title: Synthesis, cloning, and expression of Mycoplasma suis inorganic pyrophosphatase gene using PCR-based accurate synthesis and overlap-extension PCR, and its immunogenicity analysis.
abstract: Mycoplasma suis (M. suis), a hemotrophic pathogen of pigs, causes economic losses in swine production throughout the world. Inorganic pyrophosphatase (ppa) is a very important gene in M. suis. The ppa gene of M. suis was synthesized by PCR-based accurate synthesis (PAS) and overlapextension PCR, inserted into vector pMD18-T, and then subcloned to the prokaryotic expression vector pET28c.The recombinant plasmid pET28c_ppa was transformed to E. coli BL21 for expression under induction of isopropyl thiogalactoside. The expressed product was identified by SDS-PAGE and Western blot, which suggested that the recombinant protein has good antigenicity. Piglets were immunised with purified recombinant protein, and specific antibodies to the recombinant protein were detected in piglet serum. The results show that the ppa gene can be efficiently expressed in E. coli and that the expressed recombinant protein can elicit a specific serum antibody response in piglets. PAS and overlap-extension PCR were first used to synthesize the ppa of M. suis. They provide simple, rapid, reliable and relatively inexpensive methods to synthesize, clone, and express genes. The experiment conducted in this paper will enable future research into the role and function of the ppa gene.
authors.affiliation: College of Veterinary Medicine and Animal Science, Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Agricultural University, Tai'an 271018, China. liujz@sdau.edu.cn
authors.forename: Jianzhu
authors.initials: J
authors.lastname: Liu
authors.forename: Ziqiang
authors.initials: Z
authors.lastname: Cheng
authors.forename: Dong
authors.initials: D
authors.lastname: Zhou
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.forename: Zhengui
authors.initials: Z
authors.lastname: Yan
authors.forename: Zhenyong
authors.initials: Z
authors.lastname: Wang
authors.forename: Dubao
authors.initials: D
authors.lastname: Yang
authors.forename: Yongxia
authors.initials: Y
authors.lastname: Liu
authors.forename: Tongjie
authors.initials: T
authors.lastname: Chai
mesh_terms: D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D043564:Inorganic Pyrophosphatase / Q000235:genetics / Q000378:metabolism*; D009174:Mycoplasma / Q000201:enzymology* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D043564:Inorganic Pyrophosphatase