass-produced by automated equipment. It is less expensive and relatively simple to operate, and is an effective method for detecting anti-CN-1A antibodies. However, this method requires high-purity CN-1A for detection, and CN-1A is a nucleoside protease with a molecular weight of 43kd that exists in the cytoplasm. It is very difficult to obtain high-purity proteins by cell secretion expression and purification. If CN-1A truncated protein detection is used for detection, the risk of antibody missed detection and misdetection will increase due to conformational differences and insufficient epitopes. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a method for preparing CN-1A recombinant protein, and develops a blotting kit for rapid detection of human autologous myositis-related CN-1A IgG antibodies based on the produced CN-1A protein. Tests show that it has good correlation with the CBA detection method, the results are clear, and it can be mass-produced, which also solves the problems of expensive CBA detection and high operating requirements.

[0009] In one aspect, the present invention discloses a method for preparing a CN-1A recombinant protein, the method comprising the following steps:

[0010] S1, Construction of CN-1A stable cell line;

[0011] A 6X his tag and a Strep-tag II tag were introduced into the C-terminus of the gene sequence CN-1A to form a recombinant protein sequence with a length of 1158 bp;

[0012] The recombinant protein sequence is connected to the PLVX-mCMV-ZsGreen1-Puro lentiviral vector to obtain the PLVX-CN-1A-zsgreen-puro lentiviral plasmid; the lentiviral plasmid contains zsgreen fluorescent protein and puro resistance; the lentiviral plasmid is configured to construct a CN-1A stable cell line;

[0013] Among them, the weight ratio of PLVX-CN-1A-zsgreen-puro, psPAX2 and pMD2.0G in the configured lentiviral plasmid is 3:2:1;

[0014] S2, Serum-free suspension culture acclimatization and identification of CN-1A stable cell line;

[0015] The constructed CN-1A stable cell line was cultured, and after the cell growth was stable, suspension adaptation was started; the digested cells were inoculated into serum-free SMM 293-TII suspension culture medium and placed in a shaking flask for shaking culture; the cell density was cultured to 1*107 cells / mL to obtain the CN-1A stable cell line, which was frozen;

[0016] S3, cell recovery, expansion and purification of CN-1A recombinant protein;

[0017] After the frozen CN-1A stable cell line was revived; preheated culture medium was added to the cell suspension, gently mixed, centrifuged, and the cryoprotectant was removed; after the supernatant was discarded, the cells were gently resuspended with preheated culture medium to obtain a uniform cell suspension; the cells were cultured to obtain a cell fermentation liquid with a cell density of 8*106 cells / mL; the cell fermentation liquid was centrifuged at 1000rpm, the culture medium was discarded, cell lysis solution and PSMF were added, and the cells were shaken with a vortex mixer to break the cells, centrifuged, and the supernatant was collected; the CN-1A protein was fully combined with the magnetic beads; incubated After the incubation, place it on the magnetic rack for 5 minutes, remove the supernatant after the magnetic beads are completely adsorbed, add PBS, wash, place it on the magnetic rack again, remove the supernatant after the magnetic beads are completely adsorbed, repeat the addition of PBS twice; use biotin solution to elute the CN-1A protein bound to the magnetic beads, collect the eluate and dialyze it in PBS at 4°C overnight to obtain the purified CN-1A recombinant protein, and identify the CN-1A recombinant protein by SDS-PAGE electrophoresis. To ensure the consistency of the protein, further use anti-polyhistidine antibody for WB detection. .

[0018] On the other hand, the present invention also discloses a CN-1A antibody blotting kit, which contains the above-mentioned CN-1A recombinant protein and is used for rapid detection of human automyositis-related CN-1A IgG antibodies.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention simultaneously introduces his tag and Strep-tag II into the C-terminal sequence of the CN-1A sequence to construct a lentiviral plasmid and package the lentivirus, and then constructs a HEK293F cell line that stably expresses the recombinant CN-1A protein by lentivirus, forms a stable cell line that can stably express and culture in suspension after serum-free suspension acclimation, and after large-scale culture and collection of stable cells, uses the Strep-tag II label to purify the CN-1A protein in the lysate after collecting the stable cells, and after WB verification of the specificity of the protein, transfers the protein to the NC membrane to prepare an immunoblot membrane strip, and finally successfully uses the purified CN-1A protein to prepare an anti-CN-1A antibody immunoblot detection kit by optimizing the kit composition and detection method. By comparing the test results of the CBA method in parallel, it is found that the trends of the test results of the two are consistent and have a clearer interpretation effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the sequence composition of the CN-1A recombinant protein in Example 1 of the present invention;

