Analysis Of Biological Functions Of Mouse Anti Human CD52 Monoclonal Antibody-HI186 And Construction Of ScFv Based On HI186

Objective:To study the biological functions of mouse anti human monoclonal antibody-HI186 and desire to construct anti-CD52 single chain fragment variable based on HI186 monoclonal antibody (HI186-ScFv).Methods:1. To analyze the affection on some CD52+ leukemia cell lines caused by anti-CD52 monoclonal antibody in vitro, we use trypan-blue dye, MTT and Annexin V antibody to detect the growing inhibition and apoptosis of the cells.2. Amplified both heavy chain and light chain variable region gene of HI186 McAb by RT-PCR, than splice them into ScFv in form of VH-linker-VL.3. That recombinant gene fragment was cloned into plasmid pET22b and induced to express the recombinant protein, HI186-ScFv, by IPTG in E. coli BL21. 4. We purified the output by Ni Sepharose^TM, denatured/re-natured and detected it binding activity through Flow cytometry and western blot.Result:1. HI186 McAb can depress the growth of some CD52⁺ leukemia cell lines such as CEM, HPB-ALL, LCL-H and REH and, also, can induce apoptosis.2. The variable region gene of both HI186 heavy chain (342bp) and light chain (339bp) was cloned.3. The spliced ScFv gene is 759bp long and successfully cloned into pET2b plasmid to construct pET22b/HI186-ScFv expression vector. That can express recombinant protein in E. coli induced by IPTG.4. The recombinant ScFv protein can bind both nature and denature CD52 molecular. It can inhibit the binding of HI186 with the CD52 molecular.Conclusion:HI186 monoclonal antibody can inhibit growth of some CD52⁺ leukemia cell line, through detection of dying by Annexin V, it can induce those cells' apoptosis. After construction of HI186-ScFv, we should keep moving to study its potential usage and reform it for future clinical application.