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Anti-human CD25 chimeric monoclonal antibody and preparation method and application thereof

A monoclonal antibody, chimeric antibody technology, applied in the preparation of antibodies, anti-human CD25 chimeric monoclonal antibody and its preparation field, can solve problems such as inability to regulate T cell proliferation

Active Publication Date: 2018-05-29
SUZHOU BRIGHT SCISTAR ANTIBODY BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The cytoplasmic region of the IL-2 receptor α chain is short, and the dimer form of IL-2Rβ and IL-2Rγ is a necessary structure for the IL-2 signaling pathway, but only the dimer form of IL-2Rβ and IL-2Rγ In the presence of IL-2, IL-2 cannot effectively regulate the proliferation of T cells through the IL

Method used

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  • Anti-human CD25 chimeric monoclonal antibody and preparation method and application thereof
  • Anti-human CD25 chimeric monoclonal antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Obtaining of Hybridomas Producing CD25 Monoclonal Antibodies

[0053] (1) Immune mice

[0054] Mice were immunized with fusion protein or transgenic cells, immunized four times with an interval of 21 days each time, and the orbital blood titer of the mice was measured 7-10 days after the fourth immunization.

[0055] (2) Cell culture

[0056] 1. One day before fusion, take one BALB / c mouse aged 6-7 weeks and place it in 75% ethanol solution for 2 minutes.

[0057] 2. The mouse spleen was aseptically taken out, placed in a 200-mesh stainless steel screen, and ground to obtain a single cell suspension. Wash twice with 1640 basal medium (1400rpm, 5min) for later use. Use 15% FBS 1640 medium to adjust the cell concentration to 2×10 5 / ml, drop into 96-well culture plate, 100μL per well, 37℃, 5%CO 2 cultured in an incubator.

[0058] 3. Cultivate overnight, and observe under a low-power microscope the next day. And spread subcloned cells 150ul.

[0059] (3)...

Embodiment 2

[0073] Example 2 CD25 mouse / human chimeric antibody acquisition method

[0074] 1. Extract the cDNA of hybridoma cells: extract RNA from the hybridoma cell line, and use RT-PCR technology to reverse-transcribe the obtained RNA into cDNA; use specially designed upstream and downstream primers to PCR clone the hybridoma cells chain variable region (mVH) and light chain variable region (mVL);

[0075] 2. mVH and mVL were respectively connected to the cloning vector (pJET cloning vector), and the connection product was transformed into competent bacteria DH5a. Since the pJET vector carries the ampicillin (Amp+) resistance gene, the transformed bacteria can be coated on the Amp-resistant LB solid culture medium, 37 ° overnight culture;

[0076] 3. The bacteria to be plated grow scattered colonies, select colonies with clear edges and good growth, and further sequence and identify them;

[0077] 4. According to the sequencing results, the candidate heavy and light chain variable r...

Embodiment 3

[0087] 1. Isolation and extraction of PBMC

[0088] Aseptically draw the blood in the apheresis platelet leukocyte filter, hereinafter referred to as filter blood, and dilute it with PBS 1:20; take normal heparin anticoagulated peripheral blood from the same individual, hereinafter referred to as peripheral blood, and dilute it with PBS 1:2; The above-mentioned diluted filter blood and peripheral blood were separated by conventional Ficoll to obtain PBMC, washed 2 times with PBS, counted, and the cell density was adjusted to 1x106 / ml with RPMI-1640 containing 10% FBS for later use, respectively, whole blood PBMC Cells and filter blood PBMC cells.

[0089] 2. CFSE-labeled cells

[0090] The method of using CFSE labeling to detect the expansion of human peripheral blood T cells in vitro is to take an appropriate amount of separated human peripheral blood mononuclear cells (PBMC) in an appropriate amount of CFSE working solution, mix them gently, and then place them in a water b...

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Abstract

The invention discloses an anti-human CD25 chimeric monoclonal antibody and a preparation method thereof. An antibody variable region coding amino acid sequence, a preparation method and antibody functional identification are included. A heavy chain variable region (mVH) and a light chain variable region (mVL) are extracted from anti-human CD25 chimeric monoclonal antibody secreting hybridoma cells and connected with a pJET cloning vector to transform competent bacteria DH5a, candidate heavy and light chain variable region sequences are retained according to sequencing results, heavy chain andlight chain variable region sequences matched with an expression vector are amplified again through PCR (polymerase chain reaction) amplification, a PCR product is connected with a linear expressionvector subjected to double enzyme digestion pretreatment, a connection product transforms the competent bacteria DH5a, and plasmid extraction is performed after amplification culture. Cotransfection of eukaryotic expression cell strains 293 is realized through the expression vector in connection with target monoclonal antibody heavy chain and light chain variable region genes. Supernatant obtainedin culture contains the target antibody, a flow detection expression monoclonal antibody and PBMC (peripheral blood mononuclear cell) are well combined, the positive rate reaches 95% or above, and the CD25 mouse/human chimeric antibody is obtained.

Description

technical field [0001] The invention relates to a preparation method of an antibody, in particular to an anti-human CD25 chimeric monoclonal antibody and its preparation method and application, belonging to the field of biotechnology. Background technique [0002] CD25 is the α chain of the interleukin 2 (interleukin 2, IL-2) heterotrimeric receptor complex, which forms an IL-2 receptor complex with the IL-2 receptor β chain (CD122) and γ chain (CD132) . IL-2 is interleukin-2 (interleukin-2, IL-2), also known as T cell growth factor (T cell growth factor, TCRF). Activated CD4 + Cytokines produced by Th1 cells with a wide range of biological activities. It can promote the proliferation of Th0 and CTL, so it is an important factor in regulating immune response, and also participates in antibody response, hematopoiesis and tumor surveillance. [0003] The cytoplasmic region of the IL-2 receptor α chain is short, and the dimer form of IL-2Rβ and IL-2Rγ is a necessary structu...

Claims

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Application Information

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IPC IPC(8): C07K16/28A61K39/395A61P35/00
CPCA61K39/00C07K16/2866C07K2317/24C07K2317/56C07K2317/73
Inventor 张学光
Owner SUZHOU BRIGHT SCISTAR ANTIBODY BIOTECHNOLOGY CO LTD
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