Construction And Expression Of Human-Mouse Chimeric Antibody Against Human CD86 And Characterization Of Its Biological Function

On the basis of obtained murine hybridoma cell 1D1 secreting anti-CD86 monoclonal antibody(mAb),genetic technology was used in the study for transformation of mouse anti-CD86 mAb.Expression plasmid of Human -mouse chimeric antibody against CD 86 had been established and successfully been transfected into CHO cells.Cell line which can constantly and stably secret anti-CD86 chimeric antibody has been obtained.Through purification of cell culture supernatant,purified chimeric antibody has been harvested which is necessary for the study of tumor immunology therapy on targeted CD86 molecule.The study is expected to make a breakthrough in the R&D field of therapic mAb.In the partⅠ,total RNA was extracted from the murine hybridoma cell line(1D1)secreting anti-CD86 monoclonal antibody.After the V_H and V_L genes were amplified by RT-PCR, V_H and V_L genes containing signal peptide sequences were amplified by SMART-PCR using a pair of specific primers.Then V_H,V_L genes containing signal peptide sequences and constant regions of human immunoglobulin IgG1 were fused to construct chimeric heavy chain and light chain respectively by TP-PCR.To investigate correction of gene sequence,all sequences were sequenced and analyzed by automatic DNA sequencer.The genes of chimeric heavy chain and light chain were subcloned into plasmid pIRES to construct co-expressing recombinant plasmid.The restriction endonuclease digestion and PCR showed that the recombinant genes were cloned into vector pIRES.Using LipofectAMIN kit,the recombinant plasmid and mock plasmid were transfected into 293T cell line respectively,supernatants of cells culture were collected to be characterized through FACS using human CD86-L929 and mock-L929 cell line,the data showed that the human-mouse chimeric antibody against CD86 was expressed in 293T cell line successfully,human-mouse chimeric antibody against CD86 maintained the binding activity and specificity to human CD86 molecule.To express the chimeric antibody in eukaryotic expression system,we chose the CHO cell as parental cell,which could ensure natural spatial conformations and biological functions of protein.We expressed a chimeric antibody against CD86 molecule in CHO cell stably and effectively.First,Human-mouse chimeric antibody against CD86 recombinant plasmid and mock plasmid were transfected into CHO cell line using LipofectAMIN kit, respectively.The results of RT-PCR showed target CHO cells contained chimeric heavy chain and chimeric light chain gene.The combination rate between expressed anti-CD86 chimeric antibody and CD86 gene-transfected cells was 98.5%.The concentration of human-mouse chimeric antibody against CD86 in cell supernatants was 3.0mg/L.The human-mouse chimeric antibody against CD86 was expressed in CHO stably and effectively,and was humanized successfully.In the partⅡ,through indirect immunofluorescence and FCM,we analyzed on various tumor cell lines,including Daudi,Sub-T,U937,Jurkat,HO8910,H1299 and WERI-RB1.On the basis of analysis results,we choose B lymphoma cell line Daudi with natural high expression of CD86 and CD80 as targeted cell.We observed and analyzed the effectiveness on growing and proliferation of Daudi using anti-CD86 chimeric antibody alone and in combination with anti-CD80.Through observation of microscope and MTT assay,the chimeric antibody has inhibitive function on growing and proliferation of Daudi cells.PBMCs from two health people were added into 96-cell plates with anti-CD86chimeric antibody of 5μg/mL.MTT assay was used to determine proliferation of lymphocytes after 72 hours of cultivation.The results showed that anti-CD86 chimeric antibody can inhibit the proliferation effectiveness of mixed lymphocytes.In conclusion,we constructed and expressed a human-mouse chimeric antibody against human CD86 in CHO cell successfully,which has inhibitive function on proliferation of Daudi cells and mixed lymphocytes proliferation effectiveness.