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High efficient method for obtaining antigen-specific B cells rapidly

A B cell, specific technology, applied in the biological field, can solve problems such as large randomness

Inactive Publication Date: 2012-12-26
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

But the "abundance" of antigen-specific B cells in spleen cells makes the isolation of antigen-specific clones highly random

Method used

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  • High efficient method for obtaining antigen-specific B cells rapidly
  • High efficient method for obtaining antigen-specific B cells rapidly
  • High efficient method for obtaining antigen-specific B cells rapidly

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preparation example Construction

[0058] In the antibody production method of the present invention, the antibody is preferably produced after the step of enriching the clonal population of antigen-specific B cells and the step of culturing. The method includes the steps of amplifying and sequencing antibody variable region genes from one or more isolated antigen-specific cells. The heavy and light chain variable regions, including complementarity determining regions (CDRs), can be sequenced using any method known in the art for sequencing.

[0059] In one embodiment, the invention provides a method comprising the steps of:

[0060] a) preparing a population of cells comprising at least one antigen-specific B cell;

[0061] b) enriching the cell population, for example by flow cytometric sorting, to form an enriched cell population comprising at least one antigen-specific B cell;

[0062] c) isolating a single B cell from the enriched B cell population; and

[0063] d) Determining whether individual B cells...

Embodiment 1

[0073] Embodiment 1, HA protein expression and purification

[0074] According to the 2009 influenza A H1N1 virus (A / California / 7 / 2009 (H1N1), purchased from Hualan Bioengineering Co., Ltd.) HA protein genome sequence, an expression vector was constructed using baculovirus insect cells (Bac-to- Bac) protein expression system for expression. details as follows:

[0075] 1. Obtain the target gene template

[0076] The GenBank number of HA is FJ966082.1 (the target gene template was synthesized by TAKARA Company).

[0077] 2. Design and synthesis of primers, adding enzyme cutting sites (EcoRI / XhoI);

[0078] According to the principle of primer design, primers were designed using Primer premier5.0 software, and the primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The sequences are:

[0079] HA-SP: 5'-CCG TACTTCCGTTATGATCATC-3' (SEQ ID NO: 1)

[0080] HA-AS: 5'-CCG TTAAATACATATTCTAC-3' (SEQ ID NO: 2)

[0081] The boxes in the primer sequences represen...

Embodiment 2

[0132] Embodiment 2, HA protein biotin labeling and detection

[0133] 1. HA protein biotin labeling

[0134] (1) Calculate the amount of biotin reagent added according to the Sulfo-NHS-LC-Biotin manual (Pierce): According to the calculation of 20 mol of biotin per mol of protein, 1ml of 0.3mg / ml HA protein requires the amount of biotin (10mM) reagent:

[0135]

[0136] Prepare biotin (Biotin Reagent) reagent: add 180 μl ddH to Biotin Reagent (Pierce) powder 2 O, obtain the Biotin Reagent of 10mM;

[0137] (2) Add 8.6μl Biotin Reagent to 0.3mg / ml 1ml HA protein, react at 4℃ for 2h;

[0138] (3) Use a 10KD ultrafiltration tube to remove excess Biotin Reagent, 4°C, 600g, 5min;

[0139] (4) Congruently label the SARS S protein receptor binding domain (RBD) protein (purchased from Shanghai Zhongke Yingmu Biotechnology Co., Ltd.; its amino acid sequence can be found in GenBank accession number: GenBank: FJ211860.1), as an isotype control.

[0140] 2. ELISA to detect whether ...

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Abstract

The invention relates to a high efficient method for obtaining antigen-specific B cells rapidly. Surface markers especially suitable for indentifying B cells are provided. B cells having the antibody which can combine with the antigen by using the specific antigen are screened out. By using the method of the invention, antigen-specific B cells can be rapidly obtained in high efficiency.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to a method for rapidly and efficiently obtaining antigen-specific B cells. Background technique [0002] Methods of isolating, culturing and identifying B cells that produce antibodies that specifically bind to a target antigen are very important in research in this field. Such B cells can be used to prepare antigen-specific antibodies and recover nucleic acid sequences encoding such antibodies, and can also be used for functional assays such as analysis of antigen-binding properties. [0003] Antibodies are produced by B cells and can be used to identify exogenous or endogenous antigens, such as toxins, bacteria, viruses, cytokines, enzymes, etc. Each antibody binds to a specific epitope of an antigen. The ability of antibodies to recognize and bind to specific epitopes makes antibodies useful therapeutic and diagnostic tools. [0004] The method currentl...

Claims

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Application Information

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IPC IPC(8): C12N5/0781C12N15/13C12N15/63C07K16/08C07K16/10
Inventor 孙兵陈爱中边超王凌凌
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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