Human monoclonal antibody cloning and expression with overlap extension PCR and short DNA fragments

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods Pub Date : 2024-10-22 DOI:10.1016/j.jim.2024.113768

Zachary Ende , Margarita Mishina , Robert C. Kauffman , Amrita Kumar , Rashmi Kumari , Paul R. Knight , Suryaprakash Sambhara

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引用次数: 0

Abstract

Monoclonal antibodies are powerful therapeutic, diagnostic, and research tools. Methods utilized to generate monoclonal antibodies are evolving rapidly. We created a transfectable linear antibody expression cassette from a 2-h high-fidelity overlapping PCR reaction from synthesized DNA fragments. We coupled heavy and light chains into a single linear sequence with a promoter, self-cleaving peptide, and poly(A) signal to increase the flexibility of swapping variable regions from any sequence available in silico. Transfection of the linear cassette tended to generate similar levels to the two-plasmid system and generated an average of 47 μg (14–98 μg) after 5 days in 2 ml cultures with 15 unique antibody sequences. The levels of antibodies produced were sufficient for most downstream applications in less than a week. The method presented here reduces the time, cost, and complexity of cloning steps.

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利用重叠延伸 PCR 和短 DNA 片段克隆和表达人类单克隆抗体。

单克隆抗体是强大的治疗、诊断和研究工具。生成单克隆抗体的方法发展迅速。我们利用合成的 DNA 片段，通过 2 小时的高保真重叠 PCR 反应创建了可转染的线性抗体表达盒。我们将重链和轻链与启动子、自裂肽和聚（A）信号耦合到一个线性序列中，以增加从任何可用序列中交换可变区的灵活性。线性盒转染产生的抗体水平与双质粒系统相似，在 2 毫升培养基中培养 5 天后，平均产生 47 微克（14-98 微克）的 15 种独特抗体序列。在不到一周的时间内，所产生的抗体水平足以满足大多数下游应用的需要。这里介绍的方法减少了克隆步骤的时间、成本和复杂性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of immunological methods 医学-免疫学

CiteScore

4.10

自引率

0.00%

发文量

120

审稿时长

3 months

期刊介绍： The Journal of Immunological Methods is devoted to covering techniques for: (1) Quantitating and detecting antibodies and/or antigens. (2) Purifying immunoglobulins, lymphokines and other molecules of the immune system. (3) Isolating antigens and other substances important in immunological processes. (4) Labelling antigens and antibodies. (5) Localizing antigens and/or antibodies in tissues and cells. (6) Detecting, and fractionating immunocompetent cells. (7) Assaying for cellular immunity. (8) Documenting cell-cell interactions. (9) Initiating immunity and unresponsiveness. (10) Transplanting tissues. (11) Studying items closely related to immunity such as complement, reticuloendothelial system and others. (12) Molecular techniques for studying immune cells and their receptors. (13) Imaging of the immune system. (14) Methods for production or their fragments in eukaryotic and prokaryotic cells. In addition the journal will publish articles on novel methods for analysing the organization, structure and expression of genes for immunologically important molecules such as immunoglobulins, T cell receptors and accessory molecules involved in antigen recognition, processing and presentation. Submitted full length manuscripts should describe new methods of broad applicability to immunology and not simply the application of an established method to a particular substance - although papers describing such applications may be considered for publication as a short Technical Note. Review articles will also be published by the Journal of Immunological Methods. In general these manuscripts are by solicitation however anyone interested in submitting a review can contact the Reviews Editor and provide an outline of the proposed review.