Preparation And Application Of Momoclonal Antibody Specific To Human Programmed Death-1 Ligant 2(PD-L2)

Objective:In this work,we cloned programmed death ligand PD-L2(hereinafter referred to hPD-L2)diverse gene and constructed the recombinant plasmid of pET-27b-hPD-L220-208 and pET-27b-hPD-L220-123.The recombinant plasmid was transformed into E.coli RosettaTM.Recombinant proteins were induced by IPTG.Balb/c mouse were immunized with the purified and refolding recombinant protein to generate antiserum,which compared the immune effect between the two recombinant protein.We prepared monoclonal antibodies against hPD-L2 and investaged its application on immunohistochemistry,which showed potential diagnostic value in academic research and immunohistochemistry.Methods:Finding the full-length gene fragment of hPD-L2 by NCBI,sent to Nanjing Kingsi for synthesis.We amplified hPD-L2 extracellular region and IgV domain area by PCR technology,which was linked to the vector pET-27b.The recombinant plasmid was transformed into Rosseta,and recombinant hPD-L2 proteins were induced by IPTG,identified by SDS-PAGE and Western-Blot,which purified by nitrilotriacetic acid(Ni-NTA)agarose resin and AKTA purification system.Balb/c mice were immunized with purified hPD-L2,The mice were immunized three times and taken blood to dectect antibody titer by using ELISA.IHC was used to identify and evaluate the quality of antisera specific to hPD-L2 if the titer is reached,The monoclonal antibody of hPD-L2 was prepared by hybridoma technique and characterized by ELISA and IHC techniques.The subtype of monoclonal antibody was determined by ELISA.Monium sulfate precipitation method was adopted to purify monoclonal antibodies.Antibody purity was detected by SDS-PAGE.The diagnostic value of antibody was assessed by using IHC assay and Western-Blot.Results:1.We had construct ecombinant hPD-L2 extracellular domain pET-27b-hPD-L220-208 and Igv domain pET-27-hPD-L220-123 plasmid successfully.Strain expressed high hPD-L2 proteins,which induced by the final concentration of 0.5 mM IPTG.The theoretical molecular weight of hPD-L2 proteins was nearly 22 kDa and 12 kDa.2.All the antisera titers were more than 1 × 106,Immunohistochemistry(IHC)showed that the localization of PD-L2(20-208)was clear and accurate,and hPD-L2(20-123)IgV Domain was weak and blurred.3.hPD-L2 hybridomas secreting monoclonal antibody were obtained successfully.Finally,a monoclonal cell line 10E10 was obtained,The purified antibody subtype was identified as IgG3,the light chain was ? typesubtype.IHC assay showed the monoclonal antibody had a specific localization and with less background and non-specific staining,and western-blot results also showed better specificity.Conclusions:The hPD-L2 gene of 20-208 fragment could successfully prepare hPD-L2 monoclonal antibody with high specificity and affinity,and had good effect in application of immunohistochemistry.