CHARACTERIZATION OF THE GENE ENCODING THE IMMUNODOMINANT 35 KDA PROTEIN OF MYCOBACTERIUM-LEPRAE

Analysis of the interaction between the host immune system and the int racellular parasite Mycobacterium leprae has identified a 35 kDa prote in as a dominant antigen, The native 35 kDa protein was purified from the membrane fraction of M. leprae and termed MMPI (major membrane pro tein I). As the purified protein was not amenable to N-terminal sequen cing, partial proteolysis was used to establish the sequences of 21 pe ptides, A fragment of the 35 kDa proteinen-coding gene was amplified b y the polymerase chain reaction from M. leprae chromosomal DNA with ol igonucleotide primers derived from internal peptide sequences and the whole gene was subsequently isolated from a M. leprae cosmid library. The nucleotide sequence of the gene revealed an open reading frame of 307 amino acids containing most of the peptide sequences derived from the native 35 kDa protein, The calculated subunit mass was 33.7 kDa, b ut the native protein exists as a multimer of 950 kDa, Database search es revealed no identity between the 35 kDa antigen and known protein s equences. The gene was expressed in Mycobacterium smegmatis under the control of its own promoter or at a higher level using an 'up-regulate d' promoter derived from Mycobacterium fortuitum. The gene product rea cted with monoclonal antibodies raised to the native protein, Using th e bacterial alkaline phosphatase reporter system, we observed that the 35 kDa protein was unable to be exported across the membrane of recom binant M. smegmatis. The 35 kDa protein-encoding gene is absent from m embers of the Mycobacterium tuberculosis complex, but homologous seque nces were detected in Mycobacterium avium, Mycobacterium haemophilum a nd M. smegmatis, The avaibility of the recombinant 35 kDa protein will permit dissection of both antibody- and T-cell-mediated immune respon ses in leprosy patients.