• Isotype switch to generate IgG antibodies with different effector functions • Generation of entire IgG antibodies, chimeric, humanized or fully human
pFUSE-CLIg and pFUSE-CHIg plasmids are designed to change a monoclonal antibody from one isotype to another human or murine IgG isotype therefore enabling the generation of antibodies with the same antigen affinity but with different effector functions (increased or reduced ADCC and CDC). Furthermore, they can be used to produce entire IgG antibodies from Fab or scFv fragments that are either chimeric, humanized or fully human depending on the nature of the variable region.
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Principle
pFUSE-CHIg and pFUSE-CLIg express the constant regions of the heavy (CH) and light (CL) chains, respectively. They contain a multiple cloning site (MCS) upstream of these constant regions to enable the cloning of the variable (VH and VL) regions of a given antibody. Transfection of mammalian cell lines with the recombinant pFUSE-CHIg and pFUSE-CLIg pair allows to generate an IgG antibody that can be purified from the supernatant using the appropriate Protein A, Protein G or Protein L affinity chromatography.
VH and VL sequences To obtain the cDNA sequence of the VH and VL regions from an antibody producing hybridoma, total RNA or mRNA is extracted and reverse transcribed to cDNA. PCR is performed with 5’ degenerate primers to anneal to the unknown VH and VL regions and the 3’ primers designed to anneal to the “known“ CH and CL regions. Alternatively 5’ RACE can be used. The resulting amplicons are sequenced.
Cloning into pFUSE-CHIg and pFUSE-CLIg Once the VH and VL sequence are known, inserts for cloning into the plasmids can be generated. When generating the insert for VH, a Nhe I site must be introduced at the 3’ end to maintain the integrity of the constant region. Similarly, when generating the inser t for VL, a Bsi WI (human VL) or Bst API (mouse VL) site must be introduced at the 3’ end. There is a choice of restriction sites at the 5’ end.
Plasmid Features
pFUSE2-CLIg plasmids feature the constant region of the human kappa or lambda light chain, or mouse kappa light chain. They are selectable with blasticidin.
pFUSE-CHIg plasmids feature the heavy chain constant region of the human or mouse IgG isotypes. - Human isotypes: IgG1, IgG2, IgG3 and IgG4 - Murine isotypes: IgG1, IgG2A, IgG2B and IgG3 pFUSE-CHIg plasmids are selectable with Zeocin™.
Antibody generation using pFUSE-CHIg and pFUSE-CLIg
Contents and Storage
pFUSE-CLIg and pFUSE-CHIg plasmids are provided as 20 μg of lyophilized DNA. Product is shipped at room temperature and should be stored at -20°C. Plasmid is stable up to one year when properly stored. Each plasmid is provided with 4 pouches of E. coliFast-Media® Zeo (2 TB and 2 Agar).
Disclaimer: Our products are provided for research purpose only. Commercial applications may require licensing from third parties.
Note that the sequence of available ORFs provided by InvivoGen can differ from a given reference Genbank record due to genetic variations
and/or alternative splicing. Customers should verify that the version of a gene sold by InvivoGen is suitable for the customer needs.
