title: Identification and characterization of paramyosin from cyst wall of metacercariae implicated protective efficacy against Clonorchis sinensis infection.
abstract: Human clonorchiasis has been increasingly prevalent in recent years and results in a threat to the public health in epidemic regions, motivating current strategies of vaccines to combat Clonorchis sinensis (C. sinensis). In this study, we identified C. sinensis paramyosin (CsPmy) from the cyst wall proteins of metacercariae by proteomic approaches and characterized the expressed recombinant pET-26b-CsPmy protein (101 kDa). Bioinformatics analysis indicated that full-length sequences of paramyosin are conserved in helminthes and numerous B-cell/T-cell epitopes were predicted in amino acid sequence of CsPmy. Western blot analysis showed that CsPmy was expressed at four life stages of C. sinensis, both cyst wall proteins and soluble tegumental components could be probed by anti-CsPmy serum. Moreover, immunolocalization results revealed that CsPmy was specifically localized at cyst wall and excretory bladder of metacercaria, as well as the tegument, oral sucker and vitellarium of adult worm. Both immunoblot and immunolocalization results demonstrated that CsPmy was highly expressed at the stage of adult worm, metacercariae and cercaria, which could be supported by real-time PCR analysis. Both recombinant protein and nucleic acid of CsPmy showed strong immunogenicity in rats and induced combined Th1/Th2 immune responses, which were reflected by continuous high level of antibody titers and increased level of IgG1/IgG2a subtypes in serum. In vaccine trials, comparing with control groups, both CsPmy protein and DNA vaccine exhibited protective effect with significant worm reduction rate of 54.3% (p<0.05) and 36.1% (p<0.05), respectively. In consistence with immune responses in sera, elevated level of cytokines IFN-γ and IL-4 in splenocytes suggested that CsPmy could induce combined cellular immunity and humoral immunity in host. Taken together, CsPmy could be a promising vaccine candidate in the prevention of C. sinensis regarding its high immunogenicity and surface localization.
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, People's Republic of China.
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Wang
authors.forename: Wenjun
authors.initials: W
authors.lastname: Chen
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Lv
authors.forename: Yanli
authors.initials: Y
authors.lastname: Tian
authors.forename: Jingtao
authors.initials: J
authors.lastname: Men
authors.forename: Xifeng
authors.initials: X
authors.lastname: Zhang
authors.forename: Huali
authors.initials: H
authors.lastname: Lei
authors.forename: Chenhui
authors.initials: C
authors.lastname: Zhou
authors.forename: Fangli
authors.initials: F
authors.lastname: Lu
authors.forename: Chi
authors.initials: C
authors.lastname: Liang
authors.forename: Xuchu
authors.initials: X
authors.lastname: Hu
authors.forename: Jin
authors.initials: J
authors.lastname: Xu
authors.forename: Zhongdao
authors.initials: Z
authors.lastname: Wu
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; D000906:Antibodies / Q000032:analysis / Q000276:immunology; D003003:Clonorchiasis / Q000469:parasitology / Q000517:prevention & control*; D003004:Clonorchis sinensis / Q000276:immunology*; D007111:Immunity, Cellular; D007074:Immunoglobulin G / Q000097:blood; D007371:Interferon-gamma / Q000378:metabolism; D015847:Interleukin-4 / Q000378:metabolism; D058488:Metacercariae / Q000378:metabolism*; D008969:Molecular Sequence Data; D020543:Proteome / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016415:Sequence Alignment; D014335:Tropomyosin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D019444:Vaccines, DNA / Q000276:immunology
chemical_list: D000906:Antibodies; D007074:Immunoglobulin G; D020543:Proteome; D011994:Recombinant Proteins; D014335:Tropomyosin; D019444:Vaccines, DNA; D015847:Interleukin-4; D007371:Interferon-gamma