Adenovirus-mediated Transient Protein Expression

Adenovirus is a kind of non-enveloping virus with linear double-stranded DNA. Its tremendous potential on foreign DNA delivery has been witnessed in both research and therapeutic applications. As a tool vector of DNA delivery, the adenovirus has the following advantages:

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A wide range of host cells
Infectious to both replicative and non-replicative mammalian cells
Suitable for those cell lines that have low transfection efficiency with liposomes.
The virus remains epichromosomal thus only mediating transient protein expression

Creative BioMart's adenovirus-mediated transient protein expression service is based on the E 1 and E 3 gene defective adenovirus vector. The virus package lack of E 1 and E 3 is replication-incompetent, and thus, has superior biosafety.

Technology Description

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We co-transfect HEK293 cells with adenovirus shuttle plasmid carrying foreign sequences and packaging plasmid carrying adenovirus genome. Recombinant adenovirus is produced by Cre-LoxP or FLP/ FRT site-specific recombination system. The adenovirus will eventually be used to infect divided or non-divided cells

Obtain adenovirus shuttle plasmid	Timeline	Deliverable
Obtain codon-optimized foreign DNA sequence through PCR Recombination of DNA and Adenovirus shuttle plasmid in vitro Plasmid transformed into E. coli to amplify Sequencing and plasmid extraction	3-4 weeks	Target DNA synthesis report Recombinant shuttle vector sequencing report E. coli that contains shuttle vector Recombinant shuttle vector
Virus packaging generation
Co-transfection of the shuttle plasmid and adenovirus genomic plasmid into HEK293 cell line by liposome method Cre-LoxP or FLP/ FRT recombination within mammalian to form expression vector Adenovirus stock harvest, amplification and purification Virus quality test	2-3 weeks	Recombinant adenovirus stock Virus quality test report
Protein expression and purification
Recombinant virus transfected host cells Small-scale protein expression Affinity purification Identification and detection of recombinant protein	2-3 weeks	Protein sample
Scale-up and protein purification
Scale-up according to the requirement Affinity purification PAGE assay Product delivery	2-5 weeks	Protein QC report Protein product

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Features of Our Adenovirus-mediated Expression System

The shutter plasmid backbone we use is engineered to exhibit a high copy number in E. coli.
The E 1 and E 3 gene on the adenovirus genomic plasmid backbone is delated to prevent cell replication
Cre-LoxP or FLP/ FRT recombination is adopted to construct the expression vector efficiently and accurately
Suitable to study gene expression of primary non-proliferative cells —— The cells infected by adenovirus can start expression in 1-2 days.
High virus titer can be obtained —— The adenovirus system can self-replicate in the E 1 gene transfected HEK293 cells and produce viruses with titers of 1010 to 1011 PFU/ml.
Large carrier capacity —— The virus vector can hold foreign fragments of up to 5 kb.
Biosafety —— Adenoviruses are not integrated into mammalian chromosomes thus potential gene mutations and random effects caused by integration are avoided.

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