Abstract
Bacterial ghosts that are generated using the regulated PhiX174 lysis gene E offer a new avenue for the study of inactivated vaccines. Here, we constructed a library of mutant gene E using a gene-shuffling technique. After screening and recombination with the prokaryotic non-fusion expression vector pBV220, two lysis plasmids were selected. Among which, a novel mutant E gene (named mE), consisting of a 74-bp non-encoding sequence at 5'-end and a 201-bp gene ΔE, significantly increased the lysis effect on prokaryotic Escherichia coli and Salmonella enteritidis. Moreover, lysis efficiency, as measured by the OD600 value, reached 1.0 (109 CFU), avoiding the bottleneck problem observed with other bacterial lysis procedures, which results in a low concentration of bacteria in suspension, and consequent low production of bacterial ghosts. Our results may provide a promising avenue for the development of bacterial ghost vaccines.
Highlights
The lysis gene E of coliphage PhiX174 encodes a 91amino acid residue protein of approximately 10 kDa that mediates inhibition of peptidoglycan biosynthesis of Gram-negative bacteria membrane[1]
Low lysis efficiency and ghost production have been the key technical obstacles that have prevented the large-scale production and industrialization of bacterial ghost (BG) vaccines. To address these technical problems, the current study focused on the function of the bacteriophage PhiX174 lysis gene E
Shuffled gene E lysis plasmids, and screening of high lysis efficiency plasmids Gel-purified mutants fragments were digested with EcoR I and Bam H I and inserted into the vector pBV220, and transformed into E. coli DH5a
Summary
The lysis gene E of coliphage PhiX174 encodes a 91amino acid (aa) residue protein of approximately 10 kDa that mediates inhibition of peptidoglycan biosynthesis of Gram-negative bacteria membrane[1]. This facilitates lysis from changes in osmotic pressure, leaving an empty bacterial body lacking cytoplasm and nucleic acids, which is called a bacterial ghost (BG) [2]. BGs retain an intact bacterial surface structure and integrated antigen proteins, and can be generated without harsh physical or chemical inactivation methods. Inserting the exogenous antigen protein into the outer, inner, or periplasmic membranesis is relatively simple, and genetic engineering can be used to construct recombinant multivalent BG vaccines. BGs have been widely applied in the development of new vaccines [3,4,5]
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