OBJECTIVE: To express and purify polyphosphate kinase (PPK) from Proteus mirabilis and prepare the polyclonal antibody against PPK. METHODS: 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. RESULTS AND CONCLUSION: 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 1:512 000, 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.
OBJECTIVE: To express and purify polyphosphate kinase (PPK) from Proteus mirabilis and prepare the polyclonal antibody against PPK. METHODS: 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. RESULTS AND CONCLUSION: 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 1:512 000, 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.
Authors: M H Rashid; K Rumbaugh; L Passador; D G Davies; A N Hamood; B H Iglewski; A Kornberg Journal: Proc Natl Acad Sci U S A Date: 2000-08-15 Impact factor: 11.205
Authors: Stephanie D Himpsl; C Virginia Lockatell; J Richard Hebel; David E Johnson; Harry L T Mobley Journal: J Med Microbiol Date: 2008-09 Impact factor: 2.472
Authors: Laura E Batten; Alice E Parnell; Neil J Wells; Amber L Murch; Petra C F Oyston; Peter L Roach Journal: Biosci Rep Date: 2015-11-18 Impact factor: 3.840