Non-radioactive label immunoprecipitation method for detecting specific self-antibody MDA5 of inflammatory myopathy

Abstract

This application discloses a non-radiolabeled immunoprecipitation method for detecting inflammatory myopathy-specific autoantibody MDA5. The present invention provides the following method: constructing a plasmid containing the MDA5 C-terminus (447‑1025aa) and the N-terminus with a Myc tag HEK293 cells were transiently transfected with pCMV‑myc‑MDA5C, and serum from patients with inflammatory myopathy was immunoprecipitated with HEK293 cell lysates overexpressing the MDA5 C-terminus, followed by SDS‑PAGE gel electrophoresis and detection with c‑myc antibody Precipitated MDA5C-terminus solves the problem that there are no commercial ELISA kits for detecting MDA5 antibodies and western blot strips in China, as well as the high cost, high false positive rate and radiolabeling of ELISA kits that self-coat MDA5 antigens The safety of immunoprecipitation and other issues.

Description

technical field

[0001] The application belongs to the field of biotechnology, and specifically relates to a non-radiolabeled immunoprecipitation method and application for detecting inflammatory myopathy-specific autoantibody MDA5. 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), non-specific muscle inflammation (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...

Examples

Embodiment 1

[0027] Example 1 Non-radiolabeled immunoprecipitation method for detecting inflammatory myopathy-specific autoantibody MDA5

[0028] (1) Design primers MDA5-F (5'-TTCGGTCGACCAACAAAGAAGCAGTGTA-3') and MDA5-R (5'-GCGGTACCCTAATCCTCATCACTAAATAAACAG-3') to amplify the C-terminus of human MDA5 from the pENTER-MDA5 plasmid (Genbank: NM022168, 1743bp- 3479bp)( figure 1 a).

[0029] (2) Recover and purify the PCR fragment, perform double digestion with SalI and KpnI restriction endonucleases, connect overnight with the pCMV-myc vector that has been double-digested with SalI and KpnI, and transform JM109 bacteria competently, in the presence of 50ug / ml ampicillin Positive clones were screened on a penicillin plate, and the plasmid DNA of the positive clone was extracted in a small amount, and the plasmid DNA was digested with SalI and KpnI, and the results showed that the plasmid contained a 1.7kb insert ( figure 1 b).

[0030] (3) The plasmid DNA is sequenced to confirm the accuracy...

Embodiment 2

[0033] Example 2 detects the positive rate of the MDA5 antibody in the serum of 50 patients with inflammatory myopathy

[0034] (1) Inoculate 30% HEK293 cells on 75cm 2 37 culture dishes for less than 24 hours;

[0035] (2) Transfect HEK293 cells with 30ug pCMV-myc-MDA5C plasmid for 48 hours;

[0036] (3) HEK293 cells were lysed with 1 ml of RIPA lysate containing protease inhibitors, and the protein was quantified;

[0037](4) The c-myc antibody was used to confirm the overexpressed MDA5 C-terminus in HEK293 cells.

[0038] Take 20ul of the test serum, positive serum, negative serum and cell lysate overexpressing the MDA5 C-terminus of patients with inflammatory myopathy for immunoprecipitation, perform SDS-PAGE gel electrophoresis, and then detect with c-myc antibody to determine the MDA5 antibody The positive rate in patients with inflammatory myopathy is about 10% ( Figure 6 For some patients with inflammatory myopathy detected).

technical field

[0001] The application belongs to the field of biotechnology, and specifically relates to a non-radiolabeled immunoprecipitation method and application for detecting inflammatory myopathy-specific autoantibody MDA5. 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), non-specific muscle inflammation (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...

Examples

Embodiment 1

[0027] Example 1 Non-radiolabeled immunoprecipitation method for detecting inflammatory myopathy-specific autoantibody MDA5

[0028] (1) Design primers MDA5-F (5'-TTCGGTCGACCAACAAAGAAGCAGTGTA-3') and MDA5-R (5'-GCGGTACCCTAATCCTCATCACTAAATAAACAG-3') to amplify the C-terminus of human MDA5 from the pENTER-MDA5 plasmid (Genbank: NM022168, 1743bp- 3479bp)( figure 1 a).

[0029] (2) Recover and purify the PCR fragment, perform double digestion with SalI and KpnI restriction endonucleases, connect overnight with the pCMV-myc vector that has been double-digested with SalI and KpnI, and transform JM109 bacteria competently, in the presence of 50ug / ml ampicillin Positive clones were screened on a penicillin plate, and the plasmid DNA of the positive clone was extracted in a small amount, and the plasmid DNA was digested with SalI and KpnI, and the results showed that the plasmid contained a 1.7kb insert ( figure 1 b).

[0030] (3) The plasmid DNA is sequenced to confirm the accuracy...

Embodiment 2

[0033] Example 2 detects the positive rate of the MDA5 antibody in the serum of 50 patients with inflammatory myopathy

[0034] (1) Inoculate 30% HEK293 cells on 75cm 2 37 culture dishes for less than 24 hours;

[0035] (2) Transfect HEK293 cells with 30ug pCMV-myc-MDA5C plasmid for 48 hours;

[0036] (3) HEK293 cells were lysed with 1 ml of RIPA lysate containing protease inhibitors, and the protein was quantified;

[0037](4) The c-myc antibody was used to confirm the overexpressed MDA5 C-terminus in HEK293 cells.

[0038] Take 20ul of the test serum, positive serum, negative serum and cell lysate overexpressing the MDA5 C-terminus of patients with inflammatory myopathy for immunoprecipitation, perform SDS-PAGE gel electrophoresis, and then detect with c-myc antibody to determine the MDA5 antibody The positive rate in patients with inflammatory myopathy is about 10% ( Figure 6 For some patients with inflammatory myopathy detected).

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