Expression ofProteins ofMycobacterium tuberculosis in Escherichia coli andPotential ofRecombinant GenesandProteins forDevelopment ofDiagnostic Reagents

Recombinant plasmids containing DNA fromMycobacterium tuberculosis weretransformed intoEscherichia coli, andthree colonies wereselected bytheir reactivity withpolyclonal antisera toM.tuberculosis. Thethree recombinant vectors contained DNA inserts ofdifferent sizes flanking a common 4.7-kilobase (kb) sequence. Eachrecombinant produced 35-and53-kilodalton proteins (35Kand53Kproteins, respectively) whichwere absent inthecontrol E.coli. InWesternblotting experiments, bothproteins boundseveral antisera toM. tuberculosis butnotantisera toothercommonlyisolated mycobacteria. Rabbitsimmunized withthe recombinant 35Kprotein produced antisera whichboundtoboththe35Kand53Kprotein bands, asingle 35K protein bandpresent inaculture filtrate ofM.tuberculosis, andsingle protein bandswithdiffering molecular weights inwhole-cell homogenates fromotherMycobacterium spp.An additional recombinant vector containing a2.2-kb subclone ofthe4.7-kb sequencewasconstructed and,whenusedasaprobe, demonstrated homology withvarious fragments ofchromosomal digests ofselected mycobacteria. Reactivity ofthisprobeto Mycobacterium bovis andM. bovis BCG was indistinguishable fromreactivity toM. tuberculosis. Immunoglobulin G reactivity tothe35Kantigen was detected inantisera from8of20personswithactive tuberculosis, 4of18personswithleprosy, andnoneof14healthy controls. Incontrast, reactivity tovarious proteins inM. tuberculosis culture filtrate was present in18of20patients withtuberculosis, 16to18patients withleprosy, and5of14controls. Theproduction ofM. tuberculosis proteins byE.colicircumvents many difficulties encountered inthegrowth andmanipulation ofM.tuberculosis andmay facilitate thedevelopment ofbetter diagnostic andimmunizing reagents. Tuberculosis continues topresent diagnostic problems for physicians andcontrol problems forpublic health officials. Thedisease mostcommonly affects thelungs, butitmay involve almost any tissue andproduce diverse, difficult-todiagnose clinical syndromes. SinceKochidentified Mycobacterium tuberculosis asthecausative agentoftuberculosis in1882, many scientific studies andpublic health efforts have beendirected atdiagnosis, treatment, andcontrol ofthis disease. Inthedeveloped world, theseefforts havebeen largely successful, yettransmission oftuberculosis continues,with over20,000 new casesbeing diagnosed annually in theUnited States alone(2). IntheUnited States, recent reports indicate thattuberculosis may soon beon theincrease,withnew cases associated withhumanimmunodeficiency virus infections andotherfactors (3).Inthe developing world, tuberculosis isan even more important causeofmorbidity andmortality inbothchildren andadults, andthere islittle evidence thatdisease rates aredeclining (10). Control oftuberculosis indeveloped anddeveloping countries involves different problems, many ofwhichpertain to health careresourcesanddelivery. However, thecharacteristics ofM.tuberculosis havehampered research toimprove diagnosis andtodevelop more effective vaccines. The organism ispotentially infectious tolaboratorians, andseveral weeks areusually required toisolate theorganism from clinical specimens. Inaddition, thebiochemical composition oftheorganism hasmadeidentification andpurification of cellular constituents difficult, andmany ofthesematerials,