pFUSE-Lucia-CHIg-hG3

Heavy chain constant region expression plasmid - Human IgG31 - Lucia tag

pFUSE-Lucia-CHIg-hG3 is a cloning plasmid that expresses the   constant region of the human IgG3 heavy chain.  It contains a   multiple cloning site upstream of the constant region  to enable cloning  of the heavy chain variable region.

This plasmid is  designed for antibody generation when co-transfected into mammalian cells with the light chain variable region cloned into pFUSE-CLIg.

pFUSE-Lucia-CHIg-hG3 contain the secreted luciferase (Lucia) gene upstream of the MCS and the CH region to serve as a tag to facilitate the detection and quantification of recombinant antibodies.

Specifications

• Constant region of the human IgG3 heavy chain
• Lucia secreted luciferase tag

Lucia luciferase is a secreted protein, therefore, the VH sequence should not include a signal sequence.

Plasmids is selectable with Zeocin™ in E.coli and mammalian cells.

These products are covered by a Limited Use License (See Terms and Conditions).

pFUSE Contents

• 20 µg of lyophilized DNA
• 1 ml of Zeocin® (100 mg/ml)

Product is shipped at room temperature

Description

Antibody generation using pFUSE-Lucia-CHIg & pFUSE-CLIg

1- Obtaining 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.

2- Cloning into pFUSE-CHIg and pFUSE2-CLIg

Once the VH and VL sequence are known, inserts for cloning into the   plasmids can be generated. In pFUSE-Lucia-CHIg-hG3, the constant region of the human IgG3 heavy chain is preceded by a multiple cloning site containing four restriction sites: Acc 651, Eco RI, Xho I and Nhe I. The first three restriction sites can be used for insertion of the 5’end of the variable region, taking care to clone in frame with the Lucia luciferase sequence. In pFUSE-Lucia-CHIg-hG3, Nhe I must be used for insertion of the 3’end of the variable region. Nhe I must be reconstituted to maintain the integrity of the constant region. Therefore we recommend to introduce by PCR an Nhe I site at the 3’ end of the variable region in frame with the constant region.

3- Antibody production

Cotransfect mammalian cells, such as 293 and CHO cells, with the   recombinant plasmids pFUSE2-CLIg encoding the light chain and pFUSE-CHIg   encoding the heavy chain. Antibody production depends greatly on the   ratio of heavy chain and light chain expression. Typically, pFUSE-CHIg   to pFUSE2-CLIg ratio of 2:3 is used to cotransfect mammalian cells. Use   blasticidin and Zeocin™ to select pFUSE2-CLIg and pFUSE-CHIg   respectively.
Antibody production can be analyzed by different   techniques including SDS-PAGE, flow cytometry, ELISA, or a bioactivity   assay: the luciferase levels in the supernatant could be determined   using QUANTI-Luc™.

4- Antibody purification

Many antibody purification methods are available, among them human IgG3 isotype-specific affinity chromatography using Protein G or Protein L.

Details

1- Schematic representation of a Lucia luciferase-tagged antibody

2- Luciferase activity of Lucia-tagged anti-hTNF-α antibodies.

CHO cells were stably co-transfected with a heavy chain expressing plasmid, pFUSE-Lucia-TNF-CHIg-hA2m1 (TNF-CHIg-hA2m1) or pFUSE-Lucia-TNF-CHIg-hG1 (TNF-CHIg-hG1) and a light chain expressing plasmid, pFUSE2-TNF-CLIg-hk (TNF-CLIg-hk) to generate Lucia-tagged anti-hTNF-α antibodies, Lucia-anti-hTNF-α-hIgA2m1and Lucia-anti-hTNF-α-hIgG1, of human IgAm2 and human IgG1 isotypes, respectively. pFUSE2-CLIg-hk (CLIg-hk), which expresses no VL, and pSELECT-blasti-mcs (mcs) were used as negative controls. Supernatants
were collected and the luciferase levels determined using QUANTI-Luc™. Only the cells co-producing a Lucia-anti-hTNF-α heavy-chain and an anti-hTNF-α light chain displayed luciferase activity.

3- Neutralizing activity of anti-hTNF-a antibodies

The activity of anti-hTNF-α-hIgA2m1 and Lucia-anti-hTNF-α-hIgA2m1 antibodies, purified from the supernatants of CHO transfected cells, was determined by performing an TNF-α neutralizing assay. Both antibodies display similar neutralizing activities, thus fusion of the Lucia luciferase tag at the N-terminus of the heavy chain does not alter the functionality of the antibody.