pFUSE-Lucia-CHIg-hG1

Heavy chain constant region expression plasmid - Human IgG1 - Lucia tag

pFUSE-Lucia-CHIg-hG1 is a cloning plasmid that expresses the   constant region of the human IgG1 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-hG1 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 IgG1 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-hG1, the constant region  of the human IgG1 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-hG1, 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 IgG1 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.