The carcinoembryonic antigen（CEA） is an oncofetal glycoprotein known as an important clinical tumor marker and is overexpressed in several types of tumors, including colorectal and lung carcinomas. We constructed a ...The carcinoembryonic antigen（CEA） is an oncofetal glycoprotein known as an important clinical tumor marker and is overexpressed in several types of tumors, including colorectal and lung carcinomas. We constructed a chimeric protein that exhibits both specific binding and immune stimulating activities, by fusing staphylococcal enterotoxin A（SEA） to the C-terminus of an anti-CEA single-chain disulfide-stabilized Fv（scdsFv） antibody （single-chain-C-terminus/SEA, SC-C/SEA）. The SC-C/SEA protein was expressed in Escherichia coli（E. coli）, refolded, and purified on an immobilized Ni2＋ affinity chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） and Western blot analysis reveal that the target protein was expressed sufficiently. We used immunofluorescence assays to demonstrate that SC-C/SEA could bind specifically to human lung carcinoma cells（A549）, but almost human uterine cervix cells（HeLa）. We also used the L-lactate dehydrogenase（LDH） release assay to show that SC-C/SEA elicits a strong A549 tumor-specific cytotoxic T lymphocyte（CTL） response in vitro. The results suggest that SC-C/SEA shows specific activity against CEA-positive cells and has potential application in CEA-targeted cancer immunotherapy.展开更多
基金Supported by the Innovation Fund of Graduate of Jilin University, China(No.20101035)the China Postdoctoral ScienceFoundation Project(No.20100481057)
文摘The carcinoembryonic antigen（CEA） is an oncofetal glycoprotein known as an important clinical tumor marker and is overexpressed in several types of tumors, including colorectal and lung carcinomas. We constructed a chimeric protein that exhibits both specific binding and immune stimulating activities, by fusing staphylococcal enterotoxin A（SEA） to the C-terminus of an anti-CEA single-chain disulfide-stabilized Fv（scdsFv） antibody （single-chain-C-terminus/SEA, SC-C/SEA）. The SC-C/SEA protein was expressed in Escherichia coli（E. coli）, refolded, and purified on an immobilized Ni2＋ affinity chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） and Western blot analysis reveal that the target protein was expressed sufficiently. We used immunofluorescence assays to demonstrate that SC-C/SEA could bind specifically to human lung carcinoma cells（A549）, but almost human uterine cervix cells（HeLa）. We also used the L-lactate dehydrogenase（LDH） release assay to show that SC-C/SEA elicits a strong A549 tumor-specific cytotoxic T lymphocyte（CTL） response in vitro. The results suggest that SC-C/SEA shows specific activity against CEA-positive cells and has potential application in CEA-targeted cancer immunotherapy.