[0022] Figure 2 Schematic diagram of the composition of the detection film strip and the detection results in Example 2 of the present invention. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0024] Example 1

[0025] This embodiment discloses a method for preparing a CN-1A recombinant protein. The method uses Snapgene software to design primers, introduces a 6X his tag and a Strep-tag II tag at the C-terminus of CN-1A according to the gene sequence of CN-1A in the applicant's own gene library, and finally forms a protein expression sequence with a length of 1158 bp. The CN-1A recombinant protein sequence is as follows: Figure 1 shown.

[0026] The gene sequence of CN-1A is:

[0027]

[0028] The recombinant protein sequence was recombined and connected to the PLVX-mCMV-ZsGreen1-Puro lentiviral vector using a seamless cloning method, and finally the PLVX-CN-1A-zsgreen-puro lentiviral plasmid was obtained. The plasmid contained zsgreen fluorescent protein and puro resistance, which facilitated the subsequent pressure screening of stable transfectants.

[0029] Before packaging the lentiviral plasmid, HEK293T cells were revived and passaged twice until the cells were in good condition (culture conditions: 37°C, 5% CO2, 10% fetal bovine serum DMEM), and then the cells were divided into 10 cm dishes so that the density could reach 70-80% on the next day. The original culture medium was removed from the cells, and the cells were washed twice with blank culture medium, and then DMEM was added and placed in the incubator for half an hour. At this time, start to configure the chronic disease plasmids and calculate the use of each plasmid PLVX-CN-1A-zsgreen-puro: psPAX2: pMD2.0G = 3: 2: 1, and the total amount of the three plasmids is 15μg; prepare an EP tube, according to the ratio of DMEM: plasmid: PEI = 100μL: 1μg: 2μL, first add the required pure DMEM culture medium, then add the three plasmids calculated in advance, and finally add PEI. After preparing, vortex it slightly to mix it evenly and let it stand at room temperature, then add it dropwise to the cells to be transfected. After transfection for 6h, replace it with a new DMEM culture medium containing 10% serum. After transfection for 48h, collect the virus-containing cell culture fluid into a 15mL centrifuge tube, centrifuge it at 4000rpm for 15min in a 4℃ centrifuge, and then filter the virus supernatant with a 0.45μm filter. Digest and count the 293T cells in good growth state, dilute to 1×105cell / ml, add to 96-well plate, 100μl / well (1×104 cells), a total of 6 wells. Incubate in a 37°C, 5% CO2 incubator overnight. Filter the virus supernatant in an EP tube and make a 10-fold gradient dilution, with 6 consecutive dilutions. Then incubate the diluted virus and cells overnight. Aspirate the culture medium with the virus, add 100μL of complete culture medium to each well, observe the results under a fluorescence microscope, count the cells in the wells with a suitable fluorescence ratio (between 10-30%), and calculate the virus titer [titer (TU / ml) = number of cells × fluorescence percentage × 103 / volume of virus stock solution (μL)]. The 293F cells to be constructed that were revived and passaged for 3 generations in advance and in good cell condition were divided into 6-well plates so that their density could reach 60% on the second day. The culture medium in the 6-well plates was discarded and the lentiviral supernatant was added at MOI=2. In order to facilitate infection, 8 μg / mL of infection aid polybrene was added at the same time. After infection for 12 hours, the virus solution was replaced with a new SMM 293-TII culture medium containing 10% fetal bovine serum and 1% double antibody for continued culture; after further culture for 36 hours, 6 μg / mL of puromycin was added to the culture medium to culture the cells. When the cells were full, normal subculture was performed. Puromycin was used to culture the cells throughout the process. After 4 days of pressure culture with puromycin, it was replaced with SMM 293-TII containing 10% fetal bovine serum containing 1 μg / mL puromycin for subculture to the sixth generation for cryopreservation.

