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Objective: To investigate the subcellular localization of ATP synthase b subunit from Clonorchis sinensis under different conditions of Hela cell cycling, and the effect of this protein on the expression of its encoding-gene and homologous host genes., Methods: pEGFP-N1-CsATP-synt_B and the vector pEGFP-N1 were transfected into Hela cells, respectively. Transfected cells were synchronized in G0/G1 by serum starvation, G1/S, S cells by double thymidine block, and G2/M cells by thymidine-Nocodozale block. After synchronization, the subcellular localization of the expressed fusion protein was observed with a laser confocal microscope. The expression level of this fusion protein in cells was detected by flow cytometry (FCM). The expression of CsATP-synt_B and HomoATP-synt_B in different cell cycle phases accessed by RT-PCR., Results: FCM results indicated that in the G0/G1 phase the expression of pEGFP-N1 vector was decreased significantly, while pEGFP-N1-CsATP-synt_B expression showed an upward trend. In the other phases of cell cycle, the protein expression was similar in the above two kinds of plasmids. The intact CsATP-synt_B was expressed in mitochondria in the G0/G1, S, and G2/M phases and nucleus during G1/S phase. After the fusion proteins entered the nucleus, the mRNA expression of CsATP-synt_B and HomoATP-synt_B increased significantly., Conclusion: CsATP-synt_B can be expressed in the nucleus during G1/S phase, and regulated by the cell cycle and energy requirements.

Objective: To illustrate the distribution of ATP synthase b subunit in the tissue of Clonorchis sinensis adult and its subcellular mimical localization in HeLa cells., Methods: With the antiserum against recombinant CsATP-synt_B protein raised from SD rats as primary antibody, paraffin sections of the adult of C. sinensis were processed by the method of fluorescent immunohistochemistry to observe the distribution of CsATP-synt_B protein in adult worm. According to the prediction by bioinformatics of the definite mitochondrial targeting sequence (MTS) and probable Bipartite nuclear localization signals (NLS_BP)in CsATP-syntB sequence, recombinant pEGFP-N1 plasmids containing the intact and three defective CsATP-synt_B sequence with single defect of MTS or NLS_BP or double defect respectively were constructed. The recombinant plasmids and the control plasmid-pEGFP-N1, pEYFP-Mito and H2B-CFP, were transfected into the HeLa cells by Lipofectamine 2000 reagent and the subcellular location of the GFP fusion protein was observed with confocal microscopy., Results: The CsATP-synt_B protein appeared to distribute all over the adult worm, especially abundant on the acetabulum, ovary, vitellarium and tegument. The intact CsATP-synt_B was definitely expressed in mitochondria and/or nucleus of infected HeLa cells, whereas the MTS-deleted mutant only in cytoplasma and nucleus, the NLS_BP-deleted mutant in mitochondria and cytoplasm, and the double defect mutant only in cytoplasm., Conclusion: The distribution of CsATP-synt_B in adult is accord with that of mitochondria, and mainly exits in the organs and the tissues of active energy metabolism. This study first predicted and confirmed that CsATP-synt_B can be expressed in the nucleus.

Objective: To clone and express the Clonorchis sinensis F0-ATP synthase b chain (CsF0-ATP-synt_B) gene and analyze immunogenicity of the recombinant protein., Methods: The coding region F0-ATP synthase b chain gene with the mitochondrial targeting sequence (MTS) removed was amplified with PCR using the cloned plasmid as template, and the product was cloned into the prokaryotic expression vector pET-28a(+), transformed into E. coli BL21 (DE3) and induced with IPTG. The expressed product was purified by Ni-IDA affinity chromatography,and analyzed by SDS-PAGE for its expression and identified by Western blotting for its immunogenicity., Results: The coding sequence of the F0-ATP synthase b-chain like gene removed off the MTS contains 813 base pairs encoding 271 amino acids with a theoretical molecular weight of 31,171.9. PCR, double enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET-28a (+)-CsF0-ATP-synt_B was constructed successfully, and the resolvable expression was obtained in E.coli BL21. Highly purified recombinant protein was prepared through affinity chromatography. The recombinant protein could be recognized by the immune serum of the SD rat immunized with the recombinant protein., Conclusion: The CsF0-ATP-synt_B like gene has been efficiently expressed in prokaryotic expression system with immunogenicity.