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| Expression and Purification of EpCAM Protein and Preparation of the Monoclonal Antibodies |
| KONG Juan, GAO Hui-ping, ZHANG Peng-wei, SHI Bi-zhi, JIANG Hua, YAN Jin, LI Zong-hai |
| State Key Laboratory of Oncogenes and Related Genes, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Cancer Institute, Shanghai 200032, China |
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Abstract Objective: Prokaryotic expression,purification of the EpCAM protein,preparation and characterization of the monoclonal antibodies against the protein. Methods:The gene was cloned into pET-28a(+) plasmid. The recombinant plasmid EpCAM/PET28a was transformed into E.coli BL21 and induced with IPTG. The recombinant protein was purified with Ni-NTA resin. The monoclonal antibodies were prepared by fusion of the purified EpCAM protein immunized BALB/c mouse’s spleen cells with SP2/0 myeloma cells,positive cells were screened with indirect ELISA and the positive cells were used to immune BALB/c mice to obtain the monoclonal antibodies against EpCAM protein. The binding specificity of the purified monoclonal antibodies were analysised by Western blot and FACS. Results: The recombinant plasmid EpCAM/PET28a and purified protein were obtained, two hybridoma cell lines secreting anti-EpCAM IgG McAbs were established and named as 4B2, 2F2.FACS analysis showed that the two McAbs showed high specificity to FaDu cell line,while Western blotting analysis showed that only 4B2 can recognize the denatured EpCAM protein in FaDu cell line. Conclusions:The anti-EpCAM IgG McAbs were prepared which had high specificity to FaDu cell line, and it could be used to develop the anti-EpCAM antibody in cancer therapy.
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Received: 30 January 2012
Published: 25 May 2012
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