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Updated: Jul 24, 2025
Defining Substrate Specificities for Lipase and Phospholipase Candidates
Published on: November 23, 2016
[Bioinformatics analysis, prokaryotic expression and purification of phospholipase A from Neisseria gonorrhoeae]
Feng Dong1, Jia-Qi Chen1, Ying Liu1
1Laboratory of Pathogenic Biology, Dali University School of Basic Medicine, Dali, Yunnan 671000, China.
Objective:
To bioinformatically analyze the phospholipase A protein of Neisseria gonorrhoeae, predict its immunogenicity, and express and purify the protein using the prokaryotic system.
Methods:
Bioinformatics software was used for sequence analysis and prediction of T cell and B cell epitopes. The phospholipase A gene was amplified by PCR. The target gene was connected to the prokaryotic expression vector pET28a and induced with isopropyl thiogalactose (IPTG) in the host Rosetta (DE3). The expression was determined by SDS-PAGE electrophoresis and Western blot, and the immunoreactivity detected with the polyclonal antibody against Neisseria gonorrhoeae.
Results:
Peptides 12－26 and 236－250 of the phospholipase A protein were the best dominant T cell epitopes, while peptides 281－296, 331－346 and 354－369 were the best dominant B cell epitopes. The recombinant plasmid pET28a containing the phospholipase A gene was successfully constructed, and expressed and purified in the host bacteria. The results of Western blot showed that the recombinant protein had good immunoreactivity with specific immune serum.
Conclusions:
The results of prediction and successful preparation of the recombinant protein provide a basis and some reference for the development of Neisseria gonorrhoeae-related epitope vaccine and molecular diagnosis technology.
