pTRAkc::MTED抗体植物高表达载体 BioVector NTCC质粒载体菌种细胞基因保藏中心

pTRAkc::MTED抗体植物高表达载体 BioVector NTCC质粒载体菌种细胞基因保藏中心
Tobacco BY‐2 suspension cells producing DsRed (targeted to plastids) and the human monoclonal antibody M12 (retained in the endoplasmic reticulum) were generated by stable transformation with Agrobacterium tumefaciens carrying the vector pTRAkc::MTED. The expression cassettes for the M12 heavy and light chains and the DsRed protein were linked on the same T‐DNA, allowing DsRed to be used as an easily scorable marker for the identification of cells producing high levels of the antibody.
The coding sequences for the heavy and light chains of antibody M12 (originally isolated from a human naive scFv library based on its ability to bind the breast adenocarcinoma cell line MCF‐7 (Wong et al., 2001)) and DsRed were inserted into the plant expression vector pTRAkc::MTED (a derivative of pPAM (GenBank: AY027531) (Sack et al., 2007)) under the control of the double‐enhanced Cauliflower mosaic virus 35S promoter (Kay et al., 1987), and the vector was introduced into A. tumefaciens strain GV3101::pMP90RK (Koncz and Schell, 1986). As shown in Figure 1, the M12 heavy chain contains a C‐terminal KDEL motif to retrieve the antibody to the endoplasmic reticulum, and DsRed is targeted to the plastids using the GBSS transit peptide (Vanderleij et al., 1991). The nptII gene (GenBank: EU048865) was included in a nos expression cassette on the T‐DNA, allowing transformed cells to be selected using kanamycin.

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