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.
Highlights
Lactobacillus casei is a member of the lactic acid bacteria (LAB) that can be found in various environments, including that of animal and human intestines (Kandler and Weiss 1986)
Sequencing, sequence comparison and assemblage of pRCEID7.6 Initially, the contig2310 with a total length of 6814 bases showed a nucleotide identity of 96 and 87 % of the segments embraced by the positions 696–3639 and 5944–6814 respectively to sequences of plasmid pCD01 (AY662330.1) from Lactobacillus paracasei NFBC338, strongly suggesting that the contig may be part of pRCEID7.6, the missing plasmid of L. casei Thailand Institute of Scientific and Technological Research (TISTR) 1341 (Panya et al 2012)
To verify this possibility and to complete the nucleotide plasmid sequence, primers in opposite orientation were designed based on the sequence flanking both sides of the contig and used in PCR amplifications using as a template plasmid pRCEID7.6 isolated from a gel
Summary
Lactobacillus casei is a member of the lactic acid bacteria (LAB) that can be found in various environments, including that of animal and human intestines (Kandler and Weiss 1986). Besides generally recognized as safe (GRAS) status, many strains of LAB including a number of L. casei strains are recognized as probiotics, defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” There is a growing trend in the development of LAB as mucosal vaccines, as well as vehicles for the delivery of therapeutic and prophylactic molecules. A major advantage of using LAB as mucosal delivery vehicles is their ability to be engineered to express both homologous and heterologous proteins. One critical step to develop L. casei and other LAB as mucosal delivery vehicles is to construct the expression systems for these bacteria.
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