The Prokaryotic Expression And Preliminary Function Research Of Echinococcus Granulosus IAP?BIRP?ADK1?ADK8 Genes

The larval stage of Echinococcus granulosus sensu lato(s.l.),resulting in cystic echinococcosis(CE)-a parasitic zoonosis,causes huge economic losses to the livestock industry and poses a threat to public health.The disease occurs in most parts of the world,and China is a high incidence area.In this study,we screened two inhibitor of apoptosis protein genes(Eg-IAP,Eg-BIRP)and two adenylate kinase genes(Eg-ADK1,Eg-ADK8)in the transcriptome database of Echinococcus granulosus,combined with bioinformatic analysis to describe the structure prediction of the proteins.The relative transcriptional levels of four genes in different developmental stages of Echinococcus granulosus were analyzed by relative fluorescent quantitative PCR(qRT-PCR).And the recombinant proteins were subjected to western blotting and fluorescence immunolocalization.Meanwhile,the pan IAPs family inhibitor-LCL161 drug was used to induce apoptosis in the protoscoleces(PSCs)in vitro.The morphological changes of PSCs were observed by light microscopy and transmission electron microscopy(TEM),the apoptosis rate of the PSCs was detected by terminal deoxynucleotidyl transferase dUTP nick end labelling(TUNEL)assays.And the transcriptional differences of Eg-IAP and Eg-BIRP genes after PSCs were treated with the LCL161 drug.The results of this study are as follows: 1.The prokaryotic expression and preliminary function reserach of Echinococcus granulosus Inhibitor of Apoptosis Protein genes(Eg-IAP,Eg-BIRP)Inhibitor of apoptosis proteins(IAPs),a family identified in various species,plays a pivotal role in inhibiting cell apoptosis.In this study,two Eg-IAPs(Eg-IAP,Eg-BIRP)were cloned and prokaryotically expressed.Eg-IAP and Eg-BIRP encoded putative 331 and 168 residue proteins,respectively.Bioinformatic analysis showed both proteins contained a BIR domain-the essential functional domain of IAPs.The result of western blotting showed that the two rEg-IAPs could be identified by the CE-positive sheep sera,which showed a good immunogenicity profile.Fluorescence immunohistochemistry revealed that except infertile cysts,both proteins were ubiquitously localized in the tegument and hooks of PSCs,the tegument and parenchyma of adult worms(18 d),and the germinal layer of fertile cysts.And through qRT-PCR,we found that relatively higher transcription levels of two Eg-IAPs in PSCs compared to the adult worms(18 d).We further used different concentrations of LCL161,a pan-IAPs inhibitor,to induce the apoptosis in PSCs in vitro,and revealed that the transcription levels of both genes were negatively correlated with the concentration of LCL161.While the results of light microscopy,TEM,and TUNEL assay also showed a higher apoptotic rate in PSCs with the increasing concentrations of LCL161 after 8 h.Taken together,our findings provide the reasonable evidence that both Eg-IAP and Eg-BIRP have potential implication in critical anti-apoptotic roles during the development of E.granulosus.2.The prokaryotic expression and preliminary function reserach of Echinococcus granulosus Adenylate Kinases genes(Eg-ADK1,Eg-ADK8)Adenylate kinases(ADKs),a family of phosphotransferases,is widely present in various organisms.It is mainly found in tissues with high energy consumption and plays a pivotal role in the growth and development of organisms.In this study,two adenylate kinases genes of E.granulosus(Eg-ADK1,Eg-ADK8)were cloned and prokaryotically expressed.The Eg-ADK1 gene encoded 197 amino acids,and the Eg-ADK8 gene encoded 344 amino acids.The result of western blotting showed that the two rEg-ADKs could be identified by the CE-positive sheep sera,which showed a good immunogenicity profile.Fluorescence immunohistochemistry showed that Eg-ADK1 and Eg-ADK8 proteins were widely distributed in the hooks and tegument of the PSCs,the germinal layer of the fertile cysts and infertile cysts,and the tegument and parenchymal region of the adult worms(18 d).The qRT-PCR showed that the transcription levels of Eg-ADK1 and Eg-ADK8 genes were similar in the PSCs and adult worms(18 d).The above results showed that Eg-ADK1 and Eg-ADK8 genes play important roles in the growth and development of Echinococcus granulosus.