title: Molecular cloning and bioinformatic analysis of the Streptococcus agalactiae neuA gene isolated from tilapia.
abstract: Cytidine monophosphate (CMP) N-acetylneuraminic acid (NeuNAc) synthetase, which is encoded by the neuA gene, can catalyze the activation of sialic acid with CMP, and plays an important role in Streptococcus agalactiae infection pathogenesis. To study the structure and function of the S. agalactiae neuA gene, we isolated it from diseased tilapia, amplified it using polymerase chain reaction (PCR) with specific primers, and cloned it into a pMD19-T vector. The recombinant plasmid was confirmed by PCR and restriction enzyme digestion, and identified by sequencing. Molecular characterization analyses of the neuA nucleotide amino acid sequence were performed using bioinformatic tools and an online server. The results showed that the neuA nucleotide sequence contained a complete coding region, which comprised 1242 bp, encoding 413 amino acids (aa). The aa sequence was highly conserved and contained a Glyco_tranf_GTA_type superfamily and an SGNH_hydrolase superfamily conserved domain, which are related to sialic acid activation catalysis. The NeuA protein possessed many important sites related to post-translational modification, including 28 potential phosphorylation sites and 2 potential N-glycosylation sites, had no signal peptides or transmembrane regions, and was predicted to reside in the cytoplasm. Moreover, the protein had some B-cell epitopes, which suggests its potential in development of a vaccine against S. agalactiae infection. The codon usage frequency of neuA differed greatly in Escherichia coli and Homo sapiens genes, and neuA may be more efficiently expressed in eukaryotes (yeast). S. agalactiae neuA from tilapia maintains high structural homology and sequence identity with CMP-NeuNAc synthetases from other bacteria.
authors.affiliation: Department of Basic Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Ya'an, Sichuan, China.
authors.forename: E L
authors.initials: EL
authors.lastname: Wang
authors.affiliation: Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Ya'an, Sichuan, China.
authors.forename: K Y
authors.initials: KY
authors.lastname: Wang
authors.affiliation: Department of Aquaculture, College of Animal Science and Technology, Sichuan Agricultural University, Ya'an, Sichuan, China.
authors.forename: D F
authors.initials: DF
authors.lastname: Chen
authors.affiliation: Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Ya'an, Sichuan, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Geng
authors.affiliation: Department of Basic Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Ya'an, Sichuan, China.
authors.forename: L Y
authors.initials: LY
authors.lastname: Huang
authors.affiliation: Department of Basic Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Ya'an, Sichuan, China.
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Basic Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Ya'an, Sichuan, China.
authors.forename: Y
authors.initials: Y
authors.lastname: He
mesh_terms: D000123:Acetyltransferases / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D019295:Computational Biology; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D006801:Humans; D016366:Open Reading Frames; D017386:Sequence Homology, Amino Acid; D013292:Streptococcus agalactiae / Q000201:enzymology* / Q000235:genetics; D017210:Tilapia / Q000382:microbiology
chemical_list: D029968:Escherichia coli Proteins; D000123:Acetyltransferases; C513827:NeuA protein, E coli