| Budget Amount *help |
¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1987: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1986: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1985: ¥5,200,000 (Direct Cost: ¥5,200,000)
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| Research Abstract |
Plasmid fragment containing Tra T gene of R100 factor was introduced into the vector plasmid, pUC19,and introduced into Escherichia coli K12,W3110/SM through the courtesy of Dr.E.Otsubo, Institute of Applied Microbiology,University of Tokyo.This strain is more resistant to complementmediated bactericidal activity than E. coli K12 W3110/SM carrying pUC19 without Tra fragment.
From the known DNA sequence of Tra T gene(Ogata et al.J.Bacteriol.151:819.1982)a peptide fragment(86-99)was chosen and synthesized chemically in Peptide Institute,Inc.This synthetic peptide was coupled with keyhole limpet hemocyanine by glutalaldehide,and was immunized into Balb/c mice.The spleen cells of the immunized mice were fused with murine myeloma cell line,X63Ag8.6.5.3 cells.Only one hybridoma produced antibody reactive with Tra T protein in Western blotting.The antibody (mAb)211-2)produced by this hybridoma was,however,could not react with Tra T protein in the membrane fraction or on the surface of living b
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acteria.But this antibody provided the method of detecting Tra T protein by Western blotting.By this method Tra T protein was traced through various isolation procedure,permitting the development of procedure of isolation and purification of Tra T protein.Purified Tra T protein was thus obtained and used for immunization and hybridoma production.Six hybridomas were obtained;all of them produced antibodies which reacted not only in Western blotting but also with Tra T protein on membrane fractions or on the living bacteria.These antibodies will facilitate the epidemiological studies of the detection and distribution or complement-resistant bacteria amoung clinically isolated bacteria.
The radioimmunoassay method was developed by using these monoclonal antibodies,and the procedure for isolation and purification of Tra T protein was developed.The membrane fraction of Tra T bacteria was dissolved by octy-glucoside and chromatographied successively on DE52-andCM52-cellulose culumns.Tra T protein thus purified will be useful for evaluating the step at which Tra T protein inhibits the complement activity. Less
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