title: Development of an Escherichia coli-Lactobacillus casei shuttle vector for heterologous protein expression in Lactobacillus casei.
abstract: There is an increasing interest to develop various lactic acid bacteria (LAB) species as mucosal delivery vehicles, for which the development of a variety of cloning and expression systems for these bacteria is of primary importance. This study reports the complete nucleotide sequence of the cryptic plasmid pRCEID7.6 derived from the chicken probiotic LAB strain Lactobacillus casei TISTR1341. Sequence analysis and comparison showed that pRCEID7.6 is composed of nine putative open reading frames. The replicon origin of pRCEID7.6 consisted of untranslated origin of replication and translated replication protein B sequences. This region was used to construct Escherichia coli/L. casei shuttle vectors carrying erythromycin and chloramphenicol resistance genes as selective markers. Segregation and structural stability of the vectors in L. casei was sufficient for most genetic applications. The feasibility of this vector for heterologous protein expression in L. casei was determined by cloning in pRCEID-LC7.6, the gene encoding the nucleocapsid protein (NP), from the influenza A virus under the control of the homologous promoter from the lactate dehydrogenase gene. L. casei carrying this recombinant plasmid was shown to successfully express the NP protein. Therefore, this shuttle vector can be used for further study in the development of mucosal delivery vehicles.
authors.affiliation: Department of Microbiology and Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002 Thailand.
authors.forename: Namfon
authors.initials: N
authors.lastname: Suebwongsa
authors.affiliation: Department of Microbiology and Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002 Thailand.
authors.forename: Viraphong
authors.initials: V
authors.lastname: Lulitanond
authors.affiliation: Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Paseo Río Linares, s/n, 33300 Villaviciosa, Asturias Spain.
authors.forename: Baltasar
authors.initials: B
authors.lastname: Mayo
authors.affiliation: Department of Microbiology and Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002 Thailand.
authors.forename: Panjamaporn
authors.initials: P
authors.lastname: Yotpanya
authors.affiliation: Department of Microbiology and Research and Diagnostic Center for Emerging Infectious Diseases, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002 Thailand.|College of Medicine and Public Health, Ubon Ratchathani University, Ubon Ratchathani, 34190 Thailand.
authors.forename: Marutpong
authors.identifier: 0000-0001-5262-573X
authors.initials: M
authors.lastname: Panya
keywords: Cloning vectors; Heterologous expression; Lactobacillus casei; Oral live vaccine; Plasmid