In vivo expression of the antimicrobial defensin and lactoferrin proteins allowed by the strategic insertion of introns adequately spliced

Gene
Nathalie BissonnetteDenis Petitclerc

Abstract

A major limitation of conventional shuttle expression system, when cloning a bactericidal gene, is the basal expression level in bacteria, which is lethal. Although the expression level is low, the bactericidal feature inherent to the molecule leads to subsequent failure to recover intact transformants when the related gene is cloned into a conventional expression vector. Contrary to popular belief, the human cytomegalovirus immediate-early region 1 promoter (CMV), which is to date one of the most powerful promoters for eukaryotic expression, is active in bacteria. In this study, bactericidal genes were cloned into a conventional shuttle eukaryote expression vector harbouring the CMV promoter, but were interrupted with a sequence independent splicing element (SISE), thus inhibiting lethal gene expression in bacteria. The insertion strategy of the intron uses a universal restriction site-free cloning approach, which has been developed to insert a DNA fragment into a specific location of a gene, through a PCR-based cloning technique. We have found that one intervening sequence, which derives from an adenovirus, can be spliced in a mammalian system without respect to its location, thus the bactericidal protein is synthesized only ...Continue Reading

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Citations

May 3, 2007·Diseases of the Colon and Rectum·Marco ScarpaImerio Angriman
Nov 11, 2009·Journal of Gastrointestinal Surgery : Official Journal of the Society for Surgery of the Alimentary Tract·Cesare RuffoloImerio Angriman

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