An anti-human cd25 chimeric monoclonal antibody and its preparation method and application
A monoclonal antibody and variable technology, applied in the field of antibody preparation, anti-human CD25 chimeric monoclonal antibody and its preparation, can solve the problem of inability to regulate T cell proliferation and achieve the effect of promoting T cell proliferation
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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 On the culture medium, cultivate overnight at 37 °;
[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 chai...
Embodiment 3
[0087] 1. Isolation and extraction of PBMC
[0088] Aseptically extract blood from 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. Cells and filter blood PBMC cells.
[0089] 2. CFSE-labeled cells
[0090] Wherein, the method for detecting human peripheral blood T cell expansion in vitro with CFSE labeling is to take an appropriate amount of isolated human peripheral blood mononuclear cells (PBMC) in an appropriate amount of CFSE working solution, mix gently, and then place in a 37°C water bath for 10 Min, after centrifugation, remove the...
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