Cloning, sequencing, expression, and characterization of the tsh gene from an avian pathogenic Escherichia coli strain

Abstract

The temperature-sensitive hemagglutinin (Tsh) belongs to a family of high-molecular-weight serine protease autotransporters of Enterobacteriaceae (SPATEs), which can cleave different substrates. We isolated and characterised the tsh gene from an avian pathogenic Escherichia coli (APEC) strain, APEC13 serotype O2:H9, which was cloned in pET101. The 4.2 kb region of cloned DNA coded one protein of approximately 140 kDa (r-Tsh). The recombinant plasmid pET101-tsh conferred to E. coli BL21 strain (tsh) the hemagglutination-positive phenotype against chicken erythrocytes. The r-Tsh was purified by Ni-NTA column and used to produce antibody anti-Tsh. A 1.6 kb fragment of the tsh sequence was also amplified and cloned in pCR4, and a partial sequence showed high homology with other sequence analysed. The anti-Tsh reacted with the protein r-Tsh and native Tsh of APEC13, as demonstrated by Western blot, showing that r-Tsh has conserved epitopes and that its antigenicity was preserved. The anti-Tsh also inhibited the hemagglutinating activity of strains APEC13 and BL21/pET101-tsh.