Method for acquiring B cell antibody gene for resisting N-methyl-D-aspartic acid receptor encephalitis and immune repertoire research of B cell antibody gene

A B cell and gene technology, applied in the field of genetic engineering, can solve the problems of low cost, limited promotion of scientific research projects, and inability to obtain constant region sequences.

Pending Publication Date: 2020-10-23
INST OF PSYCHOLOGY CHINESE ACADEMY OF SCI
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  • Description
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AI Technical Summary

Problems solved by technology

However, these methods have certain applicability and limitations, which are mainly reflected in: 1. Mature commercial platforms (such as 10XGenomics, BD Rhapsody) are mainly suitable for high-throughput single-cell sequencing, and the number of B cells required is often several thousand - tens of thousands, not suitable for samples with a small number of B cells; 2. Very few commercial platforms suitable for a small number of B cells have high sequencing costs (such as C1 platform, the cost price of a single cell is 400-2000 yuan), and there are The method can only obtain the variable region sequence of the antibody, but cannot obtain the constant region sequence, which limits the promotion of some scientific research projects, so there is an urgent need for a low-cost, low-cost, single B-cell human antibody gene variable Methods for Region and Constant Region Amplification

Method used

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  • Method for acquiring B cell antibody gene for resisting N-methyl-D-aspartic acid receptor encephalitis and immune repertoire research of B cell antibody gene
  • Method for acquiring B cell antibody gene for resisting N-methyl-D-aspartic acid receptor encephalitis and immune repertoire research of B cell antibody gene
  • Method for acquiring B cell antibody gene for resisting N-methyl-D-aspartic acid receptor encephalitis and immune repertoire research of B cell antibody gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Isolation of positive single B lymph of cerebrospinal fluid

[0060] Take the freshly extracted cerebrospinal fluid and transport it at 0-4°C, centrifuge it at 1200rpm for 10 minutes immediately, resuspend the cells with 500 microliters of freezing solution (90% fetal bovine serum + 10% dimethyl sulfoxide), and put them into the program Store in a freezer box at -80°C.

[0061] Take 1ml of fetal bovine serum (4°C) + 15ml of 1640 medium (4°C) and mix well before performing flow cytometry, centrifuge at 250g for 5-12 minutes, and discard the supernatant; then add 1ml of cell staining buffer and mix well , adding fluorescent antibody (anti-CD20-dinophyllin chlorophyll protein cyanine series conjugate (Percp / Cy5.5) in proportion, anti-B cell surface molecule CD27 antibody--allophycocyanin (anti-CD27-APC) , anti-B cell surface molecule CD38 antibody -- phycoerythrin (anti-CD38-PE)); incubate at room temperature in the dark for 15-20 minutes; add 2ml of cell staini...

Embodiment 2

[0063] Example 2 Single B Cell Reverse Transcription and BCR / Antibody Gene Amplification

[0064] Place the polymerase chain reaction tube containing the lysate of the separated cells in the polymerase chain reaction apparatus, and incubate at 72°C for 3 minutes; set the heat cover to 75°C, and immediately put it on ice for 1 minute after the lysis is completed.

[0065] Prepare a 6 μl reverse transcription system: 2 μl SuperScript II First-Stand Buffer (5×) for synthesizing the first strand, 2 μl betaine (5 mol / L), 0.9 μl L magnesium chloride (100 mmol / L), 0.25 μL dithiothreitol (100 mmol / L), 0.1 μL TSO primer (100 μM / L), 0.25 μL RNase inhibitor (4U), 0.5 microliters of SuperScriptⅡReverse Transcriptase (20U). Reverse transcription was performed according to the following conditions: 42°C for 60-90 minutes; 50°C for 2 minutes, 42°C for 2 minutes, 10 cycles; 70°C for 15 minutes; 4°C or 12°C, ∞ to obtain cDNA.

[0066] Two rounds of nested polymerase chain reaction were carri...

Embodiment 3B

[0073] Example 3 Bioinformatics Analysis of BCR Sequencing Data

[0074] Using bioinformatics methods, sequence comparisons were carried out, and the first-generation sequencing data of our patients were analyzed, as well as the sequencing data of peripheral blood B cells of 4 healthy Chinese people downloaded from the sequence read archive (SRA) database.

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a method for acquiring single cell of B cell surface receptor (BCR) or an antibody gene of an anti-N-methyl-D-aspartic acid receptor encephalitis patient, and further discloses immune repertoire research of B cells of the anti-N-methyl-D-aspartic acid receptor encephalitis patient. According to a method for amplifying a variable region and a constant region of the single B cell humanized antibody gene and a method based on a single B cell reverse transcription and BCR amplification, the characteristicof antigen-antibody specific binding is used for the first time, a positive B cell combined with a pathogenic antigen NR1 protein subunit for resisting N-methyl-D-aspartic acid receptor encephalitisis screened out in advance by using a flow cytometry, the interference of other non-positive BCR/antibody information is reduced, the unnecessary workload is reduced, the correlation between the finally obtained antibody information and the disease is stronger, the efficiency of capturing the single-cell B cell antibody sequence is higher, the experiment steps are simpler and more convenient, andthe operability is stronger.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for acquiring a B cell antibody gene for anti-N-methyl-D-aspartate receptor encephalitis, and further discloses the anti-N-methyl-D-aspartic acid receptor encephalitis method. Immune repertoire study of B cells from patients with aspartate receptor encephalitis. Background technique [0002] Anti-N-methyl-D-aspartate receptor (N-methyl-D-aspartate-receptor, N-methyl-D-aspartate receptor) encephalitis referred to as anti-N-methyl-D- Aspartate receptor encephalitis, an autoimmune encephalitis, was included on May 22, 2018 by the National Health and Health Commission, the Ministry of Science and Technology, the Ministry of Industry and Information Technology, the State Drug Administration, and the National Center for Disease Control and Prevention. In the "First Batch of Rare Diseases" jointly issued by five departments including the Medical Adminis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/10C12Q1/6848C12Q1/6883G01N33/68G01N33/564
CPCC07K16/286C12Q1/6848C12Q1/6883G01N33/564G01N33/6854G01N33/6893G01N2800/28C12Q2521/107C12Q2531/113C12Q2549/119C12Q2565/125
Inventor 王晶
Owner INST OF PSYCHOLOGY CHINESE ACADEMY OF SCI
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