关键词: 日本血吸虫, SjELAV-like 1, 克隆表达, 免疫定位, RNA干扰, 电镜

Abstract: 【Objective】 Cloning and expressing of Schistosoma japonicum embryonic lethal abnormal vision like 1(SjELAV-like 1) gene and analyzing the expression status in different stages and at different infected conditions as well as its distributions in the worms of Schistosoma japonicum (S. japonicum). This study also intend to explore the effects of SjELAV-like 1 on the morphology and reproductive development of S. japonicum.【Method】 The RACE technique was used to amplify the 5′/3′ end of the SjELAV-like 1 gene, the obtained sequence was submitted to NCBI, Gene Bank accession number: MG515727. The recombinant plasmid pET28-SjELAV-like 1 was constructed and expressed in E.coli BL21 by IPTG induction, then the products were collected at different time. The SDS-PAGE was used for protein analysis. His-tag nickel column affinity chromatography was used to purify the recombinant protein. The purified recombinant protein was used to immune mouse to obtain the polyclonal antibody, which was used to analyze the immunogenicity of the recombinant protein by western blotting and detect the distributions of SjELAV-like 1 protein in S. japonicum by indirect immunofluorescence assay. The mice infected by cercaria of S. japonicum in the abdomen were used to collect mixture and the females and males separated by paired worms at different stages. The mice infected by cercaria escaped from the single nails were used to collect the unisexual worms at the 25th day. The expression level of SjELAV-like 1 were detected by quantitative real-time PCR (qRT-PCR). The gene silence was performed by tail vein injection of small interference RNA(siRNA) in infected mice. The long-term RNA interference(RNAi) assay was performed to analyze the effects of SjELAV-like 1 gene silence on the spawning and egg hatching of females. The tegument structures and reproductive organs of the females and males of S. japonicum were observed by scanning electron microscopy and transmission electron microscopy after RNAi.【Result】In this study, a 1 797 bp sequence of SjELAV-like 1 was obtained, whose coding region was 1 533 bp encoding 510 amino acids. The expression of the recombinant plasmid reached its highest after 4 h of IPTG induction and it mainly existed in the form of inclusion bodies. High quality polyclonal antibody was obtained against the purified protein. SjELAV-like 1 protein was mainly located in the tegument of the worms. SjELAV-like 1 was expressed in all stages of S. japonicum and the expression was stable in males but gradually became less in females as the maturing of the worms. Expression of SjELAV-like 1 was higher in development impaired females compared with the normal worms. In the RNAi assay, the long-term interference group showed that liver egg burden,liver egg burden by per female and egg hatching rate in interference group were reduced by 58.27% (P＜0.05), 40.59% (P＜0.01) and 74.58% (P＜0.01) compared with NC group, respectively. Electron microscopy observation found that the tegument structures of the worms in interference group were obviously different from that in NC group. The surface bubble adhere to the surface of males disappeared, the three-dimensional fold ridge collapsed, the funicular surface ridge arranged loosely and the network structure of the body wall disappeared. The body surface gap increased and the spines on the body wall of the female were dull. The number of spermatocyte in the males was decreased and the intracellular chromatin was reduced, while the cells were swollen and the intercellular space was widen. In females of S. japonicum the vitelline globules in vitelline cells were decreased, in which the vitelline droplets were also reduced, the endoplasmic reticulum was swollen and the cortical particles were scattered.【Conclusion】Part sequence of SjELAV-like 1 was successfully cloned and expressed, which was robustly expressed in development impaired females. SjELAV-like 1 was mainly expressed in the tegument of S. japonicum. SjELAV-like 1 silence led to decrease of liver egg burden, liver egg burden by per female and egg hatching rate. SjELAV-like 1 silence also altered the tegument structures of the worms and hindered its development of reproductive glands. These all suggested that SjELAV-like 1 play an important role in the development of the reproduction of S. japonicum.

Key words: Schistosoma japonicum, SjELAV-like 1, cloning expression, immunolocalization, RNAi, electron microscope