[0030] Serum-free suspension culture acclimatization and identification of CN-1A stable cell line:

[0031] The constructed stable cell line was cultured to the best state using SMM 293-TII containing 10% fetal bovine serum and 1 μg / mL puromycin. When the adherent cells grew to about 80% confluence, the cells were digested with trypsin and inoculated into a transition medium with a serum concentration reduced to 5% fetal bovine serum, and continued to be cultured. When the cells grew to about 80% again, the cells were digested with trypsin and inoculated into a medium with a serum concentration reduced to 2% fetal bovine serum, and continued to be cultured. After observing that the cell growth was stable, suspension adaptation began. The digested cells were inoculated into serum-free SMM 293-TII suspension medium and placed in a shaking bottle for shaking culture. The initial shaking speed was 50 rpm, and the culture conditions were 37°C, 5% CO2. After the cells were cultured for 48 hours, the cells were centrifuged at 1000 rpm for five minutes, half of the volume of the medium was removed, and half of the volume of fresh SMM 293-TII medium was added. The cells were gently blown away with a pipette, and continued to be cultured at 37°C, 5% CO2, and 50 rpm. After culturing for 24 hours, the rotation speed was increased to 100 rpm and continued to be cultured. After the cell state was observed to be stable, the rotation speed was increased to 130 rpm. After the cell density was cultured to 1*107 / mL, the cells were frozen.

[0032] Cell recovery, expansion and purification of CN-1A recombinant protein:

[0033] Before cell recovery, preheat the SMM 293-TII medium to 37°C in a water bath and prepare sterile culture bottles in advance. Take out the frozen CN-1A stable cell line from the liquid nitrogen tank, immediately put it in a 37°C water bath and gently shake it to quickly thaw the cells to avoid long-term exposure of the cells to high temperature. After the cells are completely thawed, quickly transfer the cell suspension to a 15mL sterile centrifuge tube, add 5mL of preheated culture medium, mix gently, centrifuge at 1000rpm for 5 minutes, and remove the cryoprotectant (such as DMSO). After carefully pouring off the supernatant, gently resuspend the cells with 2mL of preheated culture medium to ensure a uniform cell suspension. After counting the cells using a cell counter, the resuspended cells were inoculated into a culture bottle with a density of 2*106 / mL in a 10mL culture medium, placed in a carbon dioxide shaker at 37°C and 5% CO2, cultured at 100rpm in an incubator for 48 hours and counted with a cell counter, and the revived cells were subcultured into a new culture medium at a density of 1*106 / mL. After 72 hours of culture, the cells were transferred to 1L of fresh culture medium at 1*106 / mL and cultured for 72 hours, at which time 1L of cell fermentation liquid with a cell density of about 8*106 / mL was obtained. The cell fermentation liquid was centrifuged at 1000rpm for five minutes, the culture medium was discarded, 50mL of cell lysis solution and PSMF were added, and the cells were broken by shaking with a vortex mixer, and then centrifuged at 11000rpm for 10 minutes at 4°C, and the supernatant was collected in a 50mL centrifuge tube. Take 1mL Strep-Tacin magnetic beads and place them on the magnetic stand for 1 minute. After the magnetic beads are completely adsorbed, remove the supernatant, add 1mL PBS buffer, gently blow off the magnetic beads, place them on the magnetic stand again for 1 minute and remove the supernatant. Take 1mL PBS buffer and gently blow off the magnetic beads, add them to the supernatant of the lysed cells, place them on the shaker and gently shake them in an ice bath for 1 hour to allow the CN-1A protein to fully bind to the magnetic beads. After the incubation, place them on the magnetic stand for 5 minutes, remove the supernatant after the magnetic beads are completely adsorbed, add 50mL PBS, shake them gently on the shaker for 2 minutes, wash them, place them on the magnetic stand again for 5 minutes, remove the supernatant after the magnetic beads are completely adsorbed, and repeat the operation of adding 50mL PBS twice. Use 2.5mM biotin solution for elution. Add 5mL of 2.5mM biotin solution into a 50mL centrifuge tube from which PBS has been removed. Gently blow off the magnetic beads and shake gently on an oscillator for 20 minutes to elute the bound CN-1A protein. Collect the eluate and dialyze it in PBS at 4°C overnight using a 10kd dialysis membrane. Identify the protein by SDS-PAGE electrophoresis. To ensure the consistency of the protein, further use anti-polyhistidine antibody for WB detection. The final result shows an obvious imprint at 40kd, which is consistent with the theoretical molecular weight.

