Expression of Proteus mirabilis polyphosphate kinase and preparation of its polyclonal antibodies.
- Author:
Liang PENG
1
;
Jing-Yi OU
;
Jia-Yun PAN
;
Cong DENG
;
Jing-Hong CHEN
;
Hong CAO
Author Information
- Publication Type:Journal Article
- From: Journal of Southern Medical University 2017;37(3):312-316
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo express and purify polyphosphate kinase (PPK) from Proteus mirabilis and prepare the polyclonal antibody against PPK.
METHODSThe 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 CONCLUSIONPPK 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.