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Immune Blot Analysis on Expression of the Mammalian Target of Rapamycin in Goat Fetal Fibroblasts with recombinant Polyclonal antibody

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Journal of Integrative Agriculture 2012年第6期11卷 1002-1008页

作者： LIANG Yan LI Shu-yu WANG Xiao-jing WU Man-lin YANG Jiao-fu LI Jie HAO Xi-yan HAO Hui-fang BA Yin-ma CHEN Xian-wei WANG Zhi-gang College of Life Science lnner Mongolia UniversityHohhot 010021P.R.China

To detect the mammalian target of rapamycin （mTOR） expressed in Cashmere goat fetal fibroblasts （GFb）, mTOR gene was cloned from Inner Mongolia Cashmere goat （Capra hircus） and expressed in Escherichia coli followed by immunizing mice with the purified recombinant protein as an irnmunogen to produce the anti-goat mTOR recombinant polyclonal antibody. Antiserum was collected from the immunized mice after the fifth immunization and its titer was determined with enzyme-linked immunosorbent assay （ELISA）. The results showed that the recombinant polyclonal antibody had a titer 1：200000 and could react with the roTOR expressed in GFb cells with a specific and sensitive affinity. Western blot showed that mTOR expression and phospho-mTOR （Ser 2448） activity were inhibited when GFb cells were treated with CCI-779, an mTOR specific inhibitor.

关键词： prokaryotic expression recombinant antibody goat mTOR CCI-779

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Construction,expression and binding specificity of bispecific CD3×VEGFR-2 and CD3×NCAM antibodies in the single chain and diabody format

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Advances in Bioscience and Biotechnology 2013年第5期4卷 654-664页

作者： Anke Kopacek Thomas Boldicke Sarah Lergenmüller Frank Berthold Markus Jensen Peter P.Müller Ludger Grosse-Hovest Helmholtz Center for Infection Research Department of Molecular BiotechnologyBraunschweigGermany Department of Pediatric Oncology and Hematology University of CologneUniversity Hospital CologneCologneGermany Institute for Cell Biology Department of ImmunologyEberhard Karls UniversityTübingenGermany

Bispecific antibodies are recombinant proteins with novel immunological properties and therapeutic potential. recombinant protein quality and activity of several bispecific antibodies comprising different variable domain combinations with respect to the parental monospecific single chain fragments (scFv) were evaluated after expression in bacteria or mammalian cells. The parental scFv proteins humanized anti-NCAM scFv, murine anti-VEGFR-2 scFv, murine and humanized anti-CD3 scFv, respectively, could successfully be expressed in E. coli, whereas the murine anti-NCAM scFv version could not be reliably detected. Bispecific CD3 × VEGFR-2 and CD3 × NCAM anti-bodies were expressed in the bispecific single chain and the single chain diabody format. However, the diabody derived from the murine anti-NCAM scFv could not efficiently be expressed in E. coli or in mammalian cells. Significant binding of the CD3 × NCAM single chain diabody comprising the humanized version of anti-CD3 and humanized version of anti-NCAM was efficient to both antigens. Nevertheless, binding of the bispecific single chain version to the NCAM antigen was inefficient in comparison to CD3 binding. In conclusion, the data could indicate that the result of scFv expression in bacteria may be predictive for the chances of success for functional expression of more complex bispecific derivatives.

关键词： recombinant antibody Single Chain Diabody Bispecific antibody Protein Expression

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