[0034] Example 2

[0035] This example discloses a CN-1A antibody blotting kit, which contains the CN-1A recombinant protein in Example 1 and is used for rapid detection of human automyositis-related CN-1A IgG antibodies.

[0036] The test kit consists of a test strip, test-related reagents, and a sample incubation tank. The test strip is placed in the sample incubation tank. Figure 2 As shown in a, the test strip is a rectangular structure, made of NC film material, and is mainly divided into two parts: the pick-up area and the test area. The pick-up area and the test area are separated by the Maker line, and the pick-up area is shorter than the test area. The pick-up area is a blank film strip, which can be used for picking up the film strip during testing and marking the test sample. The Maker line is mainly used to distinguish the front and back of the test film strip and distinguish the position of the pick-up area and the test area. The detection zone demarcation lines include CN-1A protein and human IgG antibody. The CN-1A protein demarcation concentration is 0.05 mg / mL, which is mainly used for qualitative analysis of whether the sample contains anti-CN-1A IgG antibodies; the human IgG antibody demarcation concentration is 0.002 mg / mL, which is mainly used to detect the positive control (control) and semi-quantitatively detect the antibody concentration of the sample. When the test result shows that the control line is colored and the CN-1A line is colored stronger than the control line, it means that the anti-CN-1A IgG antibody content in the test sample is greater than 0.002 mg / mL. When the test result shows that the control line is colored and the CN-1A line is colored weaker than the control line, it means that the anti-CN-1A IgG antibody content in the test sample is less than 0.002 mg / mL. When the control line has no color, it means that the test is invalid. Figure 2 b shows a schematic diagram of detecting a positive CN-1A antibody. Figure 2 Figure c shows a schematic diagram of a negative CN-1A antibody test. Figure 2 The diagram of invalid detection is shown in d. The detection reagents include concentrated sample diluent, concentrated buffer, alkaline phosphatase (AP) labeled goat anti-human IgG secondary antibody, colorimetric solution BCIP, colorimetric solution NBT, and colorimetric solution dilution buffer. The sample incubation tank is 70 mm long, 6 mm wide, and 6 mm deep, and can accommodate at least 1 mL of test sample. The detection incubation tank can be reused after cleaning. The test kit is sealed and stored at 2-8°C.

[0037] Example 3

[0038] This example discloses the effect test of the kit of Example 2.

[0039] 30 healthy human serum samples, 5 CBA CN-1A positive samples and 10 CBA suspected positive samples were selected for testing. Use tweezers to gently clamp the test strip from the bag and carefully remove the test strip, and place the reagent strip with the Marker facing up in the sample incubation tank. Use ultrapure water to dilute the sample diluent 10 times to prepare the working buffer, and dilute the serum or cerebrospinal fluid to be tested 10 times with the working buffer as the test sample. Add 1mL of the 10-fold diluted serum or cerebrospinal fluid to be tested to the sample incubation tank containing the test strip along the picking area, gently adhere to the wall, and completely immerse the test strip. Incubate at room temperature for 40 minutes. During the incubation process, gently shake the pipette tank 2-3 times to fully incubate it. After the incubation is completed, aspirate the test sample liquid along the picking area, add 1mL of concentrated buffer diluted 10 times with ultrapure water, and gently shake and wash twice, each time for 2 to 3 minutes. Use sample diluent to dilute alkaline phosphatase (AP) labeled goat anti-human IgG secondary antibody at a ratio of 1:2000 as working secondary antibody. After aspirating the working buffer in step 3, add 1mL of working secondary antibody to the incubation tank to completely immerse the test strip and incubate at room temperature for 40 minutes. During the incubation process, gently shake the pipette plate 2-3 times to fully incubate. After the incubation is completed, aspirate the working secondary antibody solution, add 1mL of concentrated buffer diluted 10 times with ultrapure water, and gently shake and wash 3 times, 2 to 3 minutes each time. Aspirate the last washing working buffer, add 1mL of color working solution prepared according to the ratio shown in Table 1 to the incubation tank to completely immerse it. Start timing after adding, incubate in the dark for 15-20 minutes, then aspirate the color solution, add 1mL of ultrapure water and wash twice, 2 minutes each time. After washing, aspirate the ultrapure water, then carefully grasp the pick-up area with tweezers to take out the test strip, and read the results after drying at room temperature in the dark. Place the dried test strip parallel to the marked film strip and align the Maker lines to form a line. If there is a line in the test area CN-1A, it means that it contains positive antibodies. The test comparison results are statistically shown in the following table:

