Preparation and identification of mouse anti-human B7-H6 monoclonal antibodies
10.3760/cma.j.issn.0254-5101.2016.07.008
- VernacularTitle:鼠抗人B7-H6单克隆抗体的制备及其生物学特性鉴定
- Author:
Ying CHEN
;
Yang HE
- Publication Type:Journal Article
- Keywords:
B7-H6;
Eukaryotic expression;
Monoclonal antibody;
Specificity
- From:
Chinese Journal of Microbiology and Immunology
2016;36(7):519-522
- CountryChina
- Language:Chinese
-
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.
