Method for sorting and screening nano antibody through flow cytometry

Abstract

The invention discloses a method for sorting and screening nano antibody through flow cytometry. The method comprises the following steps: (1) collecting peripheral blood B lymphocyte of a camelidae animal with immune target antigen; (2) sorting the B lymphocyte as a single cell by applying target antigen through the flow cytometry; (3) directly performing reverse transcriptional reaction on the sorted single B lymphocyte to generate cDNA; (4) taking the cDNA as a template, performing PCR amplification on an antibody heavy chain sequence and recycling an amplified product; (5) taking the amplified product in the step (4) as a template, and performing PCR amplification on CH1 and CH2 sequence coding genes of the antibody; (6) taking the amplified product in the step (4) which is amplified to be negative in the step (5) as a template, and performing PCR amplification on a VHH segment coding gene of the antibody; (7) cloning a VHH segment obtained in the step (6) into an expression vector, and expressing the VHH segment in host bacteria; and (8) identifying the nano antibody which is pressed in the step (7). The method disclosed by the invention is simple to operate, is short in screening period, avoids phage pollution risk, improves screening efficiency, reduces workload greatly, is low in economic cost, and is suitable for industrial large-batch screening requirements.

Description

technical field [0001] The invention discloses a method for screening nanobodies. Background technique [0002] In 1993, Hamers-Casterman et al. found that a class of only heavy chain dimer antibody H was found in camelids (camel, dromedary and llama). 2 , which are mainly IgG2 and IgG3 types. Such antibodies are called heavy chain-only-like antibodies (HCAbs) due to the lack of light chains, and their antigen-binding sites are composed of a domain called the VHH region, so Such antibodies are also referred to as single domain antibodies or single domain antibodies (sdAbs). Because this type of antibody is the variable region sequence after the constant region is removed, the molecular weight is only 15kD, and the diameter is about 10 nanometers, so it is also called nanobody (Nbs). Additionally, this class of single-domain antibodies, known as VNARs, has also been observed in sharks. This heavy-chain-only antibody was originally recognized only as a pathological form of...

AI Technical Summary

Problems solved by technology

However, due to the steps of RNA extraction and reverse transcription, and RNA is a substance that is easily contaminated and degraded, it is difficult to ensure a high enough screening library capacity, so that subsequent reverse transcription and PCR amplification are easily affected by template specificity. and the impact of quantity, resulting in failure of screening or extension of screening cycle

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