[0040]

[0041] Among them, 1:100, 1:32, and 1:10 represent the positive dilution multiples of CBA detection; +++ represents strong positive, ++ represents moderate positive, + represents weak positive, and - represents negative.

[0042] After testing and comparison, it was found that when testing positive samples, the comparison between the immunoblotting kit and the CBA positive samples showed not only the same positive results but also the same positive intensity. When testing suspected positive samples of CBA, not all of the immunoblotting results were positive. One of the five suspected samples was positive and weakly positive. In the test results of healthy people, CBA showed two suspected positive or weakly positive results in 10 samples, while the immunoblotting results were all negative. CBA showed a small number of positive results or some suspected positive results in the detection of healthy samples, which would cause some trouble for the testers in judging the results. It is speculated that this may be related to the detection method. For example, since CBA detects intracellular CN-1A, it is easy to cause the residual fluorescent secondary antibody in the cell, and different serum samples may have antibodies against other cell targets, which may cause false positives.

[0043] The CN-1A IgG antibody blotting kit developed by the present invention uses a highly purified recombinant CN-1A protein as a detection antigen, avoiding interference from other antibodies that may non-specifically bind to cells, and is simple and controllable to operate, and can be used as an important auxiliary means for clinical diagnosis.

[0044] The above are only some embodiments of the present invention. It should be pointed out that for ordinary technicians in this field, other modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing CN-1A recombinant protein, characterized in that: The method comprises the following steps: S1, Construction of CN-1A stable cell line; A 6X his tag and a Strep-tag II tag were introduced into the C-terminus of the gene sequence CN-1A to form a recombinant protein sequence with a length of 1158 bp; The recombinant protein sequence is connected to the PLVX-mCMV-ZsGreen1-Puro lentiviral vector to obtain the PLVX-CN-1A-zsgreen-puro lentiviral plasmid; the lentiviral plasmid contains zsgreen fluorescent protein and puro resistance; the lentiviral plasmid is configured to construct a CN-1A stable cell line; Among them, the weight ratio of PLVX-CN-1A-zsgreen-puro, psPAX2 and pMD2.0G in the configured lentiviral plasmid is 3:2:1; S2, Serum-free suspension culture acclimatization and identification of CN-1A stable cell line; The constructed CN-1A stable cell line was cultured, and after the cell growth was stable, suspension adaptation was started; the digested cells were inoculated into serum-free SMM 293-TII suspension culture medium and placed in a shaking flask for shaking culture; the cell density was cultured to 1*107 cells / mL to obtain the CN-1A stable cell line, which was frozen; S3, cell recovery, expansion and purification of CN-1A recombinant protein; After the frozen CN-1A stable cell line was revived; preheated culture medium was added to the cell suspension, gently mixed, centrifuged, and the cryoprotectant was removed; after the supernatant was discarded, the cells were gently resuspended with preheated culture medium to obtain a uniform cell suspension; the cells were cultured to obtain a cell fermentation liquid with a cell density of 8*106 cells / mL; the cell fermentation liquid was centrifuged at 1000rpm, the culture medium was discarded, cell lysis solution and PSMF were added, and the cells were shaken with a vortex mixer to break the cells, centrifuged, and the supernatant was collected; the CN-1A protein was fully combined with the magnetic beads; incubated After the incubation, the cells were placed on a magnetic rack for 5 minutes. After the magnetic beads were completely adsorbed, the supernatant was removed and PBS was added for washing. The cells were placed on a magnetic rack again. After the magnetic beads were completely adsorbed, the supernatant was removed and PBS was added for washing. The cells were placed on a magnetic rack again. After the magnetic beads were completely adsorbed, the supernatant was removed and PBS was added for washing. The CN-1A protein bound to the magnetic beads was eluted with a biotin solution. The eluate was collected and dialyzed in PBS at 4°C overnight to obtain the purified CN-1A recombinant protein. The CN-1A recombinant protein was identified by SDS-PAGE electrophoresis. To ensure the consistency of the protein, an anti-polyhistidine antibody was further used for WB detection.

