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design, construction and cloning of Trastuzumab humanized monoclonal antibody: A possible biosimilar for Herceptin
Soudabeh Akbarzadeh-Sharbaf, Bagher Yakhchali, Zarrin Minuchehr, Mohammad Ali Shokrgozar, Sirous Zeinali
Adv Biomed Res
2012, 1:21 (6 July 2012)
There is a novel hypothesis in that antibodies may have specificity for two distinct antigens that have been named "dual specificity." This hypothesis was evaluated for some defined therapeutic monoclonal antibodies (mAbs) such as Trastuzumab, Pertuzumab, Bevacizumab, and Cetuximab. In silico design and construction of expression vectors for trastuzumab monoclonal antibody also in this work were performed.
Materials and Methods:
First, in bioinformatics studies the 3D structures of concerned mAbs were obtained from the Protein Data Bank (PDB). Three-dimensional structural alignments were performed with SIM and MUSTANG softwares. AutoDock4.2 software also was used for the docking analysis. Second, the suitable genes for trastuzumab heavy and light chains were designed, synthesized, and cloned in the prokaryotic vector. These fragments individually were PCR amplified and cloned into pcDNA™ 3.3-TOPO
and pOptiVEC™ TOPO
shuttle vectors, using standard methods.
First, many bioinformatics tools and softwares were applied but we did not meet any new dual specificity in the selected antibodies. In the following step, the suitable expression cascade for the heavy and light chains of Trastuzumab therapeutic mAb were designed and constructed. Gene cloning was successfully performed and created constructs were confirmed using gene mapping and sequencing.
This study was based on a recently developed technology for mAb expression in mammalian cells. The obtained constructs could be successfully used for biosimilar recombinant mAb production in CHO DG44 dihydrofolate reductase (DHFR) gene deficient cell line in the suspension culture medium.
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Online since 15 January, 2012