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Detection method of aquaporin-4 autoantibody, fusion expression virus vector and application thereof

An aquaporin, fusion expression technology, applied in the field of flow cytometry detection of aquaporin-4 autoantibodies, can solve the problems of large loss of human and material resources, unstable fluorescence efficiency, long time consumption, etc., to improve the diagnostic level, improve the Clinical diagnosis and treatment level, the effect of saving manpower and time cost

Inactive Publication Date: 2014-07-23
GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It takes a long time, consumes a lot of manpower and material resources, and the fluorescence efficiency is unstable

Method used

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  • Detection method of aquaporin-4 autoantibody, fusion expression virus vector and application thereof
  • Detection method of aquaporin-4 autoantibody, fusion expression virus vector and application thereof
  • Detection method of aquaporin-4 autoantibody, fusion expression virus vector and application thereof

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Experimental program
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Effect test

Embodiment 1

[0051] An aquaporin-4 autoantibody fusion expression virus vector, the expression virus vector is pCDH-MCS-AQP4-EF1-copGFP with the sequence shown in SEQ ID NO.2, the construction method:

[0052] 1) Vector construction: construction of a lentiviral vector mediated by a strong constitutive promoter CMV for fusion expression of human AQP-4 gene and fluorescent marker gene EGFP;

[0053] The human AQP-4 gene without a stop codon is connected to the EGFP gene with a flexible sequence (GGGGS) composed of 5 amino acids, AQP-4-GGGGS-EGFP (as shown in SEQ ID NO.1), EGFP is in the entire fusion protein The N-terminus of the AQP-4 protein to ensure the topology, subcellular location and relatively independent functions of the two proteins in the natural state of the AQP-4 protein;

[0054] The EGFP sequence was amplified on the pEGFP-N1 vector, and the primers used were as follows:

[0055] EGFPF: 5'- CTA GGATCC ATGGTGAGCAAGGGCGAGGAG-3' and EGFPR: 5'- CAG GTCGAC GAATTC GCGGCCGC CTCGAG...

Embodiment 2

[0061] A method of detecting the presence of aquaporin-4 autoantibodies in a biological sample from an individual:

[0062] 1) Vector construction: construction of a lentiviral vector mediated by a strong constitutive promoter CMV for fusion expression of human AQP-4 gene and fluorescent marker gene EGFP;

[0063] The human AQP-4 gene without a stop codon is connected to the EGFP gene with a flexible sequence (GGGGS) composed of 5 amino acids, AQP-4-GGGGS-EGFP (as shown in SEQ ID NO.1), EGFP is in the entire fusion protein The N-terminus of the AQP-4 protein to ensure the topology, subcellular location and relatively independent functions of the two proteins in the natural state of the AQP-4 protein;

[0064] The EGFP sequence was amplified on the pEGFP-N1 vector, and the primers used were as follows:

[0065] EGFPF: 5'- CTA GGATCC ATGGTGAGCAAGGGCGAGGAG-3' and EGFPR: 5'- CAG GTCGAC GAATTC GCGGCCGC CTCGAG CTGCAG TTACTTGTACAGCTCGTCCATGCCG-3', the amplified fragment was gel-cut ...

Embodiment 3

[0075] Actual application:

[0076] Research objects: 58 NMO patients, 54 MS patients, and 22 other neurological diseases (OND) patients were selected from the Department of Neurology, Tianjin Medical University General Hospital from January 2010 to December 2012.

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Abstract

The invention discloses a detection method of an aquaporin-4 autoantibody, a fusion expression virus vector and application thereof. The virus vector is pCDH-MCS-AQP4-EF1-copGFP, the nucleotide sequence of which is shown as a sequence table SEQIDNO.2. The invention further discloses application of the fusion expression virus vector in improving the clinical diagnosis and treatment level of ophthalmoneuromyelitis and multiple sclerosis. By adopting immunofluorescent cytochemical staining with higher sensitivity and specificity combined with a flow cytometric detection technology, the AQP-4 antibody is quantitively and qualitatively detection clinically, so that the detection result is more stable, and the labor and the time cost are extremely saved. The project is established to extremely improve the diagnosis and treatment level of ophthalmoneuromyelitis and multiple sclerosis, so that the labor capacity and the living quality of the patient are improved, and huge social and economic benefits are generated.

Description

technical field [0001] The invention relates to a method for detecting aquaporin-4 autoantibody by flow cytometry and its fusion expression virus vector. Aquaporin (AQP)-4 antibody positive is an important supporting condition for the diagnosis of neuromyelitis optica, and the antibody titer is correlated with the severity, prognosis, and recurrence of the patient's condition. The invention belongs to the field of molecular identification and diagnosis of diseases, combined with virus vector construction, transfection, fluorescent immunostaining and flow cytometric analysis technology, mainly applied to the clinical differential diagnosis of NMO and MS, improving the diagnostic level of NMO and MS; can assist as Medication efficacy, disease monitoring, and recurrence prediction indicators; the establishment of the AQP-4 detection platform can provide a basis for further research on the pathogenic mechanism of AQP-4 antibodies. Background technique [0002] Multiple scleros...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66G01N33/68G01N15/14
Inventor 闫亚平施福东金薇娜李敏淑齐媛
Owner GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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