Identification of protein products of the operon for leukocytosis (lymphocytosis) -stimulating factor from Bordetella pertussis cloned in Escherichia coli
Rozinov, M.N.; Daĭn, A.A.; Shumakov, I.L.; Gol'tsmaĭer, T.A.; Gershanovich, V.N.
Molekuliarnaia Genetika Mikrobiologiia i Virusologiia 12: 20-26
1987
ISSN/ISBN: 0208-0613 PMID: 2895891 Document Number: 292452
The hybrid plasmid pRH119 was constructed on the basis of vector plasmid pUC19 and shown to carry Bordetella pertussis PT operon in the same transcriptional orientation with the lac-promoter of the vector plasmid. Expression of PT genes in E. coli cells harbouring pRH119 was not registered. Weak expression of PT genes was found by immunoscreening of recombinant clones in situ with antiserum against PT when PT genes were put closer to lac-promoter. 0.95 kb SalGI fragment was deleted from pRH119. The derivative plasmid pRH122 was digested by SalGI and the ends were polymerized to "blunt" by polIK and ligated. The obtained plasmid pRH122K was deleted for 40 bp in XbaI site by Bal31 deletion. The lysate of E. coli cells harbouring the resulting plasmid pRH134 passed through Sepharose 4B with covalently bound immunoglobulins from antiserum against PT. The eluted protein contains S2 multimers identified by immunoblotting. The experiments with CHO-cells and active mice protection have shown the absence of S2 multimers protectiveness.