Preparation Of Cells Transfected B7-2 And Monoclonal Antibody Against B7-2

The B7 family mainly includes B7-1 and B7-2. B7-2 molecules express on monocytes,dendritc cells, macrophages and other APCs. The receptor of B7 molecules expressed on T cells belongs to CD28 super family.In 1989, Freeman et al first cloned the cDNA of B7-1 and analysised its sequence. Soon,the cDNA of B7-2 was cloned.B7-2 molecules locate at 3q21 with the length of 1120 which encoding 306 aa. B7-2 is involved in the induction of T cell activation and plays a role in costimulatory signals necessary for immunology effects.It also get involved in the pathology of tumor,autoimmune disease,allergic disease and graft rejection. Therefore, the preparation of B7-2 transfected cells,the obtaining of anti-B7-2 monoclonal antibodies may have significant theoretic and clinical values. 1.Cloning of human B7-2 gene and construction of B7-2 transfected cells.cDNA fragment encoding human B7-2 was obtained from the total RNA of mature DCs by RT-PCR and inserted into retrovirus expression vector pEGZ-Term with gene cloning technique. Then the derived recombinant expression vector together with helper virus expression vectors were transfected into packed cell 293T by liposomes to produce recombinant retrovirus, which was used to infect L929 cells for 72h. The transfected L929 cell expressing B7-2 stably was obtained through Zeocin selection and grew well after long time passaging in vitro and storage in liquid nitrogen. The expression of B7-2 on cell surface stably maintains above 97%.2.Preparation of monoclonal antibodies (mAb) against B7-2. 2.1Establishment of hybridoma cells linesBALB/c mice were immunized with human B7-2 transfected L929 cells as animmune antigen and the splenocytes were fused with murine myeloma SP2/0 cells by the cell fusion hybridoma technique. By means of multiple cell subcloning and repeated screening with L929/B7-2 as antibody screening positive cell while L929/mock as negative control, five hybridoma cell lines (named as 1F9, 6C8, 3A7, 2H6JH9) stably secreting anti-B7-2 monoclonal antibodies were selected out. Then the Ig isotypes were identified with test paper. The isotope of 1F9 was mouse IgGl while the isotopes of 6C8,2H6,7H9 were IgG2b and the isotope of 3A7 was IgG2a. The hybridoma cells grew well after long-term culture in vitro and storage in liquid nitrogen. 2.2Production and purification of the mAbThe ascites were produced according to the ascites-inducing procedure in mouse abdominal cavity. The ratio of ascites formation was above 95 percent and the average yield is about 4 milliliter (ml) per mouse. After the ascites were purified by protein G affinity chromatography, the protein content was between 0.8 and 5 milligram(mg)/ml with 1:10000 dilution by FACS. And the usuage of doing FACS was 2.0 microgram (ug)/5><105 cells. 3.The biological characteristics of the mAb 3.1 Antigen epitope recognized by B7-2 mAbIn the competition test with PE-conjugated standard CD86mAb(HA5.2B7),lF9^ 3A7 could completely block the binding of standard mAb to B7-2 molecule on L929/B7-2 cells.lt suggested that these mAbs recognized the same epitope from that of standard mAb.6C8 and 7H9 could not block the binding of standard mAb to B7-2 molecule on L929/B7-2 cells. It suggested that these mAbs recognized the different epitope from that of standard mAb(HA5.2B7).By the same way,it was resulted that 2H6 recognized the similar epitope. 3.2The recognization of membrane B7-2 by B7-2 mAbDaudi and Raji cells were incubated with the five mAbs respectively, washed and added PE-conjugated CD86 mAb(HA5.2B7),then analysised by FACS.L929/B7-2 cells were used as positive control and Jurkat cells were used as negative control.The results suggested that the five antibodies all can recognize the B7-2 molecules on different cells.3.3The biological effects of these mAbs on the Daudi cellsThe B7-2 mAb with different concentrations was added to the culture system of Daudi cells constantly expressing B7-2 molecules, we observed cell growth ,counted cell number and described growth at 24h,48h,72h and 96h respectively. The result showed that these mAbs caused the proliferation suppression of Daudi.After 24h hours of treatment, apoptosis was detected by using annexin-V staining.The results indicated the these mAbs(1F9,6C8) might have the capacity of killing tumor cells.