[An avirulent vibrio cholerae strain--producer of the cholera toxin B subunit: obtaining and molecular genetic analysis].

The conjugative recombinant plasmid pIEM3 (KmR TcR) was constructed in order to introduce the cloned ctxB gene encoding the cholera toxin B subunit into the Vibrio cholerae cells. The plasmid was obtained as a result of co-integration of two plasmids: a conjugative plasmid, pIEM1(KmR), carrying mini-kan transposon and IS1 element, as well as the pCTdelta27(TcR) plasmid that is a derivative of the pBR322 which carries the cloned ctxB gene. The avirulent Vibrio cholerae strain eltor biovar deprived (according to the PCR analysis) of the key structural and regulatory pathogenicity genes and carrying a mutation in a single gene of the O1 antigen was chosen as the pIEM3 plasmid carrier strain. The cointegrate uncoupling was shown to take place in 5% the cholera vibrio cells followed by retention of only the multi-copy pCTdelta7 plasmid. This event leads to the formation of the TcRKmS clones characterized by high levels of the cholera toxin secreted B subunit production (10 to 14 microg/ml), one of these (KM93) being selected as a strain-producer of the protein. Molecular-genetic and biochemical assays were used to elucidate peculiar features of inheritance and expression of the cloned ctxAB gene within the KM93 cells. The expression of the cloned ctxB gene was shown to be independent of the presence of the toxR, tcpP, tcpH, toxT regulatory genes suggesting the existence of some other mechanisms that might exert their control over the transcriptional activity of the cholera toxin B subunit gene. Effective production of the cholera toxin B subunit would be also observed if the constructed producer strain was cultured under the conditions of industrial process. This indicates a possibility of its employment as a source of this protein involved in manufacturing cholera immunodiagnostic and prophylactic preparations.