Preparation and identification of mouse anti-human B7-H6 monoclonal antibodies / 中华微生物学和免疫学杂志

ABSTRACT

Objective To prepare mouse anti-human monoclonal antibodies against B7-H6 and to identify their biological characteristics. Methods The B7-H6 gene was cloned by RT-PCR from a human lung adenocarcinoma cell line ( A549 ) and then subcloned into the eukaryote expression vector pCMV3 to construct the recombinant vector pCMV3-B7-H6. The recombinant vector pCMV3-B7-H6 that was verified with enzyme digestion and gene sequencing was transfected into NIH/3T3 cells by electroporation. BALB/c mice were immunized with the successfully transfected cells named 2H8 through intraperitoneal injection. The monoclonal antibodies against human B7-H6 with the advantages of high affinity and specificity were pre-pared by using hybridoma technology. Western blot assay and flow cytometry analysis were used to identify the specificity of prepared monoclonal antibodies. Results The recombinant eukaryotic expression vector encoding B7-H6 was successfully constructed. Two hybridoma clones that stably secreted monoclonal anti-bodies against B7-H6 were screened out by using flow cytometry analysis and the monoclonal antibodies se-creted by them were belonged to IgG2a isotype. Specific reactions between B7-H6 and the secreted mono-clonal antibodies were confirmed by Western blot assay and flow cytometry analysis. Conclusion The mon-oclonal antibodies which recognized B7-H6 specifically were prepared successfully.