Transcriptome Analysis Of The Encystation Process Of Echinococcus Granulosus Protoscoleces And The Gene Cloning And Eukaryotic Expression Of Egpka Gene

Cystic echinococcosis(CE),also called hydatid disease,is a chronic neglected zoonotic disease caused by the larvae of Echinococcus granulosus,which seriously endangers human health and causes huge economic losses in the animal husbandry.However,due to the lack of in-depth understanding of the growth,development and formation of hydatid cystic fluid,the prevention and treatment of CE have been lack of innovative methods.In this study,the RNA-seq technology was first used to detect the gene transcription expression levels of E.granulosus Protoscoleces(PSCs)at different developmental stages in vitro,and differentially expressed genes with significant up-regulation during the process of PSCs encystation into hydatid cysts were screened for enrichment analysis of KEGG and GO database.It was found that the vasopressin-regulated water reabsorption metabolic pathway is closely related to the formation of cystic fluid,and this pathway is mainly transports water and small molecular substances by protein kinase A(PKA)mediated vesicle transport.Then,Echinococcus granulosus protein kinase A(EgPKA)gene sequence was amplified and its physicochemical properties,subcellular localization,secondary and tertiary structures,epitopes and codon usage bias were predicted by multiple bioinformatics methods.The results showed that,compared with the NCBI reference sequence,the EgPKA coding region showed a deletion of 48 bp from 417–464 bp,with a total length of 1053 bp,encoding 350 amino acids.Notably,sequence comparison analysis revealed that the characteristic conserved domains of EgPKA after mutation were not changed,such as ATP binding site(GTGSFGRV),Ser/Thr activation site(RDLKPEN),and PKA regulatory subunit binding site(LCGTPEY).In addition,EgPKA has multiple epitopes and immunoreactivity.Finally,the EgPKA was cloned and the recombinant strain GS115/pPIC9K-EgPKA was constructed in Pichia pastoris.After purifying the target protein,its reactivity with serum antibodies was studied,and the results showed that the optimal protein production was achieved by cultivating the culture with 1.0 % methanol for 72 h.The recombinant protein which purified by high affinity NI-NTA resin exhibited specific reactivity with immune sera.Apart from that,the transcriptional expression level of EgPKA was detected through Quantitative real-time PCR(QPCR)detect.The results showed that EgPKA had the same rise expression trend in the culture of PSCs at different developmental time in vitro and in the echinococcus cyst wall tissues of different developmental time in mice infected with PSCs.This is the first report on the transcriptome information of E.granulosus PSCs encystation into hydatid cysts;we found and verified the EgPKA coding region sequence had a gene deletion mutation;EgPKA eukaryotic expression system was successfully constructed;The recombinant protein showed a good immunoreactivity to the sera of mice infected with hydatids,and had the potential value as a clinical diagnostic antigen against CE,which layed a foundation for the formation mechanism of cystic fluid.