Method for rapidly acquiring nano antibody by using high-flux sequencing technique

A nanobody, high-throughput technology, applied in the direction of recombinant DNA technology, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of difficult antibody libraries, and achieve good antigen binding, simple methods, and wide application Effect

Inactive Publication Date: 2017-06-20
康众(北京)生物科技有限公司
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  • Abstract
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Problems solved by technology

However, there are still some problems in the phage antibody library technology, such as the difficulty in constructing a large and diverse antibody library, long-term preservation of the antibody library, and the decline in diversity needs to be improved

Method used

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  • Method for rapidly acquiring nano antibody by using high-flux sequencing technique
  • Method for rapidly acquiring nano antibody by using high-flux sequencing technique
  • Method for rapidly acquiring nano antibody by using high-flux sequencing technique

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Embodiment Construction

[0037] The invention will be described in further detail below with specific embodiments

[0038] The present invention has been described with reference to specific embodiments, however, it will be apparent that various modifications and changes can be made without departing from the spirit and scope of the present invention. Accordingly, the description and drawings of the present invention are to be regarded as illustrative rather than restrictive.

[0039]The technical process of obtaining CD3e nanobody in the present invention is an example to illustrate the method of rapidly obtaining nanobody by flow cytometry and high-throughput sequencing technology:

[0040] 1. Isolation of PBMC cells

[0041] 1. Using conventional methods to immunize alpacas with CD3e protein, and obtain venous blood before and after immunization. The specific blood collection steps are as follows: take 30 mL of blood from the vein, and put it into a pre-prepared 50 mL centrifuge tube with anticoa...

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Abstract

The invention provides a method for rapidly acquiring a nano antibody by using a high-flux sequencing technique. With the combination of a flow cytometry separation technique and a high-flux sequencing technique, the method comprises the following steps: acquiring alpaca peripheral blood mononuclear cells (PBMC) which are immunized or unimmunized by using a specific protein W; acquiring PBMCs which are specified for the specific protein W by using a flow cytometry separation method; extracting total RNA (Ribonucleic Acid) of unimmunized, immunized and separated PBMCs, and performing inverse transcription into cDNA (Complementary Deoxyribonucleic Acid); by taking the cDNA as a template, performing two-step method PCR (Polymerase Chain Reaction) amplification so as to obtain DNA fragments of a specific protein W resistant nano antibody; establishing a DNA library of the DNA fragments of the nano antibody, and performing high-flux sequencing analysis; and analyzing the abundance of the DNA fragments of the nano antibody; establishing nucleotide sequences of the DNA fragments of the nano antibody into an expression vector, and expressing the DNA fragments of the nano antibody in appropriate hosts.

Description

technical field [0001] The invention belongs to the technical field of antibodies, in particular to a method for rapidly obtaining nanobodies by using high-throughput sequencing technology. Background technique [0002] Antibodies are immunoglobulins produced by plasma cells differentiated from B cells under the stimulation of antigenic substances, and can specifically bind and react with corresponding antigens. [0003] In 1993, Hamers Casterman of Belgium reported that not only IgG1 conventional antibodies composed of 2 heavy chains (H chain) and 2 light chains (L chain) existed in camel serum, but also IgG2 and IgG3 subtypes lacking light chains The heavy-chain antibody (heavy-chain antibody, hcAb), IgG2 and IgG3 subtypes have full antigen-binding ability. These heavy chain antibodies (HcAbs) account for approximately 50% of the total Ig in Lama serum and approximately 75% of total Ig in Camelus serum. Heavy chain antibodies can bind some epitopes that are inaccessible ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/70C12N15/10C12Q1/68
CPCC12Q1/6869C07K16/18C07K2317/569C12N15/1096C12N15/70C12N2800/101C12Q2535/122
Inventor 李福胜庄文超李洪利覃海兰
Owner 康众(北京)生物科技有限公司
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