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Cloning and Expression of Foreign Genes in Mycobacteria.

Dellagostin, Odir Antonio. (1994) Cloning and Expression of Foreign Genes in Mycobacteria. Doctoral thesis, University of Surrey (United Kingdom)..

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Abstract

In this thesis, the construction and use of integrative vectors to express foreign genes in mycobacteria is described. These vectors are based on the transposition properties of the IS900 insertion sequence. Transformation by stable integration of multiple copies of the vector into the chromosome of Mycobacterium smegmatis, M. bovis BCG and M. vaccae was achieved. The M. leprae gene encoding the 18-kDa antigen was used to drive the expression of foreign antigens in mycobacteria. The major immunogenic site of Foot-and-Mouth disease virus (FMDV) comprising amino acids 140-160 of the virus protein 1 (VP1) was inserted in the 18kD gene and expressed as a fusion protein. Guinea pigs were vaccinated with the recombinant BCG expressing the FMDV epitope and immune responses specific for the FMDV were detected. A system for testing mycobacterial promoter strength in both extracellularly and intracellularly growing mycobacteria, as well as in E. coli was developed. It consists of a shuttle vector containing a promoterless reporter gene (lacZ). A promoter library was constructed using M. bovis BCG chromosomal DNA and screened in M. smegmatis and E. coli and a number of clones containing DNA inserts with promoter activity were isolated. The activity of each individual clone was measured in both, E. coli and M. smegmatis. Several previously characterized mycobacterial promoters including those of the Mycobacterium bovis BCG hsp60, the M. leprae 18kD and the putative iron regulated M. leprae 28kD gene were also cloned in front of the promoterless lacZ gene and the promoter strength was determined. To study intracellular expression, rBCG was used to infect murine macrophages and the activity of the reporter gene was measured using a fluorescent substrate and a FACS can system. Relative differences in the expression of the reporter gene during intracellular and extracellular growth were detected for some promoters. The BCG hsp60 promoter was shown to be the strongest of the characterized promoters also during intracellular growth. To obtain a higher level of expression of the 18kD/FMDV fusion, the 18kD promoter was replaced by the hsp60 promoter. A level of expression of the fused gene that in vitro was at least 10 fold higher was obtained with the hsp60 promoter in BCG.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Dellagostin, Odir Antonio.
Date : 1994
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 1994.
Depositing User : EPrints Services
Date Deposited : 24 Apr 2020 15:27
Last Modified : 24 Apr 2020 15:27
URI: http://epubs.surrey.ac.uk/id/eprint/854999

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