title: Identification, immunolocalization, and immunological characterization of nitric oxide synthase-interacting protein from Clonorchis sinensis.
abstract: Recently, accumulating evidences indicate that nitric oxide (NO) is a potent mediator with diverse roles in regulating cellular functions, signaling pathways, and variety of pathological processes. In the present study, using data from the published genomic for Clonorchis sinensis (C. sinensis), we investigated a gene encoding nitric oxide synthase-interacting protein (NOSIP) of C. sinensis. Recombinant CsNOSIP (rCsNOSIP) was expressed and purified from Escherichia coli BL21. The open reading frame of CsNOSIP comprises 867 bp which encodes 289 amino acids and shares 72.9, 45.2, 47, 46.4, and 45.8% identity with NOSIP from Schistosoma mansoni, Xenopus laevis, Rattus norvegicus, Mus musculus, and Homo sapiens, respectively. Bioinformatics analysis suggested that the full-length sequence contains an eNOS-interacting domain and numerous B-cell epitopes. Quantitative RT-PCR indicated that CsNOSIP differentially transcribed throughout the adult worms, metacercariae, and egg stages of C. sinensis, and were highly expressed in the adult worms. Moreover, western blot analysis showed that the rCsNOSIP could be detected by the serum from BALB/c mice infected with C. sinensis and the serum from BALB/c mice immunized with excretory/secretory products (ESPs). Furthermore, immunolocalization assay showed that CsNOSIP was specifically localized in the intestine, vitellarium, and eggs of adult worm. Both immunoblot and immunolocalization results demonstrated that CsNOSIP was one component of ESPs of C. sinensis, which could be supported by SignalP analysis. Moreover, analysis of the antibody subclass and cytokine profile demonstrated that subcutaneously immunized BALB/c mice with rCsNOSIP could significantly enhance serum IgG1 level and up-regulate expression of IL-4 and IL-6 in the splenocytes. Our results suggested that CsNOSIP was an important antigen exposed to host immune system and probably involved in immune regulation of host by inducing Th2-polarized immune response.
authors.affiliation: Department of Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510080, People's Republic of China.
authors.forename: Meng
authors.initials: M
authors.lastname: Bian
authors.forename: Shan
authors.initials: S
authors.lastname: Li
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Wang
authors.forename: Yanquan
authors.initials: Y
authors.lastname: Xu
authors.forename: Wenjun
authors.initials: W
authors.lastname: Chen
authors.forename: Chenhui
authors.initials: C
authors.lastname: Zhou
authors.forename: Xueqing
authors.initials: X
authors.lastname: Chen
authors.forename: Lei
authors.initials: L
authors.lastname: He
authors.forename: Jin
authors.initials: J
authors.lastname: Xu
authors.forename: Chi
authors.initials: C
authors.lastname: Liang
authors.forename: Zhongdao
authors.initials: Z
authors.lastname: Wu
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.forename: Xuerong
authors.initials: X
authors.lastname: Li
authors.forename: Xinbing
authors.initials: X
authors.lastname: Yu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D003004:Clonorchis sinensis / Q000235:genetics / Q000378:metabolism*; D018985:Epitopes, B-Lymphocyte / Q000276:immunology; D004926:Escherichia coli; D005260:Female; D015801:Helminth Proteins / Q000235:genetics / Q000276:immunology*; D007074:Immunoglobulin G / Q000097:blood; D015847:Interleukin-4 / Q000276:immunology; D015850:Interleukin-6 / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016366:Open Reading Frames
chemical_list: D000909:Antibodies, Helminth; D002352:Carrier Proteins; D018985:Epitopes, B-Lymphocyte; D015801:Helminth Proteins; D007074:Immunoglobulin G; D015850:Interleukin-6; D015847:Interleukin-4