Preparation Of Mouse Anti-human CD86 Monoclonal Antibody And Research Of Its Biological Function

The CD86(B7-2)gene is located at the 3q21.1 site of human chromosome,with the total length of 1120bp and the molecular weight of approximately 50KD.CD86 molecule is mainly a transmembrane glycoprotein expressed on the surface of antigen-presenting cells(APC)including macrophages,dendritic cells and B lymphocytes.Under normal physiological conditions,it acts as a ligand molecule that binds to the receptor CD28 molecule on the surface of T cells,mediates the second signal of T cell activation,promotes T cell activation,proliferation and exerts an immune response.With the deepening of the research,it was found that CD86 molecules are also abnormally highly expressed on many tumors,and it has been confirmed that they are involved in the development and prognosis of tumors.At present,research has shown that CD86 molecules on the surface of tumor cells can be induced by specific antibodies to induce apoptosis,which has obvious anti-tumor effect.In order to further explore the biological significance of the expression of CD86 molecules on tumor cells,the main functions of the CD86 molecules in the cells can be studied by up-regulat:ing or down-regulating the expression of the molecules at the gene level which finds new target molecules for tumor therapy.In view of this,the aim of this paper is to carry out the following research:?.Using the gene transfected cell line L929-CD86 which can be stably expressed in the undergraduate laboratory as the immunogen,Hybridoma cell lines stably secreting mouse anti-human CD86 monoclonal antibody were prepared by B lymphocyte hybridoma technique.The ascites-type antibody was produced,and the antibody was purified by Protein A affinity immunochromatography,and the subclass,titer and specificity of the antibody were identified.?.The self-prepared monoclonal antibody was co-incubated with tumor cells with high expression of CD86 molecule,and the effects on tumor cell growth,proliferation,migration and apoptosis were observed in vitro.?.Based on the above research,RNAi technology was used to down-regulate the expression of CD86 on the surface of tumor cells,and then the effect of the molecule on the proliferation and migration of tumor cells was observed.And compared with the above studies,to explore the role of CD86 molecules in the development of related tumors.Finally,we will jointly search for new targeting molecules with anti-tumor effects.Part ? Preparation of mouse anti-human CD86 monoclonal antibody and identification of its biological characteristicsObjective:To prepare mouse anti-human CD86 monoclonal antibody and to identify its biological characteristics.Methods:The BALB/c mice were immunized with the gene transfected cell line L929-CD86,which was constructed by the laboratory and stably expressed the molecule of interest.The fused cells were prepared using the B lymphocyte hybridoma technique.Hybridoma cells capable of stably secreting the antibody of interest are screened by immunofluorescence labeling and continuous subcloning.Subclasses of antibodies were determined using antibody subclass rapid qualitative test strips and Elisa kits.After the hybridoma cells were expanded and cultured,the ascites-type antibody was prepared by inducing ascites in nude mice,and purified by Protein A affinity immunochromatography.The titer of the purified antibody was detected by flow cytometry(FCM).FCM analysis of this antibody competes with the site of anti-human CD86 commercial antibody to inhibit binding.Immunofluorescence staining was used to detect the recognition effect of antibodies on surface membrane molecules of L929-mock,L929-CD86,Raji,Daudi,8266,Jeko,U266,DOHH2,HCT116,A375,7721,TMD8,U937,A549,HepG-2 and MCF-7 cell.Results:Cell fusion was performed by the above method,and 24 96-well cell culture plates were co-fused with a fusion rate of 70%.A hybridoma cell line stably secreting CD86 antibody was successfully screened by immunofluorescence labeling and continuous subcloning and named as 12G4.The subclass rapid identification test strips and kits have the same identification results.The heavy chain of 12G4 is IgG2b and the light chain is K.Monoclonal antibodies were prepared by inducing ascites in nude mice.The ascites formation rate was 100%,and the average delivery volume was 4 ml/mouse.The yield of the antibody protein obtained by purifying the ascites-type antibody was 1.61 mg/ml.The titer of the purified antibody was 0.1 ?g/s × 105 cells.Site competition inhibition experiments indicated that 12G4 and anti-human CD86 commercial antibody(TT2.2)can recognize different epitopes.The mouse anti-human CD86 mAb obtained in this experiment can recognize cell membrane type imolecules well,and it is related to L929-mock,L929-CD86,Raji,Daudi,8266,Jeko,U266,DOHH2,HCT116,A375,7721,TMD8,U937,The positive binding rates of A549,HepG-2 and MCF-7 cells were 0.51%,100%,96.5%,99.0%,99.8%,33.6%,25.2%,15.7%,2.87%,1.37%,1.00%,1.76,respectively.