A MONOCLONAL-ANTIBODY GENERATED AGAINST A RECOMBINANT PEPTIDE FRAGMENT OF THE B3 DOMAIN OF CARCINOEMBRYONIC ANTIGEN REACTS WITH INTACT CARCINOEMBRYONIC ANTIGEN

作者：HAGENDORFF G; HANES J; VONDERKAMMER H; SCHEIT KH

来源：Biochimica et Biophysica Acta-Gene Structure and Expression, 1995, 1260(3): 259-268.

DOI：10.1016/0167-4781(94)00204-G

• CARCINOEMBRYONIC ANTIGEN
• RECOMBINANT PEPTIDE FRAGMENT
• MONOCLONAL ANTIBODY

The chemical synthesis of a gene coding for a polypeptide of 77 amino acid residues (designated ceaB3) representing a fragment of the CEA-BS domain of carcinoembryonic antigen (CEA) was achieved. The ceaB3 fragment was cloned into the plasmid pLZPWB1 at the C-terminus of a derivative lacZMF of the lacZ gene, devoid of methionine and cysteine amino acid residues. The fusion protein lacZMF-ceaB3 represented approx. 30% of total proteins expressed after induction. The fusion protein was formed as inclusion bodies. Simple washing steps led to an insoluble fusion protein which was of approx. 80% purity. Another fusion gene was generated by inserting ceaB3 between the malE gene encoding maltose binding protein (mbp) and lacZ alpha of the pmal-c2 vector. Expression of the resulting pmal-c2-ceaB3-lacZ alpha yielded the fusion protein mbpceaB3-lacZ alpha with a molecular mass of 57.94 kDa, which was obtained as a soluble protein in almost homogenous form after affinity chromatography employing amylopectin. Polyclonal sheep anti-CEA antiserum specifically reacted with fusion proteins lacZMF-ceaB3 and mbp-ceaB3-lacZ alpha, A monoclonal antibody CEA/HK2 was generated employing lacZMF-ceaB3 for immunization and CEA for screening purposes. The mAB CEA/HK2 specifically recognized CEA in immunoblots. The described experimental strategy should be generally applicable for generation of fusion proteins. These fusion proteins are suitable for epitope characterization of existing antibodies, production of regiospecific polyclonal or monoclonal antibodies.

• 出版日期1995-2-21