2. The method for preparing CN-1A recombinant protein according to claim 1, characterized in that: In step S1, before configuring the lentiviral plasmid, the HEK293T cells were revived and passaged twice. When the cells were in good condition, the cells were divided into 10 cm dishes so that the density could reach 70-80% on the next day. The original culture medium was removed from the cells, and the cells were washed twice with blank culture medium, and then DMEM was added and placed in an incubator for half an hour.

3. The method for preparing CN-1A recombinant protein according to claim 2, characterized in that: The method for configuring lentiviral plasmid is: The total amount of plasmids PLVX-CN-1A-zsgreen-puro, psPAX2 and pMD2.0G is 15 μg, which are prepared in an EP tube according to the ratio of DMEM: plasmid: PEI of 100 μL: 1 μg: 2 μL. First, pure DMEM medium is added, then the three plasmids are added, and finally PEI is added. After mixing evenly and standing at room temperature, it is added dropwise to the cells to be transfected. After transfection for 6 hours, it is replaced with a new DMEM medium containing 10% serum; 48 hours after transfection, the cell culture fluid containing the virus is collected and centrifuged in a centrifuge tube, and the virus supernatant is filtered; Digest and count the 293T cells in good growth state, dilute to 1×105cell / ml, add to 96-well plate, 100μl / well, 6 wells in total; culture overnight in a 37°C, 5% CO2 incubator; filter the virus supernatant in an EP tube and make a 10-fold gradient dilution for 6 consecutive dilutions; then incubate the diluted virus and cells overnight; remove the culture medium containing the virus, add 100μL of complete culture medium to each well, observe the results under a fluorescence microscope, count the cells in the wells with a fluorescence ratio between 10-30%, and calculate the virus titer; The 293F cells to be constructed that were revived and passaged for 3 generations in advance and in good cell condition were divided into 6-well plates so that their density could reach 60% on the second day. The culture medium in the 6-well plates was discarded and the lentiviral supernatant was added. In order to facilitate infection, 8 μg / mL of the infection aid polybrene was added at the same time. After infection for 12 hours, the virus liquid was replaced with a new SMM293-TII culture medium containing 10% fetal bovine serum and 1% double antibody for continued culture; after continuing to culture for 36 hours, 6 μg / mL of puromycin was added to the culture medium to culture the cells. When the cells were full, normal subculture was performed. Puromycin was always used to culture the cells throughout the process. After 4 days of pressure culture with puromycin, it was replaced with 1 μg / mL of puromycin containing 10% fetal bovine serum SMM 293-TII culture and passaged to the sixth generation for cryopreservation.

4. The method for preparing CN-1A recombinant protein according to claim 1, characterized in that: In step S2, the method for culturing the constructed CN-1A stable cell line is as follows: using SMM 293-TII containing 10% fetal bovine serum and 1 μg / mL puromycin for culturing, when the adherent cells grow to a confluence of about 80%, digesting the cells with trypsin, and inoculating them into a transition medium with a serum concentration reduced to 5% fetal bovine serum, and continuing the adherent culture, when the cells grow to about 80% again, digesting the cells with trypsin, and inoculating them into a medium with a serum concentration reduced to 2% fetal bovine serum, and continuing the adherent culture, and after observing that the cell growth is stable, starting the suspension adaptation.