%,3.69%,1.07%,1.97%,and 1.76%.Conclusion:The mouse anti-human CD86 monoclonal antibody was successfully prepared,which provided a material basis for subsequent research.Part ? Effect of antibody blocked CD86 molecule on growth and apoptosis of tumor cellObjective:To observe the effects of antibodies on cell proliferation,migration and apoptosis in vitro with blocking the CD86 molecules on the surface of tumor cells by self-prepared monoclonal antibody,Methods:Raji,Daudi and 8266 cell lines with high expression of CD86 molecules were used as observation objects.Added concentrations of 5 ?g/ml,10 ?g/ml,20 ?g/ml CD86 mAb respectively to co-incubate.When cultured to 24 h,FCM was used to detect the apoptotic cells by antibody induction.When co-incubated for 48 h,MTT was used to detcct the effect of antibodies on proliferation of cell and TranswellTM was used to study the effect of antibodies on migration of cell.Results:Raji,Daudi and 8266 cell were incubated with the antibody for 24 hours.Compared with the isotype control group,the cells of antibody group were severely aggregated,the refractive index and the stereoscopic effect were decreased.MTT results showed that 20pg/ml antibody had a significant inhibitory effect on proliferation of cell(P<0.05),and the inhibition rate was 23±1.28%,25±1.48%and 20±2.25%respectively.The FCM results showed that the apoptosis rates of Raji,Daudi and 8266 cells were 16±1.15%,18±1.53%and 14±2.52%respectively,when the antibody concentration was 20 ?g/ml.which was significantly higher than that of the isotype control group(P<0.05).At 48h,TranswelITM results showed that the mobility of Raji,Daudi and 8266 cells in the 20?g/ml antibody group was 26±0.25%,23±2.08%and 24±1.52%respectively,which was compared with the isotype control group and the migration rate of the cells in the control group was 36±2.52%,33±0.58%and 34±0.76%respectively.The antibody group was significantly lower than the isotype control group(P<0.05).Conclusion:The monoclonal antibody prepared in this study has an inhibitory effect on the proliferation and migration of tumor cells expressing CD86 molecules,and it can induce cells apoptosis.It is suggested that the antibody has a certain anti-tumor effect on tumors expressing the corresponding antigen molecules.Part ? Effect of RNAi inhibitied expression of CD86 molecule on proliferation and migration of tumor cellObjective:To explore the possible role of this molecule in cell proliferation and migration,the expression of CD86 on the surface of tumor cells was down-regulated by RNAi technology.Methods:The coding sequence of human CD86 mRNA was searched from GenBank,and the template DNA sequence of siRNA was designed and synthesized,which was introduced into the transfer vector PGLV3(Hl/GFP).The transfer vector PGLV3(H1/GFP),packaging helper plasmid PG(Gag/Pol),PG(Rev)and envelope plasmid PG(VSV-G)were co-transfected into 293T cells by liposome method.Packaging produces recombinant lentiviruses.The virus solution of infecting cells was obtained by concentrated.Infected the 293T cells of logarithmic growth phase by diluting the virus solution in 10 gradients respectively,the titer of the recombinant lentivirus solution was measured by a fluorescence microscope.Raji,Daudi and 8266 cells in logarithmic growth phase were infected with 1×108 TU/mL,1×107 TU/mL and 1×106 TU/mL virus solution respectively.FCM and Western blot were used to detect the effect of lentivirus on infection of cell and the interference on the surface of CD86 molecule.MTT and TranswellTM were used to detect the effect of lentiviral-mediated RNAi down-regulation CD86 molecule on proliferation and migration of tumor cellResults:Three RNAi target sites were selected in the CDS region of human CD86 molecule.After sequence design,the oligo sequence of the shRNA template was synthesized by GenePharma,the recombinant human CD86 shRNA lentiviral expression vector was successfully prepared and named as LV3-142,LV3-406 and LV3-644 respectively.Their titers were 1 × 109 TU/mL,2 × 109 TU/mL and 1 ×109 TU/mL respectively.FCM showed that the expression of green fluorescent protein in the cells gradually increased with the increase of the concentration of virus dilution.The optimal concentration for infection is 1 × 108 TU/mL.The recombinant lentivirus with the best effect on the above cell infection was LV3-142,the infection efficiency was 86±3.93%,88± 1.64%and 88±2.87%respectively(P<0.05).After three different sequences of recombinant lentivirus infected,the mean value of CD86 molecules on the cell surface was significantly reduced.The interference effect of LV3-142 was the most obvious,the CD86 mean value of the cell surface was decreased by 67±3.99%,41±2.5g%and 48±4.78%(P<0.001).Western Blot showed that the best interference effect was also LV3-142,its interference efficiencies for Raji,Daudi and 8266 cells were 58±1.05%,41±3.55%and 42±0.42%respectively(P<0.001).MTT showed that the proliferation of Raji,Daudi and 8266 cells was significantly inhibited by LV3-142(P<0.05),the inhibition rate of proliferation was 24±2.13%,18±1.59%and 15±1.76%respectively.TranswellTM results showed that the migration rates of Raji,Daudi and 8266 cells were 28±2.04%,30±0.85%and 28±0.17%respectively.After infected with LV3-142,which were lower than those of LV3-NC control(35±1.89%,36±1.63%and 33±1.92%),the difference was statistically significant(P<0.05).Conclusion:Recombinant human CD86 shRNA lentivirus can inhibit proliferation and migration by interfering with the expression of CD86 molecules on the surface of tumor cells.It is further suggested that CD86 molecules on the surface of tumor cells may be involved in the development and prognosis of tumors.