Developing an Anti-human AEG-1 Monoclonal Antibody Secreting Hybridoma and Its Variable Region Fragment Amplification
Author(s): LONG Min, DONG Ke, WANG Xi, CHEN Xi, LIU Chong, LIU Li, ZHANG Hui-zhong, Department of Clinical Research and Diagnosis, Tangdu Hospital, Fourth Military Medical University
Pages: 10-15
Year: 2014 Issue: 12
Journal: China Biotechnology
Keyword: AEG-1 Mc Ab Variable Region Clone;
Abstract: Human Astrocyte elevated gene-1( AEG-1) encodes 582 amino acid residues and locates at chromosome 8q22. AEG-1 involves in multiple signal pathways and represents an important genetic determinant regulating multiple events in tumorigenesis and development. In order to further explore its role in the development of malignant tumors,recombinant AEG-1 protein was used to immunize the BALB / c mice. At the end of immunization,the mouse spleen cells were fused with SP /20 cells to develop hybridoma cells,and after ELISA screening with AEG-1 protein coated plate, an anti-human AEG-1 monoclonal antibody secreting hybridoma cell line was obtained and named as 1E3. The results of Western blot and Immunocellchemistry /Immunohistochemistry detecting showed that this AEG-1 monoclonal antibody could special reacted with AEG-1protein in cancer cells. Then,the variable region fragment genes of anti-AEG-1 antibody from 1E3 cells were amplified by RT-PCR method,and both the mRNA and protein sequences were analyzed and compared in BLAST,which indentified them as mouse-derived Ig G variable heavy and light chains( VH and VL). Moreover,structural analysis of these VH and VL genes were also did by using Kabat online analysis system,and confirmed that both VH( encoding 117 amino acids) and VL( encoding 119 amino acids) genes had complete FWRs and CDRs regions. All these results have perhaps laid a foundation for further study of AEG-1 function in the development of malignant tumors and the potential use of this bio-marker in the clinical diagnosis of malignant tumors.
Pages: 10-15
Year: 2014 Issue: 12
Journal: China Biotechnology
Keyword: AEG-1 Mc Ab Variable Region Clone;
Abstract: Human Astrocyte elevated gene-1( AEG-1) encodes 582 amino acid residues and locates at chromosome 8q22. AEG-1 involves in multiple signal pathways and represents an important genetic determinant regulating multiple events in tumorigenesis and development. In order to further explore its role in the development of malignant tumors,recombinant AEG-1 protein was used to immunize the BALB / c mice. At the end of immunization,the mouse spleen cells were fused with SP /20 cells to develop hybridoma cells,and after ELISA screening with AEG-1 protein coated plate, an anti-human AEG-1 monoclonal antibody secreting hybridoma cell line was obtained and named as 1E3. The results of Western blot and Immunocellchemistry /Immunohistochemistry detecting showed that this AEG-1 monoclonal antibody could special reacted with AEG-1protein in cancer cells. Then,the variable region fragment genes of anti-AEG-1 antibody from 1E3 cells were amplified by RT-PCR method,and both the mRNA and protein sequences were analyzed and compared in BLAST,which indentified them as mouse-derived Ig G variable heavy and light chains( VH and VL). Moreover,structural analysis of these VH and VL genes were also did by using Kabat online analysis system,and confirmed that both VH( encoding 117 amino acids) and VL( encoding 119 amino acids) genes had complete FWRs and CDRs regions. All these results have perhaps laid a foundation for further study of AEG-1 function in the development of malignant tumors and the potential use of this bio-marker in the clinical diagnosis of malignant tumors.
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