Dellagostin OA, Cruz FW, Medeiros MA, Armoa GR, McFadden JJ; American Society for Microbiology. General Meeting.

Abstr Gen Meet Am Soc Microbiol. 1999 May 30-Jun 3; 99: 650 (abstract no. U-84).

Universidade Federal de Pelotas, Brazil.

BCG offers unique advantages for development as a multivalent vaccine vehicle for other pathogens. It is a potent adjuvant, can be administered as an oral vaccine, it is heat stable, it is safe and inexpensive to produce. The production of a recombinant vaccine requires an effective gene cloning system that has the ability to introduce DNA into the host and stably maintain and express the inserted DNA. The use of a strong promoter to drive the expression of foreign genes may have a detrimental effect on plasmid stability due to its interference with the plasmid replication mechanism. In addition, downstream genes may also be affected. This effect can be minimised by the use of transcription terminators downstream of the foreign gene. We have performed a systematic study on transcription terminator activity in mycobacteria. A terminator probe vector was constructed by cloning the M. tuberculosis hsp60 gene promoter in front of a promoterless lacZ gene, in a shuttle vector. A BstEII restriction site localised just before the ribossomal binding site (RBS) of the hsp60 promoter was used as the cloning site for potential terminator sequences. A terminator library was constructed by cloning partially digested M. bovis BCG DNA fragments into the BstEII site. After transformation of M. smegmatis, colonies that had lost the ability to produce beta- galactosidase were selected and the inserted fragment was sequenced. In the same way, known E. coli terminators like the T4 and the TrpA terminators were cloned and their ability to end transcription was assessed. The results obtained showed that the T4 is the most effective terminator, and that the TrpA terminator does not act as terminator in mycobacteria. None of the clones isolated from the terminator library so far had a strong terminator activity.

Publication Types:

• Meeting Abstracts

Keywords:

• BCG Vaccine
• Base Sequence
• Cloning, Molecular
• Cloning, Organism
• Genes, Regulator
• Genetic Vectors
• Lac Operon
• Mycobacterium
• Mycobacterium bovis
• Plasmids
• Promoter Regions (Genetics)
• Terminator Regions (Genetics)
• Transcription, Genetic
• beta-Galactosidase
• genetics
• methods

Other ID:

• 20712292

UI: 102195822

From Meeting Abstracts