Cloning,Expressing And Preliminary Function Exploration Of ELAV(embryonic Lethal Abnormal Vision System)-like 1 Of Schistosoma Japonicu

Schistosomiasis is a zoonotic parasitic disease caused by schistosomiasis infection that is a serious threat to social and economic development.Eggs produced by females are the main cause of pathological damage,and it is also the original cause of the disease.Therefore,it is of great significance to study the gene functions related to the reproductive development of schistosomes in order to control the occurrence of schistosomiasis from the reproductive development direction of schistosomiasis.In this study,Schistosoma japonicumembryonic lethal abnormal vision like 1(Sj ELAV-like 1)gene was cloned and expressed and we analyzed the expressing situations at different stages and infected conditions,and its distributions in Schistosoma japonicum(S.japonicum).This study also intend to explore the effects of Sj ELAV-like 1 on the form and reproductive development of S.japonicum.The PCR technique was used to obtain the sequence of the Sj ELAV-like 1 gene(Gene Bank accession number: MG515727)and a 1797 bp sequence of Sj ELAV-like 1 was obtained,which coding region was 1533 bp encoding 510 amino acids.The recombinant plasmid p ET28-Sj ELAV-like 1 was constructed and expressed in E.coli BL21 induceing by IPTG,then the products were collected after 0h,1h,2h,3h,4h,6h and 8h.The SDS-PAGE method was used for protein electrophoresis.We found that the the expression of the recombinantplasmid reached its highest amount after 4h induced by IPTG and it mainly existed in inclusion bodies.His-tag nickel column affinity chromatography method was used to purify the recombinant protein,which was used to immune mouse to obtain the polyclonal antibody and it showed good immunogenicity analyzed by western blotting.Indirect immunofluorescence assay was applied to detect the distributions of Sj ELAV-like 1 protein in S.japonicumand the result showed that it was mainly located in the tegument of the worms..The mice were infected by cercaria in the abdomen,and the worms at different stages as well as the females and males separated by the paired worms of different stages were collected,respectively.The cercaria escaped from single nail was used to infect mice to obtain the unisexual worms at 25 th day,and the cercaria escaped from mixture nails were used to infect mice to obtain the females and males separated by the paired worms at the same time.The total RNA was extracted by using Trizol.The expressing situations of Sj ELAV-like 1 were detected by real-time 61 quantitative PCR(q RT-PCR).The result showed that Sj ELAV-like 1 was expressed at all different stages in S.japonicum and the expressing was stable in males but gradually down in females as the maturing of the worms.The expressing of Sj ELAV-like 1 was significantly high in developmental repressing females while there was no significantly different in males,compared with the normal worms.The best small interfering RNA(si RNA)selection was performed by mice tail vein injection,and it was used to perform the long-term RNA interference(RNAi)assay to analysis the effects of Sj ELAV-like 1 gene silence on the spawning and egg hatching of females.In the RNAi assay,S2 si RNA showed a better interference effect compared with the control group,and 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.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.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,the cells were swollen and the intercellular space was widen.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 in the females of S.japonicum.Part sequence of Sj ELAV-like 1was successfully cloned and expressed,which was highly expressed in developmental repressing females.Sj ELAV-like 1 was mainly expressed in the tegument of S.japonicum.Sj ELAV-like 1 silence led to liver egg burden,liver egg burden by per female and egg hatching rate being significantly decreased.The tegument structures of the worms have been distinctly altered and the development of reproductive glands also has been hindered.These all suggesting that Sj ELAV-like 1 plays an important role in the development of the reproduction of S.japonicum.