Expression of Escherichia coliβ‐galactosidase in Mycobacterium bovis BCG using an expression system isolated from Mycobacterium paratuberculosis which induced humoral and cellular immune responses

Abstract

A promoter sequence, PAN, was isolated from Mycobacterium paratuberculosis and characterized. This promoter lies adjacent to, and outside, the 3' end of an IS900 insertion element. IS900 contains an open reading frame, ORF2, on the complementary strand which codes for the putative transposase of this insertion sequence. A DNA fragment containing PAN and part of ORF2 was fused to the lacZ gene and inserted into the replicative shuttle vector pRR3. Mycobacterium smegmatis and Mycobacterium bovis BCG (BCG) transformed with this plasmid exhibited beta-galactosidase activity. However, lacZ was only expressed in Escherichia coli under the control of PAN, when ORF2 was deleted. Immunization of mice with the recombinant M. bovis BCG expressing lacZ resulted in the induction of a high humoral and cellular response directed against beta-galactosidase. The PAN-ORF2 expression system may prove to be particularly useful for cloning and expression of heterologous genes in the BCG vaccine strain.

The Mycobacterium tuberculosis katG promoter region contains a novel upstream activator

“…The experiment was therefore performed using RNA isolated from M. smegmatis cells harbouring various katG promoter clones. The determination of transcription start sites for M. tuberculosis genes, using this heterologous host, has been shown to correlate well with results obtained from native mRNA transcripts (Levin & Hatfull, 1993 ;Dhandayuthapani et al, 1997 ;Movahedzadeh et al, 1997), and has successfully been used as a substitute in many cases (Murray et al, 1992 ;Kremer et al, 1995 ;Nesbit et al, 1995).…”

“…2. The construct pANIIIW (a gift from Karen Kempsall, Glaxo-Wellcome), consisting of a 170 bp PCR fragment containing the P AN promoter cloned into the T-tailed EcoRV restriction site of the vector pT7Blue (Novagen), was used as a source of the M. paratuberculosis promoter (Murray et al, 1992). The 218 bp HindIII-BamHI fragment from pANIIIW was cloned into the corresponding restriction sites of pJCluc to form pLPan.…”

<i>Mycobacterium bovis</i> BCG as a Delivery System for the <i>dtb</i> Gene Antigen from Diphtheria Toxin

“…In the present study, the promoter P AN of M. paratuberculosis [43] subunit of pertussis toxin [30]. Our results showed that, contrary to DTP, the response to the rBCGpUS977dtbpw8 was strong, especially in the 12 -20 week interval.…”

Plasmid instability when the <i>hsp</i>60 gene promoter is used to express the protective non-toxic fragment B of the diphtheria toxin in recombinant BCG

“…Our best results came out with the BCG transformed with the pUS977dtb PW8 construct in which the expression of DTB is driven by the P AN promoter, a regulatory sequence derived from M. paratuberculosis. The P AN sequence, a weak mycobacterial promoter [18], has been successfully used by others for the expression of the α-galactosidade of E. coli [30], the LacZ of E. coli [31], the Gp63 surface antigen of Leishmania [32], the Nef antigen of Simian Immunodeficiency Virus [33], the Nef, Gag, Env antigens of Simian Immunodeficiency Virus [34], the S1 subunit of the Bordetella pertussis toxin [24], the hepatitis B surface antigens [35] and the LipL32 antigen of Leptospira interrogans [28]. In our study all rBCGpUS977dtb PW8 clones stably expressed significant amounts of DTB while rBCGpUS973dtb PW8 clones were unstable and, consequently, unsuitable for DTB expression.…”