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.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
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.
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com