Molecular characterization and serological reactivity of a vacuolar ATP synthase subunit ε-like protein from Clonorchis sinensis

• Published in: Parasitology research (1987) 2014-04, Vol.113 (4), p.1545-1554
• Main Authors: Lv, Xiaoli, Huang, Lisi, Chen, Wenjun, Wang, Xiaoyun, Huang, Yan, Deng, Chuanhuan, Sun, Jiufeng, Tian, Yanli, Mao, Qiang, Lei, Huali, Yu, Xinbing
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibodies, Helminth - blood; Biomedical and Life Sciences; Biomedicine; Cloning, Molecular; Clonorchis sinensis; Clonorchis sinensis - enzymology; Clonorchis sinensis - genetics; Clonorchis sinensis - immunology; Epitopes, B-Lymphocyte - immunology; Escherichia coli; Ethenoadenosine Triphosphate - immunology; Humans; Immunity, Cellular; Immunoglobulin G - blood; Immunology; Medical Microbiology; Microbiology; Molecular Sequence Data; Original Paper; Rats; Rats, Sprague-Dawley; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Schistosoma japonicum; Vacuolar Proton-Translocating ATPases - immunology
• ISSN: 0932-0113; 1432-1955
• DOI: 10.1007/s00436-014-3799-7

The vacuolar ATPase enzyme complex (V-ATPase) pumps protons across membranes, energized by hydrolysis of ATP. Extensive investigations on structural and biochemical features of these molecules have implied their importance in the physiological process. In this study, a full-length sequence encoding a vacuolar ATP synthase subunit ε-like protein of Clonorchis sinensis ( Cs ATP-ε) was isolated from our cDNA library. The hypothetical 226 amino acid sequence shared 76 % identity with ATP-ε proteins of Schistosoma japonicum and above 55 % identity with ATP-ε proteins from human and other eukaryotes. Characteristic Asp 140 amino acid residues and seven B-cell epitopes were predicted in this sequence. The complete coding sequence of the gene was expressed in Escherichia coli . Recombinant Cs ATP-ε (r Cs ATP-ε) protein could be probed by anti-r Cs ATP-ε rat serum and C.sinensis -infected human serum in Western blotting experiment, indicating that it is an antigen of strong antigenicity. The high level of antibody titers (1:204,800) showed that Cs ATP-ε has a powerful immunogenicity. Both the increased level and the change trend of IgG1/IgG2a subtypes in serum showed that the r Cs ATP-ε can induce strong combined Th1/Th2 immune responses in rats and stimulate the immune response changes to the dominant Th2 from Th1 along with long time infection. The results of immunoblot and immunolocalization demonstrated that Cs ATP-ε was consecutively expressed at various developmental stages of the parasite, which was supported by real-time PCR analysis. In immunohistochemistry, Cs ATP-ε was localized on the intestine, vitellarium, and testicle of an adult worm and excretory bladder of metacercaria, implying that Cs ATP-ε may relate to energy intake and metabolism. This fundamental study would contribute to further researches that are related to growth and development and immunomodulation of C. sinensis .