Anti human CD19 mouse immune globulin variable zone gene and uses
An immunoglobulin, variable technology, applied in the biological field, which can solve problems such as weak antigenicity
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Embodiment 1
[0018] Nucleotide sequence and amino acid sequence of two genes of the present invention:
[0019] 1. Anti-human CD19 mouse immunoglobulin heavy chain variable region gene (mAb ZCH-4-2E8 VH 2E8 ) nucleotide sequence (SEQ ID NO 1) and amino acid sequence (SEQ ID NO 3).
[0020] 2. Anti-human CD19 mouse immunoglobulin light chain variable region gene (antigen monoclonal antibody ZCH-4-2E8 VL 2E8 ) nucleotide (SEQ ID NO 2) and amino acid sequence (SEQ ID NO 4).
Embodiment 2
[0022] The two anti-human CD19 mouse immunoglobulin variable region genes provided by the present invention are obtained through the following steps:
[0023] 1. The development of ZCH-4-2E8 monoclonal antibody: basically according to the classical method of mouse-mouse hybridoma reported by Koller & Milstein, using children's acute undifferentiated lymphocytic leukemia (uALL) cells (the immunophenotype of which is HLA-DR+, CD19+, CD10-, CD33-, TdT+, CD7-, CD41a-) as immunogen, 10 7Leukemic cells were injected intraperitoneally into 8-week-old female Balb / C mice, once a week, 4 times in total, and on the 3rd day after the fourth injection, the mice were killed by dislocation. The NS-1 cells of the mouse myeloma cell line at stage 1 were mixed at a ratio of 6:1, and the cells were fused with 50% polyethylene glycol (PEG, Sigma Company, USA, molecular weight 3350 Daltons) solution as a fusion medium, and placed in a 96-well plate. (Falcon, USA) for selective culture. On the 9t...
Embodiment 3
[0080] In order to confirm the potential clinical application prospect of 2E8 antibody, we coupled the purified 2E8 mouse-derived intact antibody with trihydroxyisoflavone (Genistein, Gen for short) to make the immunotoxin (2E8-Gen) of 2E8 antibody, and observed its effect on 2E8 antibody. Positive (acute B-lymphoblastic leukemia, Nalm-6) and negative (acute T-cell leukemia cell line Mplt-3 and myeloid leukemia cell line) cells were targeted for killing, and it was found that 2E8-Gen had no effect on Nalm-6 cells (B-cell leukemia / lymphoma cell line) has a strong selective killing effect. After incubation at 37°C for 48 hours, the cell survival rate of the 2E8-Gen group was only 5%, which was significantly higher than that of the control group, and the 2E8-Gen -Gen has no killing effect on 2E8-negative Molt-3 and K562 cells, indicating that 2E8-Gen has a good targeting effect on killing B-cell leukemia cells (see Figure 7 ), where PBS is phosphate buffered saline, Gen is free ...
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