5. The method for preparing CN-1A recombinant protein according to claim 4, characterized in that: The shaking culture method is as follows: the initial shaker speed is 50 rpm, the culture conditions are 37°C, 5% CO2, after the cells are cultured for 48 hours, the cells are centrifuged at 1000 rpm for five minutes, half the volume of the culture medium is removed, half the volume of fresh SMM 293-TII culture medium is added, the cells are gently blown off with a pipette, and the culture is continued at 37°C, 5% CO2, 50 rpm; after culturing for 24 hours, the speed is increased to 100 rpm and the culture is continued.

6. The method for preparing CN-1A recombinant protein according to claim 1, characterized in that: In step S3, before cell recovery, the SMM 293-TII medium was preheated to 37°C in a water bath, a sterile culture bottle was prepared in advance, and the frozen CN-1A stable cell line was taken out from the liquid nitrogen tank, and immediately placed in a 37°C water bath and gently shaken to quickly thaw the cells to avoid prolonged exposure of the cells to a high temperature environment.

7. The method for preparing CN-1A recombinant protein according to claim 6, characterized in that: In step S3, the preparation method of the cell fermentation broth is as follows: after counting the cells using a cell counter, the resuspended cells are inoculated in a culture bottle with 10 mL of culture medium at a density of 2*106 cells / mL, placed in a carbon dioxide shaker at 37°C and 5% CO2, cultured in an incubator at 100 rpm for 48 hours and counted with a cell counter, and the revived cells are passaged into a new culture medium at a density of 1*106 cells / mL. After culturing for 72 hours, the cells are transferred to 1 L of fresh culture medium at a density of 1*106 cells / mL and cultured for 72 hours.

8. The method for preparing CN-1A recombinant protein according to claim 7, characterized in that: The method for binding CN-1A protein to magnetic beads is as follows: 1 mL of Strep-Tacin magnetic beads are placed on a magnetic rack. After the magnetic beads are completely adsorbed, the supernatant is aspirated, 1 mL of PBS buffer is added to gently blow away the magnetic beads, and then the beads are placed on a magnetic rack again, and the supernatant is aspirated; 1 mL of PBS buffer is taken to gently blow away the magnetic beads and then added to the supernatant of lysed cells, and then placed on an oscillator and gently shaken in an ice bath to incubate, so that the CN-1A protein and the magnetic beads are fully bound.

9. A CN-1A antibody blotting kit, comprising the CN-1A recombinant protein according to any one of claims 1 to 8, characterized in that: Used for rapid detection of human automyositis-related CN-1A IgG antibodies, the kit is composed of a test strip, detection-related reagents and a sample incubation tank, the test strip is placed in the sample incubation tank, the test strip includes a taking area and a detection area, the taking area and the detection area are separated by a Maker line, and the length of the taking area is shorter than the detection area; the taking area is a blank film strip, which is used for taking the film strip during detection and marking the detection sample; the detection area has CN-1A protein and human IgG antibody, and the concentration of the CN-1A protein line is 0.05 mg / mL, which is used for qualitative analysis of whether the sample contains anti-CN-1A IgG antibodies; The human IgG antibody line concentration is 0.002 mg / mL, which is used to detect the positive control and semi-quantitatively detect the antibody concentration of the sample. When the test results show that the human IgG antibody control line is colored and the CN-1A line is stronger than the human IgG antibody control line, it means that the anti-CN-1A IgG antibody content in the test sample is greater than 0.002 mg / mL. When the test results show that the human IgG antibody control line is colored and the CN-1A line is weaker than the human IgG antibody control line, it means that the anti-CN-1A IgG antibody content in the test sample is less than 0.002 mg / mL. When the human IgG antibody control line has no color, it means that the test is invalid.

10. The CN-1A antibody blotting kit according to claim 9, characterized in that The detection-related reagents include concentrated sample diluent, concentrated buffer, alkaline phosphatase-labeled goat anti-human IgG secondary antibody, color developing solution BCIP, color developing solution NBT, and color developing solution dilution buffer.

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