Dissection of Mycobacterium tuberculosis antigens using recombinant DNA.

Abstract

A recombinant DNA strategy has been used systematically to survey the Mycobacterium tuberculosis genome for sequences that encode specific antigens detected by monoclonal antibodies. M. tuberculosis genomic DNA fragments with randomly generated endpoints were used to construct a large Xgtll recombinant DNA expression library. Sufficient numbers of recombinants were produced to contain inserts whose endpoints occur at nearly every base pair in the pathogen genome. Protein antigens specified by linear segments of pathogen DNA and produced by the recombinant phage of Escherichia coli were screened with monoclonal antibody probes. This approach was coupled with an improved detection method for gene isolation using antibodies to clonally isolate DNA sequences that specify polypeptide components of M. tuberculosis. The methodology described here, which is applicable to other pathogens, offers possibilities for the development of more sensitive and specific immunodiagnostic and seroepidemiological tests for tuberculosis and, ultimately, for the development of more effective vaccines.

Construction of an Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp

“…In addition, this plasmid transformation and molecular cloning system may have broader applicability for cloning actinomycete genes of interest, including genes from the medically important pathogens Nocardia and Mycobacterium spp. Mycobacterium DNA has been cloned in both E. coli (10,25,28,44,50) and Streptomyces spp. (25).…”

Characterization of a major polymorphic tandem repeat in Mycobacterium tuberculosis and its potential use in the epidemiology of Mycobacterium kansasii and Mycobacterium gordonae

“…The M. tuberculosis lambda gt-11 gene library from R. A. Young was screened as described by Young et al (45). Plaques were transferred to Colony/Plaque Screen Membranes (Dupont, Boston, Mass.)…”

Cloning and Sequence Analysis of the 10 kDa Antigen Gene of Mycobacterium tuberculosis

“…tuberculosis antigens that bear epitopes recognized by T lymphocytes (T cell epitopes) (Kingston et al, 1987 ;Young et al, 1986;Kadival et al, 1987;Oftung et al, 1987) are those defined by the 'TB' series of monoclonal antibodies (mAbs), (Coates et al, 1981). The gene for one of these antigens, of molecular mass 65 kDa, has been cloned (Young et al, 1985) and sequenced (Shinnick, 1987): the antigen appears to be a heat-shock protein.…”