Cloning and Sequencing of a Unique Antigen MPT70 from Mycobacterium tuberculosis H37Rv and Expression in BCG Using E. coli‐Mycobacteria Shuttle Vector
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.
Available from: Izumi Asahina
- "Infection and growth of live BCG in the host cell and released BCG-related cytokine were estimated as the reasons for the depression of the tumor cells. MPB70  (α antigen) known to be an immunogenic mycobacterial protein secreted in large amounts from culture filtrated of Mycobacterium bovis Bacillus Calmette-Guérin(BCG) Tokyo. This protein is thought to be crucial for binding phagocytic cell having fibronectin receptors and this function might be a direct effect of BCG immunotherapy[10,11]. BCG is thought to bind to the bladder wall via interaction between the bacterial antigen complex and fibronectin . "
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ABSTRACT: Intravesical BCG immunotherapy is effective for preventing recurrence and progression in none muscle-invasive bladder cancer but the dosing schedule and duration of treatment remain empirical. The mechanisms by which intravesical BCG treatment mediates antitumor activity are currently poorly understood.
HeLa cell infected with Mycobacterium bovis Bacillus Calmette-Guérin(BCG) Tokyo which were different multiplicity of infection(MOI). Proliferation of HeLa cell reduced in a dose-dependent manner by live BCG. The cytoplasm of the HeLa cell showed variety lysosomal stages by internalized and interacted BCG.
Proliferated Live BCG secreted the protein and depressed the growth of tumor. The possibility for clinical introduction of BCG therapy for carcinoma reported with review of literature.
Available from: Harald Wiker
- "To explore the reasons underlying these differences in production, sequence-based analysis has been performed , but no mutations in the encoding genes or their upstream promoter regions have been detected (Hewinson et al ., 1996; Vosloo et al ., 1997). Complementation of BCG Pasteur with mpb70 from BCG Tokyo did not restore levels of MPB70 to those observed with BCG Tokyo, suggesting differences in expression inherent to the parent BCG strain (Matsumoto et al ., 1995). By targeted expression analysis, using reverse transcription polymerase chain reaction (RT-PCR) and Northern blots, an obvious difference in mpb70 transcription was observed between BCG Tokyo (high-producer) and BCG Pasteur (low-producer ) (Matsuo et al ., 1995). "
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ABSTRACT: Mycobacterium bovis Bacille Calmette-Guerin (BCG) strains are genetically and phenotypically heterogeneous. Expression of the antigenic proteins MPB70 and MPB83 is known to vary considerably across BCG strains; however, the reason for this phenotypic difference has remained unknown. By immunoblot, we separated BCG into high- and low-producing strains. By quantitative reverse transcription polymerase chain reaction (RT-PCR), we determined that transcription of the antigen-encoding genes, mpb70 and mpb83, follows the same strain pattern with mRNA levels reduced over 50-fold in low-producing strains. Transcriptome comparison of the same BCG strains by DNA microarray revealed two gene regions consistently downregulated in low-producing strains compared with high-producing strains, one including mpb70 (Rv2875) and mpb83 (Rv2873) and a second that includes the predicted sigma factor, sigK. DNA sequence analysis revealed a point mutation in the start codon of sigK in all low-producing BCG strains. Complementation of a low-producing strain, BCG Pasteur, with wild-type sigK fully restored MPB70 and MPB83 production. Microarray-based analysis and confirmatory RT-PCR of the complemented strains revealed an upregulation in gene transcription limited to the sigK and the mpb83/mpb70 gene regions. These data demonstrate that a mutation of sigK is responsible for decreased expression of MPB70 and MPB83 in low-producing BCG strains and provide clues into the role of Mycobacterium tuberculosis SigK.
Available from: Hideki Kitaura
- "(1990) reported the sequence of an analogous gene from M. bovis AN5 from position -81 to 582, including a signal region. Matsumoto e t al. (1995) reported the nucleotide sequence from position -157 to 726 in Mycobacteritlm ttlberctllosis. Epitope mapping of MPB70 from M. bovis was reported by Radford e t al. "
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ABSTRACT: Substrains of Mycobacterium bovis BCG (BCG) have been divided into two major groups, high and low producers, on the basis of the amount of secretion of the MPB70 protein. The antigen is produced in high concentration by BCG Tokyo, Moreau, Russia and Sweden (high-producer substrains), whereas in BCG Pasteur, Copenhagen and Tice (low-producer substrains) it is detected at 1% (w/w) or less of the concentration of BCG Tokyo. To investigate why this protein is secreted differently, the MPB70 genes of BCG Tokyo and Pasteur were cloned, sequenced and compared. The MPB70 genes in two substrains showed exactly the same sequence. Even the upstream and downstream regions of the MPB70 gene were identical. MPB70 gene expression was assessed by means of Northern hybridization analysis and reverse transcriptase polymerase chain reaction. The mRNA was clearly detected in BCG Tokyo, but a very low level in BCG Pasteur. On the basis of these results, the difference in the secretion of the MPB70 protein between BCG Tokyo and Pasteur was attributed to differential transcription efficiencies.
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