pTRIOZ-mIgG2a

pTRIOZ General plasmid map

High-yield production of recombinant murine IgG2a kappa mAbs 

pTRIOZ-mIgG2a is designed for high-yield production of whole monoclonal antibodies (mAbs) with the use of a single plasmid. It consists of three cassettes encoding for the expression of the mAb heavy chain, light chain, and antibiotic selection with Zeocin®. 

pTRIOZ-mIgG2a expresses the constant regions of:

• The murine IgG2a heavy chain
• The murine light chain (kappa or lambda-2)

Upstream of both of these regions are unique multiple cloning sites (MCS) that enable the insertion of the variable regions of any given mAb. 

Specifications

Heavy chain cassette:

• Composite promoter: Aldehyde dehydrogenase ( AldA ) enhancer/ Human ferritin heavy chain gene ( hFerH )
• Cloning sites: 5’- AgeI, MluI, EcoRV, NheI, and Eco47III -3’
• Constant region: Murine IgG2a

Light chain cassette:

• Composite promoter: Human cytomegalovirus immediate-early gene 1 ( hCMV ) enhancer / Human ferritin light chain gene ( hFerL )
• Cloning sites: 5’- SgrAI, AscI, PmeI, and NcoI -3’.
• Constant region: Murine kappa or lambda-2 

Antibiotic resistance: Zeocin® (selectable in both bacterial and mammalian cells)

Quality control:

• Plasmid construct has been confirmed using DNA sequencing and restriction analysis
• Purified by ion exchange chromatography

Contents

• 20 µg of lyophilized pTRIOZ-mIgG2a plasmid DNA
• 1 ml of Zeocin® (100 mg/ml)

Product is shipped at room temperature

Upon receipt product should be stored at -20°C

Resuspended DNA should be stored at -20 ̊C and is stable up to 1 year.

Details

PLASMID FEATURES

Cassette 1: mAb Heavy chain

• AldA enh/ hFerH: This composite promoter combines the human aldehyde dehydrogenase (aldA) enhancer and the core promoter of the human ferritin heavy chain gene. 
• MCS1: To facilitate cloning of the variable heavy (VH) chain, the multiple cloning site contains restriction sites that are compatible with many different enzymes, 
• mIgG2a: The constant region of the murine immunoglobulin IgG2a heavy chain.
• βGlo pAn: The human beta-globin 3’UTR and polyadenylation sequence allow the efficient arrest of the transgene transcription.

Cassette 2: mAb Light chain

• hCMV enh / hFerL prom: This composite promoter combines the human cytomegalovirus (CMV) immediate-early gene 1 enhancer and the core promoter of the human ferritin light chain gene. 
• MCS2: To facilitate cloning of the variable light (VL) chain, the multiple cloning site contains restriction sites that are compatible with many different enzymes, 
• Murine light chain: The constant region of the murine light chain (kappa or lambda-2)
• SV40 pAn: The Simian Virus 40 late polyadenylation signal enables efficient cleavage and polyadenylation reactions resulting in high levels of steady-state mRNA

Cassette 3: Zeocin® selection

• mCMV/hEF1-HTLV prom: This composite promoter combines mouse cytomegalovirus (mCMV) immediate-early gene 1 enhancer, the elongation Factor-1α (EF-1α) core promoter, as well as the R  segment and part of the U5 sequence (R-U5’) of the Human T-Cell Leukemia Virus (HTLV) type 1 long terminal repeat. The EF-1α promoter exhibits a strong activity and yields a long-lasting expression of a transgene in vivo. The R-U5’ has been coupled to the EF-1α core promoter to enhance the stability of RNA.
• EM7 prom: This is a bacterial promoter that enables the constitutive expression of the antibiotic resistance gene in E. coli. EM7 is located within an intron and is spliced out in mammalian cells.
• Sh Ble gene: Resistance to Zeocin® is conferred by the Sh ble gene from Streptoalloteichus hindustanus. The same gene confers resistance in both mammalian cells and E. coli.
• hEF-1alpha pAn: This provides a strong polyadenylation signal. InvivoGen uses a sequence that starts after the stop codon of the EF1 cDNA and finishes after a bent structure rich in GT.

General features: pTRIOZ

• 5’ UTR: The 5’ UTR enhances mRNA stability and protein translation.
• Ori: A minimal E. coli origin of replication.