Cloning and characterization of the gene for the "19 kDa" antigen of Mycobacterium bovis

Abstract

Monoclonal antibody CMA134.1 reacted with a protein antigen of apparent molecular mass 22 kDa from Mycobacterium bovis and Mycobacterium tuberculosis, and with an apparently 24 kDa antigen of Mycobacterium kansasii, but not with other mycobacteria or related species. This antibody was used to screen a gene library of M. bovis in Agtll and identified a recombinant clone that expressed a protein with an apparent molecular mass of 1% 20 kDa. Gene expression occurred from the lac promoter in lgtll, but used an unidentified vector promoter, possibly that of the replication primer RNA, in the final plasmid construct. The sequence of an 840 bp fragment was determined and shown to code for a product of 15 kDa. This sequence is identical to that, independently determined, of a gene from M. tuberculosis, usually referred to as the 19 kDa antigen. The reasons for the apparent size discrepancies are discussed.

Induction of Apoptosis and Release of Interleukin‐1β by Cell Wall–Associated 19‐kDa Lipoprotein during the Course of Mycobacterial Infection

“…In this regard, a relatively lower virulence of naturally mutated M. tuberculosis strains that do not express the 19kDa protein has been described in an in vivo murine model of M. tuberculosis infection [37]. Because it has been reported that, within the M. tuberculosis complex, the 19-kDa gene was found to be conserved [38,39], the present study aimed to determine the relevance of the mycobacterial 19-kDa lipoprotein in induction of apoptosis. We demonstrated that, after infection with 19KO M. tuberculosis and BCG, MDM cell death was reduced (by nearly 70%), compared with apoptosis induced by wt strains.…”

Identification of a Regulated Alkaline Phosphatase, a Cell Surface-Associated Lipoprotein, inMycobacterium smegmatis

“…In an effort to study mutations conferring protein export defects in mycobacteria, we constructed a Tn5370 library in a reporter strain of M. smegmatis, mc 2 1279 (Table 1). The reporter strain, mc 2 1279, was designed to constitutively express and export a recombinant fusion of the secreted M. tuberculosis 19-kDa antigen (LpqH) (15,23) and the truncated E. coli PhoA (ЈPhoA). The secreted fusion protein was detected by its extracellular alkaline phosphatase activity, assayed by blue color production on plates containing the chromogenic substrate of PhoA, BCIP.…”

“…The negative control strain used in these assays was M. smegmatis mc 2 155 phoA::hyg. The effect of inorganic phosphate on phoA induction was studied at low (500 M) and high (10 mM) concentrations of phosphate in the medium as suggested before (15,30), with some modifications. The strains were grown in 7H9 medium (Difco) to an optical density at 600 nm of 1.0, and then the cultures were washed twice with 500 M phosphate buffer and the cells were resuspended to a final optical density at 600 nm of 0.2 in a home-prepared 7H9 medium containing either 500 M or 10 mM K 2 NaH 2 PO 4 buffer, pH 7.0.…”

A novel 27 kDa lipoprotein antigen from Mycobacterium bovis

“…There is a n increasing interest in these antigens t o prepare new generation anti-TB vaccines ( O r m e 1988; Pal & Horwitz, 1992;Andersen, 1994) However, certain non-secreted proteins, such as membrane-associated, cell-wall-associated or cytoplasmic (Andersen & Brennan, 1994) (Lee e t al., 1991) are the most studied M. tuberculosis membrane antigens (Ashbridge, 1989;Collins et al, 1990;Faith et al, 1991;Booth e t al., 1993;Prestidge e t al., 1995).…”