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ABSTRACT SUBMISSION FORM for CITIM 2015 Conference Would you like an oral presentation of your research? __ Yes __ No Person to whom all communication should be addressed: Complete Mailing Address, incl telephone, Fax and e-mail address: By submitting this file, you verify that: You have the approval of all authors to submit this work for presentation; any animal studies conform with the “Guiding Principles in the Care and Use of Animals” of the American Physiological Society and any human experimentation has been conducted according to a protocol approved by the institutional committee on ethics of human investigation or if no such committee exists, that it conforms with the principles of the Declaration of Helsinki of the World Medical Association (CLINICAL RESEARCH 14:193, 1966). EXAMPLE OF ABSTRACT FORMATTING Construction and expression of anti-Tn-antigen-specific single chain antibody (scFv) genes from hybridoma producing MLS128 monoclonal antibody. Yoko Fujita-Yamaguchi1, Tomoyuki Koizumi1, Kazuhiro Tukamoto1, Haruhiko Ogawa, Ayano Matsumoto-Takasaki1, Hideki Asanuma1, Hiroshi Nakada2, and Noriaki Yuasa1 1Department of Applied Biochemistry, Tokai University School of Engineering, Kanagawa, Japan; 2Department of Molecular Bioscience, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan. Oncogenic transformation is associated with dysregulation of glycosylation processes that then leads to altered patterns of carbohydrate functionalization on the surface of cancer cells. These tumor-associated carbohydrate antigens are often involved in metastatic processes and are associated with a poor prognosis, thus representing excellent targets for cancer intervention. MLS128 is a monoclonal antibody which recongnizes an epitope consisting of three or two consecutive Tn-antigens (GalNAcα-Ser/Thr): Tn3 or Tn2. We previously published that MLS128 treatment inhibited colon and breast cancer cell growth, and that MLS128-inhibited cancer cell growth is in part mediated by down-regulation of growth factor receptors. This provided the first insights into the potential use of this particular type of anti-Tn antigen antibodies as a cancer therapeutic (Morita et al. Biosci. Trends 3:32-37, 2009). In this study, cDNA encoding VH or VL constructed from hybridoma were assembled into VL-Linker-VH, and subcloned into pCANTAB5E. Phages produced were subjected to panning and screening using Tn3-peptide. DNA sequences revealed 6 independent clones which showed similar binding activities to Tn3-peptide. One MLS128 scFv gene (F11) was subcloned into pET22b to generate a pET/MLS128-F11scFv expression vector. E. coli BL21 transformed with pET/MLS128-F11scFv was induced by IPTG to overexpress MLS128 scFv proteins. Inclusion bodies prepared from bacterial cells were solubilized in a buffer containing 3.5M Gdn-HCl. From the supernatant, denatured MLS128 scFv proteins were purified by Ni2+-Sepharose chromatography, and then subjected to refolding by stepwise dialyses. Nearly 100% of MLS128 scFv proteins were recovered as soluble proteins as judged by SDSPAGE and protein assays. The purified MLS128 scFv protein exhibited Tn3- and Tn-2 binding activities. Furthermore, to establish stable expression of scFv-Fc proteins in CHO-DHFR cells, the MLS128 scFv gene was inserted into a template plasmid signal-human Fc-pCI-neo (pCI-Fc), as described previously (Zhang et al. Biochemistry 46: 263-270, 2007). The MLS128 scFv-Fc protein purified by protein A-Sepharose chromatography exhibited Tn-antigen binding activity. These results confirmed that the cloned MLS128 variable domain genes can be used for developing cancer diagnostics and therapeutics.