| B lineage acute lymphoblastic leukemia (ALL) is the most common type of hematopoietic malignancies in children. The current primary treatment of childhood ALL is combined chemotherapy. Although the present therapies on childhood ALL are effective, severe toxicity and side effects are still the major complications due to the poor treatment selection of the chemotherapeutic agents. Since 1975 when the technology of hybridoma and monoclonal antibody was developed, many investigators have been paying close attention to antibody targeting therapy for patients with cancer. As compared to conventional chemotherapy, the use of monoclonal antibodies as "targeted" therapy on leukemia cells (biotherapy) is highly selective. It only kills leukemia cells expressing target molecules while leaving those without target antigens unaffected. Therefore targeting therapy can greatly reduce the opportunity of nonspecific toxicity and side effects on normal tissues. But at present, most McAbs with high affinity are mouse origin. The potential of eliciting human anti-mouse antibody (HAMA) responses by using murine antibodies is the major concern from a possible interference with subsequent antibody therapy. With the development of the current molecular biology techniques, the immunogenicity of murine antibodies can be reduced with the recognition capacity of antigens reserved through the DNA recombination technology. To generate the recombinant antibodies, the key point is the cloning and the sequencing of the VH and VL genes from antibody secreting hybridoma cell line.CD19 is continuously and stably expressed on all stages of B lineage differentiation, so it might be an ideal target for antibody-targeting treatment of B lineage ALL. ZCH-4-2E8, a new clone of CD 19 McAb was generated in this lab and assigned into CD 19 category by the 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA6) in 1996.In this study, we will clone and sequence the VH and VL genes from CD19 antibody secreting murine hybridoma cell line ZCH-4-2E8 by a series of molecular biology techniques. The results of this study have provided a better fundamental for generating recombinant antibody of 2E8, such as single chain fragment of variable region, human-mouse chimeric antibody or humanized antibody.Material and Methods1. Cell culture and flow cytometer evaluation of Hybridoma cell line ZCH-4-2E8: The cell lines of 2E8, Nalm-6 were cultured under routine conditions with RPMI 1640 medium supplemented with 20% heat-inactivated calf serum. Flow cytometer (B-D) evaluated the antibody isoforms and its block activity to CD19.2. Isolation of total RNA and mRNA: At lease 106~ 107 fresh hybridoma cells were harvested by centrifugation to isolate the total RNA using TRIZOL and the mRNA using mRNA PolyATtract mRNA Isolation Systems. Concentration of RNA was determined by ultraviolet spectrophotometer.3. RT-PCR amplification of the VH and VL genes from 2E8 hybridoma: First-strand cDNA was synthesized from 0.5 g mRNA using reverse transcriptase M-MLV and an oligo (dT) primer, incubated for 90 minutes at 37. Mix the first strand cDNA with the PCR constituents. Each 50ul reaction contains lug cDNA and 1 unit of high fidelity Taq DNA Polymerase. Amplification consisted of an initial denaturation step of 5 minutes at 95, followed by 35 cycles of 95 for 50 seconds, 58for 55 seconds and 72 for 1 minute. Then add 5 unit of Taq DNA Polymerase for a final extension step of10 minutes at 72. Amplified variable regions were analyzed on a 0.8% agarose gel and visualized after staining with ethidium bromide.4. Cloning and Sequencing of the VH and VL genes: The PCR products were purified by cutting the interested bands of Gel after electrophoresis. Purified products were ligated into pGEM?T Easy vector and E Coli DH5a cells were transformed and plated on LB agar plates before they were incubated overnight to form small colonies. Colonies found to contain an insert of ap... |
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