Cloning and sequencing of a unique antigen MPT70 from Mycobacterium tuberculosis H37Rv and expression in BCG using E. coli-mycobacteria shuttle vector.
ABSTRACT MPB70 is known to be an immunogenic mycobacterial protein secreted in large amounts from Mycobacterium bovis BCG (BCG) Tokyo. The analogous gene for MPT70 was cloned from Mycobacterium tuberculosis H37Rv which produces this protein in only a small amount. The gene encoding 193 amino acid residues including 30 amino acids for the signal peptide, the promoter-like sequence, and the ribosome-binding site, was completely identical to that of BCG Tokyo. Computer analysis revealed that the carboxy-terminal half of MPT70 was homologous to amino acid sequences of fasciclin I, osteoblast-specific factor 2 (OSF-2), and human transforming growth factor-beta induced gene product (beta IG-H3). Escherichia coli (E. coli) -mycobacteria shuttle vectors containing mpt70 or mpb70 genes 0.7kbp upstream of the 5' end of them were able to be expressed in BCG Pasteur which is a MPB70 low-producer, but the extent of the expression was not that of a high-producer.
SourceAvailable from: Kazuo Tomita[Show abstract] [Hide abstract]
ABSTRACT: Exogastrula-inducing peptides (EGIP) of the sea urchin Anthocidaris crassispina are endogenous peptides related to epidermal growth factor (EGF), which induce exogastrulation in the embryo. Recently, a protein(s) from sea urchin embryos that binds to one of the EGIP, EGIP-D (EGIP-D-binding protein, EBP) was purified. The isolation and characterization of the cDNA clones for two EBP proteins (EBP-α and EBP-β) is reported. The two EBP proteins were highly similar in structure to each other; both possessed putative cell-binding sites and two repeated sequences characteristically seen in the insect neuronal cell adhesion protein, fasciclin I. The EBP showed similarity with other sea urchin proteins HLC-32, Bep1, and Bep4. It has been confirmed that bacterially expressed EBP proteins associate with EGIP-D as does native EBP, suggesting the interaction between EGF-related proteins and fasciclin I-related proteins. An EBP transcript of 1.4 kb was strongly expressed in immature ovaries but not in immature testes. A somewhat lower level of the transcript existed in unfertilized eggs and the amount gradually declined to an almost undetectable level by the pluteus stage. The EBP proteins were present throughout embryonic development at nearly constant levels. Although most of the proteins were distributed rather evenly in the cytoplasm, a small portion was detected on the apical surface of blastomeres and ectodermal cells, showing that EBP are components of the hyaline layer.Embryologia 07/1999; 41(4):483 - 494. DOI:10.1046/j.1440-169x.1999.00446.x · 2.18 Impact Factor
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ABSTRACT: Like the products of the genes mpt83 and mpt70, the putative protein encoded by the gene located between these genes was undetectable in Mycobacterium tuberculosis with an antiserum raised against the recombinant protein. The protein showed 100% homology with M. tuberculosis Rv2874 and similarities with CcdA and DipZ proteins involved in cytochrome-c biogenesis in bacteria. Expression analysis by RT-PCR and transcriptional fusions of Rv2874 and their neighbor genes Rv2871, Rv2872, mpt83 and mpt70 with lacZ suggest that these genes are part of an operon and their transcription is driven by promoter regions located 5′ upstream of mpt83 and of Rv2874 genes.
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ABSTRACT: MPB70 and MPB80 (MPB70/80) and MPB83 are closely related antigens which are highly expressed in Mycobacterium bovis. MPB70/80 are soluble secreted antigens, while MPB83 is an exported lipoprotein associated with the bacterial surface. In the present study, these antigens had different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. These differences may be explained by the fact that MPB70 and MPB83 both have two internal cysteine residues which would create ring structures by disulfide bonding. We analyzed the structures of MPB70/80 and MPB83 by using monoclonal antibodies (MAbs) raised against bovine purified protein derivative or whole M. bovis cells. MAb 1-5C reacted specifically with MPB70 and MPB80, and MAb MBS43 reacted specifically with MPB83, while the other antibodies, including several previously described MAbs, bound all three antigens. MAbs and polyclonal antibodies reacted strongly with reduced protein and less well with nonreduced protein, indicating involvement of linear epitopes. Epitopes of MAbs Bov-1, 2-6B, 1-5C, and 1-1D were mapped by using synthetic peptides of MPB70. Sequence comparison showed the peptide with the 1-5C-reactive epitope to have three residues different from those in the homologous region of MPB83. Exchanges of A for S in position 112 or Q for E in position 116 abolished the reactivity of MAb 1-5C. Polyclonal rabbit antibodies to native purified MPB70 reacted strongly with peptides 6, 7, and 8 of the N-terminal half of mature MPB70. Cattle sera of experimentally M. bovis-infected animals recognized a broader spectrum of peptides. These findings indicate that there is diagnostic potential for these proteins and that there is also a possible role for antibodies in elucidation of the host-mycobacterium relationship involving a surface-bound and exposed lipoprotein, MPB83, and its highly homologous soluble secreted MPB70/80 counterparts.Infection and Immunity 04/1998; 66(4):1445-52. · 4.16 Impact Factor