|Budget Amount *help
¥2,800,000 (Direct Cost : ¥2,800,000)
Fiscal Year 1999 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1998 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1997 : ¥1,200,000 (Direct Cost : ¥1,200,000)
We focused on one of the ribosomal proteins, S20, to replace the commonly used drugresistance gene for construction and application of recombinant bacteria in clinical therapy, since the gene, rpsT, coding for S20 resides on chromosome as a single gene unit in many common bacteria such as E. coli and B. subtilis, and this feature is quite different from other ribosomal protein genes which form large operon structures. To clone the rpsT gene from S. gordonii by PCR, geveral primers were chemically synthesized based on the published E. coli and B. subtilis rpsT gene sequences. However, no positive bands were observed using S. gordonii chromosome as a template. Recently, world wide genome science has been advanced and complete as well as partial chromosomal DNA sequence data are available in many bacteria via internet service. The presence of the rpsT homologues were found in three streptococci, S. mutans, S. pyogenes, and S. pneumoniae. Unfortunately, it seemed to be impossible to amplif
y the S. gordonii rpsT genes because of the low homology of the flanking regions among three oral streptococci. On the other hand, presence of the common upstream and downstream ORFs are confirmed in S. mutans and S. pyogenes, which code for pantothenate kinase and two component histidine kinase, respectively. Several conserved regions could be found in these two genes, and it is possible to expect the presence of the same rpstream and downstream genes in S. gordonii chromosome, the rpsT gene might be amplified by using the PCR primers complementary to the conserved regions found in the flanking genes.
In the course of our research project for establishing the passive-immunization system to prevent oral diseases in human, mouse monoclonal antibody against the P. gingivalis vesicle-associated hemagglutination was constructed following establishment of hybridoma cells. Further, scFv protein was produced in E. coli cells, however, only limited amounts of scFv could be obtained. This prompted us to produce this protein in gram-positive bacteria. The gene coding for the scFv was fused with the secretion domain of the α-amylase gene from B. subtilis, transformed into B. brevis, and the scFv was successfully secreted. Less