Amyloid-beta peptides （Aβ） are believed to be .responsible for the mental decline in patients with Alzheimer＇s reported that pathology in vaccination disease （AD）. In 1999, Schenk et all immunization with Aβ at...Amyloid-beta peptides （Aβ） are believed to be .responsible for the mental decline in patients with Alzheimer＇s reported that pathology in vaccination disease （AD）. In 1999, Schenk et all immunization with Aβ attenuated AD-like the PDAPP mouse, and developed a new approach to AD. Such vaccines were successfully tested in mouse models of AD for the reduction of Aβ plaque burden and the improvement of cognitive performance. However, 6% of AD patients developed symptoms of brain inflammation after vaccination that resembled enceohalitis or meningitis.展开更多
Two vectors, pWA180 and pROH80, for expression of single-chain Fv fragments (ScFv) were con-siruciea. (?)ne anti-CD3 VH and VL genes were amplified from UCHTl cells by RT-PCR and sequenced. Both genes were cloned in p...Two vectors, pWA180 and pROH80, for expression of single-chain Fv fragments (ScFv) were con-siruciea. (?)ne anti-CD3 VH and VL genes were amplified from UCHTl cells by RT-PCR and sequenced. Both genes were cloned in pWA180 to express native ScFv and pROH80 for GST-ScFv fusion protein expression. The expression products were analysed by ELISA and Western blot. The combining site of OKT3 was modeled. Human [g LS1 and Nd were selected as acceptors of CDRs of OKT3 VL and VH to construct a reshaping antibody against CD3. By com-paring OKT3, LS1 and Nd with their own family sequences, some residues were changed and the reshaping VL and VH genes were designed. The full VH gene was assembled in three steps with eight chemically synthesized oligonu-cleotide fragments using overlapping PCR and sequenced. The VH gene was expressed as active protein in pCOMB3 and as inclusion bodies in pGEX-4T-l by ELISA and Western blot analysis.展开更多
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the grants from National Natural Science Foundation of China (No. 3040052)Natural Science and Technology Foundation of Guangdong Province (No. 04300218, 2005B10401047, 2006B36004001)Foundation of Science and Technology Bureau of Guangzhou City (No. 2004Z3-E0151, 2005Z3-E4021, 2006J1-C0101)
文摘Amyloid-beta peptides （Aβ） are believed to be .responsible for the mental decline in patients with Alzheimer＇s reported that pathology in vaccination disease （AD）. In 1999, Schenk et all immunization with Aβ attenuated AD-like the PDAPP mouse, and developed a new approach to AD. Such vaccines were successfully tested in mouse models of AD for the reduction of Aβ plaque burden and the improvement of cognitive performance. However, 6% of AD patients developed symptoms of brain inflammation after vaccination that resembled enceohalitis or meningitis.
基金Project supported by the National Natural Science Foundation of China and "863" Plan.
文摘Two vectors, pWA180 and pROH80, for expression of single-chain Fv fragments (ScFv) were con-siruciea. (?)ne anti-CD3 VH and VL genes were amplified from UCHTl cells by RT-PCR and sequenced. Both genes were cloned in pWA180 to express native ScFv and pROH80 for GST-ScFv fusion protein expression. The expression products were analysed by ELISA and Western blot. The combining site of OKT3 was modeled. Human [g LS1 and Nd were selected as acceptors of CDRs of OKT3 VL and VH to construct a reshaping antibody against CD3. By com-paring OKT3, LS1 and Nd with their own family sequences, some residues were changed and the reshaping VL and VH genes were designed. The full VH gene was assembled in three steps with eight chemically synthesized oligonu-cleotide fragments using overlapping PCR and sequenced. The VH gene was expressed as active protein in pCOMB3 and as inclusion bodies in pGEX-4T-l by ELISA and Western blot analysis.
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.
