Nonradioactive labeling immunoprecipitation method for detecting NXP2 autoantibody of inflammatory myopathies and application
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An autoantibody and immunoprecipitation technology, which is applied in the direction of viruses/bacteriophages, tumor-specific antigens, chemical instruments and methods, etc., can solve problems such as high false positive rate and high cost of ELISA kits
Inactive Publication Date: 2017-02-22
CENT SOUTH UNIV
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[0006] The purpose of the present invention is to provide a non-radiolabeled immunoprecipitation method and application for detecting NXP2 autoantibodies in inflammatory myopathy, to solve the defects of ELISA kits and wester
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Embodiment 1
[0023] Example 1 Non-radiolabeled immunoprecipitation detection method of NXP2 autoantibodies in inflammatory myopathy
[0024] A non-radiolabeled immunoprecipitation detection method for NXP2 autoantibodies in inflammatory myopathy, comprising the following steps:
[0025] (1) Design primer pair NXP2-F (5'-CGGTCGACCGCGGCGCAGCACC-3') and NXP2-R2 (5'-GCGGTACCTTAGCTTGGAGTTGAAAGAGC-3'), primer pair NXP2-F2 (5'-CGGTCGACCGAAGATACAGAAGCGTCC-3') and NXP2-R (5'-GCGGTACCTTAAGTACTACTGATTTCACTCATT-3'), and then the N-terminal (Genbank: NM_015358.2, 80-1576bp) or C-terminal (Genbank: NM_015358.2, 1274-2896bp).
[0026] (2) Recover and purify the PCR fragment with a gel recovery kit (Omega, D2500-01), perform double digestion with SalI and KpnI restriction endonucleases, and ligate overnight with the pCMV-myc vector that has been double-digested with SalI and KpnI , Transform JM109 bacteria competent, screen positive clones on a plate containing 50ug / ml ampicillin, extract a small amount...
Embodiment 2
[0030] Example 2 detects the positive rate of NXP2 antibody in serum of 50 patients with inflammatory myopathy
[0031] (1) Inoculate 30% HEK293 cells on 75cm 2 Cell culture dish, cultivated at 37°C for less than 24 hours;
[0032] (2) Transfect HEK293 cells with 20ug pCMV-myc-NXP2C plasmid for 48 hours;
[0033] (3) HEK293 cells were lysed with 1 ml of RIPA lysate containing protease inhibitors, and the protein was quantified;
[0034] (4) Using c-myc antibody to confirm the overexpressed NXP2 C-terminus in HEK293 cells.
[0035] Take 20ul of the test serum, positive serum, and negative serum from patients with inflammatory myopathy for immunoprecipitation with the cell lysate overexpressing the C-terminus of NXP2, perform SDS-PAGE gel electrophoresis, and then detect with c-myc antibody to determine the presence of NXP2 antibody in The positive rate in patients with inflammatory myopathy was about 12.8% (as attached Figure 6 As shown, (a) and (b) are respectively select...
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Abstract
The invention of the application discloses a nonradioactive labeling immunoprecipitation method for detecting an NXP2 autoantibody of inflammatory myopathies and application. A plasma pCMV-myc-NXP2N or pCMV-myc-NXP2C which comprises an NXP2 N end (1-500aa) or an NXP2 C end (400-939aa) and has a Myc tag is constructed, an HEK293 cell is transiently transfected, a serum of an inflammatory myopathies patient with positive NXP2 autoantibody is subjected to immunoprecipitation with an HEK293 cell lysis buffer with an overexpressed NXP2 N end or an NXP2 C end, the NXP2 N end or the NXP2 C end is found to be precipitated by the NXP2 autoantibody, the to-be-detected serum of the inflammatory myopathies patient is subjected to immunoprecipitation with the HEK293 cell lysis buffer with the overexpressed NXP2 C end, then SDS-PAGE gel electrophoresis is implemented, and the precipitated NXP2 C end is detected by a c-myc antibody to determine whether the NXP2 autoantibody exists in the serum of the inflammatory myopathies patient. The problem that no immunoblotting membrane strip or ELISA (Enzyme-Linked Immuno Sorbent Assay) kit for commercially detecting the NXP2 autoantibody exists in the domestic at present is solved, and the problems of high cost of the ELISA kit autonomously coating an NXP2 antigen, high false positive rate, safety of a radioactive labeling immunoprecipitation method and the like are solved.
Description
technical field [0001] The application belongs to the field of biotechnology, and in particular relates to a non-radiolabeled immunoprecipitation method and application for detecting NXP2 autoantibodies in inflammatory myopathy. Background technique [0002] Idiopathic inflammatory myopathy (idiopathic inflammatory myopathies, IIM) is a group of systemic autoimmune diseases mainly invading skeletal muscle, the abnormality of autoimmunity is the key to the occurrence and development of IIM. According to the latest diagnostic classification criteria, IIM can be divided into polymyositis (PM), dermatomyositis (DM), immune-mediated necrotizing myopathy (IMNM), nonspecific Myositis (nonspecific myositis, NSM) and inclusion body myositis (inclusion body myositis, sIBM). The clinical features of IIM are muscle weakness in the proximal extremities, characteristic rash, systemic damage, and the presence of various autoantibodies in the serum, which are closely related to the clinica...
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