pAX56b plasmid vector抗體表達(dá)質(zhì)粒載體

The Gateway? System allows rapid and highly efficient swapping of gene segments (flanked with specific recombination sequences combined with a positive selection marker) to multiple vector systems, allowing convenient subcloning. Therefore, plasmid was prepared from individual Nanobody constructs in pAX056a and used as input material for a series of parallel recombination reactions between the individual ‘entry’ clones and ‘destination’ vector pAX056b using the Gateway? LR Clonase Enzyme Mix (Invitrogen) according to the manufacturer's protocol. This recombination reaction was followed by heat shock mediated transformation of the resulting VHH in pAX56b constructs to E. coli WK6. pAX056b allows the expression of the VHH as a His6—and c-Myc-tagged fusion protein in the periplasmic space of E. coli. Due to leaky expression, supernatant of each culture can be used to screen for antigen binding in ELISA. Following this procedure, it was shown in a cell ELISA comparing signals on Her14 versus 3T3 (Example 2) that approximately 30% of individual clones expressed EGFR specific VHHs.

This method allows efficient swapping of VHH between the non-expression vector and a Gateway? compatible vector of choice optimized for functional screening. An example of such destination vector is an expression vector that allows efficient recloning of the Nanobody as multivalent constructs.
Recloning of individual Nanobody genes into expression vector pAX56b by site-specific recombination (Gateway).


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