Expression of Proteus mirabilis polyphosphate kinase and preparation of its polyclonal antibodies / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To express and purify polyphosphate kinase (PPK) from Proteus mirabilis and prepare the polyclonal antibody against PPK.</p><p><b>METHODS</b>The antigenicity and hydrophobicity of PPK were analyzed using software. The N-terminal conservative sequence containing 309 amino acids was selected as the target peptide, and its corresponding gene sequence with modification based on prokaryotic cells-preferred codon was synthesized and inserted into plasmid pET28b(+). The constructed recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and induced with IPTG. The expressed fusion protein was purified using Ni-affinity chromatography. The purified protein was injected along with adjuvant in rabbits to prepare the polyclonal antibodies against PPK.</p><p><b>RESULTS AND CONCLUSION</b>PPK fusion protein expressed by E. coli was purified successfully using Ni-affinity chromatography. ELISA result demonstrated that the harvested rabbit anti-sera against PPK had a high titer of 1512 000, and Western blotting showed a good specificity of the antibody, which can be used further study of the role of PPK in the pathogenesis of Proteus mirabilis infection.</p>