| Cerebral coenurosis,caused by the larval Coenurus cerebralis of Taenia multiceps,is a fatal central nervous system disease in sheep and other herbivores,as well as a parasitic zoonosis,occasionally happened in human beings.The life cycle of T.multiceps consists of adult,Coenurus cerebralis and oncosphere stages.And the Coenurus cerebralis is composed of cyst fluid,protoscolices and cyst wall.In order to adapt to its parasitic life,different genes of the parasites were expressed at differently developmental stages.Study of the stage-specific genes is of significance to provide insights into the mechanism of development biology and parasitic life of T.multiceps.In this study,RNA-seq was applied to identify transcriptions in the life cycle of T.multiceps,including oncosphere(Onc),protoscolices(Pro),cyst wall(Cyst)and adult(Adu),as well as scolex-neck proglottids(Snp),immature-mature proglottids(Imp)and gravid proglottids(Grp)of the adult stage.A total of 12822 genes(FPKM>0)were identified.Pair comparision of the differentially expressed genes among Onc,Pro,Cyst and Adu,as well as among Snp,Imp and Grp,showed that the number of up-regulated genes in Adu was more than other stages,and the number of up-regulated genes in Imp was more than Snp and Grp.Besides,a large number of genes related to the development and parasites-host interaction were identified.Further analysis indicated that 2437 genes were specific up-regulated in different stages,and that 1228 genes were specific up-regulated in different tissues of adult stage.GO analysis suggested that the specific up-regulated genes in stages and tissues of adult mainly participate in the function of cell process and metabolic process,binding and catalytic activity,cell and cell part.KEGG analysis showed that specific up-regulated genes are mainly involved in genetic information processing,cellular processes and environmental information processing.The result of qRT-PCR made sure that the mRNA transcription levels of the DEGs were basically consistent with the results of transcriptomics analysis,which demonstrated the reliability of RNA-seq analysis.According to the results of transcriptome analysis,the protein kinase A catalytic subunit gene family was screened for further study.The gene family contains four protein kinase A catalytic subunit genes,named TmPKA-C1,TmPKA-C2,TmPKA-C3 and TmPKA-C4,respectively.Specific primers of four TmPKA-C genes were designed based on the open reading frame(ORF)and RT-PCR was applied to amplify the full ORF from total RNA of adult.Sequence analysis showed that four TmPKA-C genes all contained typical serine/threonine protein kinase activity sites,protein kinase ATP binding sites and protein kinase domains.Based on the results of bioinformatics analysis,TmPKA-C1 and TmPKA-C2 proteins were selected to study their reactinogenicity.The TmPKA-C1 and TmPKA-C2 gene fragments were ligated into pET30 a prokaryotic expression vector to construct recombinant expression plasmids,which were transfected into Transetta(DE3)to induce their expression.SDS-PAGE analysis indicated that the recombinant proteins of TmPKA-C1 and TmPKA-C2 were highly expressed in the prokaryotic system with molecular sizes of 42 kDa and 46 kDa,respectively,mainly in the form of inclusion bodies.Western blotting showed that both of recombinant TmPKA-C1 and TmPKA-C2 proteins could recognize by sera from sheep infected with Coenurus cerebralis,but a weaker reaction was detected for TmPKA-C2.The reactinogenicity of the recombinant TmPKA-C1 protein was further identified suggesting that it could have specific reaction with the sera of artificially infected sheep with coenurosis after 1-19 weeks.The study laid the foundation for further development of new diagnosis methods for coenurosis of sheep. |
