title: Cloning and characterization of a novel sigma-like glutathione S-transferase from the giant panda parasitic nematode, Baylisascaris schroederi.
abstract: BACKGROUND
  Baylisascaris schroederi, an intestinal nematode of the giant panda, is the cause of the often fatal disease, baylisascariasis. Glutathione S-transferases (GSTs) are versatile enzymes that can affect parasite survival and parasite-host interactions and, are therefore, potential targets for the development of diagnostic tests and vaccines.
  
  
  METHODS
  In this study, we identified a full-length cDNA that encoded a novel, secretory sigma-like GST (Bsc-GSTσ) from a B. schroederi-omic dataset. Following cloning and sequencing, sequence and structural analyses and comparative modeling were performed using online-bioinformatics and proteomics tools. The recombinant Bsc-GSTσ (rBsc-GSTσ) protein was prokaryotically expressed and then used to detect antigenicity and reactivity using immunoblotting assays. In addition, the native protein in female adult B. schroederi was located via immunofluorescence techniques, while the preliminary ELISA-based serodiagnostic potential of rBsc-GSTσ was assessed in native and infected mouse sera.
  
  
  RESULTS
  Bsc-GSTσ contained a 621-bp open reading frame that encoded a polypeptide of 206 amino acids with two typical sigma GST domain profiles, including a GST_N_Sigma_like at the N-terminus and a GST_C_Sigma_like at the C-terminus. The presence of an N-terminal signal sequence indicated that Bsc-GSTσ was a secretory protein. Sequence alignment and phylogenetic analyses showed that Bsc-GSTσ was a nematode-specific member of the Sigma class GSTs and shared the closest genetic distance with its homologue in Ascaris suum. Further comparative structure analyses indicated that Bsc-GSTσ possessed the essential structural motifs (e.g., βαβαββα) and the consensus secondary or tertiary structure that is typical for other characterized GSTσs. Immunolocalization revealed strong distributions of native Bsc-GSTσ in the body hypodermis, lateral chords, gut epithelium, gut microvilli, oviduct epithelium, and ovaries of adult female worms, similar to its homologue in A. suum. Building on good immunogenic properties, rBsc-GSTσ-based ELISA exhibited a sensitivity of 79.1% and a specificity of 82.0% to detect anti-B. schroederi IgG antibodies in the sera of experimentally infected mice.
  
  
  CONCLUSIONS
  This study presents a comprehensive demonstration of sequence and structural-based analysis of a new, secretory sigma-like GST from a nematode, and its good serodiagnostic performance suggests that rBsc-GSTσ has the potential to detect B. schroederi and, therefore, could be used to develop an ELISA-based serological test to diagnose baylisascariasis in giant pandas.
authors.affiliation: Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Ya'an, 625014, China. zhandegaokandey123@163.com.
authors.forename: Yue
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Centre for Animal Diseases Control and Prevention, Dachuan Animal Husbandry Bureau, Dazhou, 623000, China. zhouxuan198866@163.com.
authors.forename: Xuan
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Ya'an, 625014, China. chenlin11222@163.com.
authors.forename: Lin
authors.initials: L
authors.lastname: Chen
authors.affiliation: Chengdu Research Base of Giant Panda Breeding, Chengdu, 610081, China. parasitology513@163.com.
authors.forename: Zhihe
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: China Conservation and Research Center for Giant Panda, Wolong, 623006, China. cake1220@126.coml.
authors.forename: Chengdong
authors.initials: C
authors.lastname: Wang
authors.affiliation: Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Ya'an, 625014, China. guxiaobin198225@126.com.
authors.forename: Xiaobin
authors.initials: X
authors.lastname: Gu
authors.affiliation: Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Ya'an, 625014, China. wangtao0614@gmail.com.
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.affiliation: Department of Chemistry, College of Life and Basic Science, Sichuan Agricultural University, Ya'an, 625014, China. pxuerong@yahoo.com.cn.
authors.forename: Xuerong
authors.initials: X
authors.lastname: Peng
authors.affiliation: Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Ya'an, 625014, China. guangyou1963@aliyun.com.
authors.forename: Guangyou
authors.initials: G
authors.lastname: Yang
mesh_terms: D000818:Animals; D001199:Ascaridoidea / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D019295:Computational Biology; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods; D017422:Sequence Analysis, DNA; D001503:Ursidae / Q000469:parasitology*
chemical_list: D018076:DNA, Complementary; D005982:Glutathione Transferase