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Humanized anti-CD25 single-chain antibody and preparation method

A single-chain antibody, humanized technology, applied in biochemical equipment and methods, botanical equipment and methods, microorganism-based methods, etc., can solve the problems of inactivity and low yield, and achieve easy mass production and purification. Effect

Inactive Publication Date: 2009-09-30
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the Pichia pastoris expression system is not necessarily suitable for the expression of all proteins, and although some proteins are successfully expressed, the yield is very low or there is no activity [Hackel BJ, Huang D, Bubolz JC, Wang XX, Shusta EV. Production of soluble and active transfer rinreceptor -targeting single-chain antibody using Saccharomyces cerevisiae. Pharm Res. 2006 Apr;23(4):790-7.]

Method used

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  • Humanized anti-CD25 single-chain antibody and preparation method
  • Humanized anti-CD25 single-chain antibody and preparation method
  • Humanized anti-CD25 single-chain antibody and preparation method

Examples

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Effect test

Embodiment 1

[0035] [Example 1] Obtaining of humanized anti-CD25 single-chain antibody Dmab (scFv)

[0036] 1. Gene design and synthesis

[0037] The gene of Dmab (scFv) is based on the heavy chain variable region (V H ) and the light chain variable region (V L ) amino acid sequence (Naoya Tsurushita, Paul R. Hinton, Shankar Kumar Design of humanized antibodies: From anti-Tac to Zenapax. Methods, 2005, 36: 69-83), with V H -(Gly 4 Ser) 3 -V L pattern designed. 6His-Tag was introduced at the 3' end of the synthetic gene for detection and purification. At the same time, in order to facilitate cloning into yeast expression vectors, EcoRI and NotI restriction sites were added to the 5' and 3' ends respectively during synthesis. The designed Dmab (scFv) nucleotide and its amino acid sequence are as follows figure 1 As shown, the total gene synthesis was carried out by Invitrogen Company.

[0038] 2. Construction of Yeast Expression Plasmids

[0039] According to the conventional mole...

Embodiment 2

[0049] [Example 2] Humanized anti-CD25 single-chain antibody Dmab (scFv) binding function detection

[0050] 1. FITC labeling of protein

[0051] To identify the binding activity of the produced Dmab(scFv), we first labeled the purified protein with FITC. Refer to Pierce EZ-Label TM FITC Protein Labeling Kit method, the target protein dissolved in PBS with 1M Na 2 CO 3 Adjust the pH to 8.5, and then add 10 mg / ml FITC (dissolved in dimethylformamide) to make the molar ratio of protein to FITC 1:24. After mixing, incubate at room temperature for 1 h in the dark. After dialysis against PBS to remove excess FITC, centrifuge at 20 kg at 4°C for 10 min to remove inactive protein that may precipitate, and the supernatant is labeled Dmab(scFv)-FITC.

[0052] 2. Binding capacity and specificity analysis

[0053] For the binding ability and specificity of Dmab (scFv) to CD25 antigen, we use CD25 positive and negative cell lines or cells with different expression levels to analyze...

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Abstract

The invention provides a single-chain antibody established based on humanized anti-CD25 monoclonal antibody daclizumab, and also provides a nucleotide molecule encoding the single-chain antibody and a method for preparing the single-chain antibody. The preparation method can obtain soluble activated proteins which are easy to produce massively and purify.

Description

technical field [0001] The present invention relates to an antibody, in particular to a single-chain antibody. [0002] The present invention also relates to expression vectors and host cells expressing the single-chain antibody. [0003] The present invention also relates to the preparation method of the single-chain antibody. Background technique [0004] CD25 is the α subunit of the IL-2 receptor. It is not expressed or expressed at a very low level on resting T cells, but it is highly expressed on activated T cells and becomes one of the signs of activated T cells, so it is also called Tac antigen (T cell activation antigen) [Robb RJ, Kutny RM. Structure-function relationships for the IL-2 receptor system. J Immunol 1987; 139: 855-62]. Anti-CD25 antibodies can block CD25, block IL-2 signal transmission, and inhibit T cell activation. For example, anti-CD25 monoclonal antibody anti-Tac can significantly reduce the incidence of transplant rejection [Kirkman RL, Saqiro ME...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N15/81C12N1/19C12R1/84
Inventor 万琳卢晓风程惊秋蔡华伟陆燕蓉李胜富张杰李幼平步宏
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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