Expression of proteins of Mycobacterium tuberculosis in Escherichia coli and potential of recombinant genes and proteins for development of diagnostic reagents.
Recombinant plasmids containing DNA from Mycobacterium tuberculosis were transformed into Escherichia coli, and three colonies were selected by their reactivity with polyclonal antisera to M. tuberculosis. The three recombinant vectors contained DNA inserts of different sizes flanking a common 4.7-kilobase (kb) sequence. Each recombinant produced 35- and 53-kilodalton proteins (35K and 53K proteins, respectively) which were absent in the control E. coli. In Western blotting experiments, both proteins bound several antisera to M. tuberculosis but not antisera to other commonly isolated mycobacteria. Rabbits immunized with the recombinant 35K protein produced antisera which bound to both the 35K and 53K protein bands, a single 35K protein band present in a culture filtrate of M. tuberculosis, and single protein bands with differing molecular weights in whole-cell homogenates from other Mycobacterium spp. An additional recombinant vector containing a 2.2-kb subclone of the 4.7-kb sequence was constructed and, when used as a probe, demonstrated homology with various fragments of chromosomal digests of selected mycobacteria. Reactivity of this probe to Mycobacterium bovis and M. bovis BCG was indistinguishable from reactivity to M. tuberculosis. Immunoglobulin G reactivity to the 35K antigen was detected in antisera from 8 of 20 persons with active tuberculosis, 4 of 18 persons with leprosy, and none of 14 healthy controls. In contrast, reactivity to various proteins in M. tuberculosis culture filtrate was present in 18 of 20 patients with tuberculosis, 16 to 18 patients with leprosy, and 5 of 14 controls. The production of M. tuberculosis proteins by E. coli circumvents many difficulties encountered in the growth and manipulation of M. tuberculosis and may facilitate the development of better diagnostic and immunizing reagents.
D000907:Antibodies, Bacterial / Q000032:analysis; D000942:Antigens, Bacterial / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D008970:Molecular Weight; D009161:Mycobacterium / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000276:immunology; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014376:Tuberculosis / Q000175:diagnosis
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004274:DNA, Recombinant; D011994:Recombinant Proteins