Transcriptomic And Proteomic Analysis Of Metorchis Orientalis And Identification Of Immune-related Genes

Metorchis orientalis inhabits in the gallbladder and bile duct of poultry and some mammals as well as humans.It can cause gallbladder enlargement,biliary tract congestion and even death.Due to the similarities between M.orientalis and Clonorchis sinensis,such as the morphology of metacercariae,endemic areas,intermediate hosts,parasitic sites and clinical symptoms.Thus,metorchiasis is likely to be misdiagnosed as clonorchiasis,which is maybe an important neglected parasitic zoonosis.M.orientalis is mainly distributed in Asia,including China,especially in Fujian,Anhui and Heilongjiang province.In recent years,the reports about infection rate of M.orientalis were uptrend,and causes serious economic losses and public health problems.Despite recently some reports about M.orientalis were published focus on morphology,life cycle,infection of experimental animals,epidemiological and molecular classification,the information about the transcriptome,proteome and immunological diagnosis of M.orientalis is still rare.Thus,we performed the transcriptome and proteome on three developmental stages,the biological and metabolic characteristics were revealed by analyzing the differences expression gene/protein between transcriptional and proteome in three developmental stages.Candidate genes related to the immunity of M.orientalis were screened based on the dataset of transcriptome and proteome,then veridied immunogenicity by prokaryotic expression systerm,and developed the indirect ELISA diagnostic methods of M.orientalis.The main results were as following:1.we performed de novo sequencing,transcriptome assembly and functional annotations of three stages of M.orientalis,including adult stage,metacercariae stage and egg stage.A total of199,723 unigenes with mean length of 1,012 nt and N50 length of 1,396 nt were obtained,and91,398 unigenes annotated by BLASTn and BLASTx searches against the available database.Among these unigenes,76,247(38.17%),34,046(17.04%),23,640(11.83%),19,141(9.58%),35,633(17.84%),50,577(25.32%)and 50,449(25.25%)unigenes were assigned to NR,NT,KOG,KEGG,Swiss-Prot,GO and Pfam databases,respectively.A total of 23,071 distinct express genes(DEGs)were found in three developmental stages.Of these DEGs,13,823 genes in adult versus metacercariae,10,343 genes in adult versus egg,and 18,348 genes in egg versus metacercariae,respectively.GO analysis showed that DEGs were mainly enriched inmicrotubule synthesis,protein metabolism and reproductive behavior in the adult stage,and DEGs were mainly enriched in larval development,embryonic development and nuclear division in the metacercaria stage,while DEGs were mainly enriched in macromolecular complex assembly,neuron development and cytoskeletal part.We identified a pathway associated with liver fibrosis by KEGG analysis,namely TGF-? signaling pathway.DEGs in three development stages were selected to validation using q PCR,the results showed that the expression trend of each gene was consistent with the results of transcriptome sequencing.2.A total of 3,616 protein were identified by the proteome among adult stage,metacercariae stage and egg stage of M.orientalis,and 2,571 differentially expressed proteins(DEPs)were found in three developmental stages.Of these DEPs,1,445 proteins in adult versus metacercariae,1,728 proteins in adult versus egg,and 1,936 proteins in egg versus metacercariae,respectively.GO and KEGG analysis showed that DEPs were significantly enriched in protein folding,muscle contraction regulation and glycolysis,and were mainly take part in protein processing in endoplasmic reticulum,ribosome and gap junction pathway in the adult stage.DEPs were significantly enriched in microtubule motor activity,ATPase activity and helicase activity,and were mainly take part in pathways such as spliceosome,RNA transport and DNA replication in egg stage.DEPs were significantly enriched in phosphotransferase activity,glucose-6-phosphate dehydrogenase activity and aconitate hydratase activity,and were mainly take part in pathways such as valine,leucine and isoleucine degradation,fatty acid degradation and glycerolipid metabolism in metacercariae stage.DEPs in three development stages were selected to validation using PRM,the results showed that the expression trend of each protein was consistent with the results of proteome sequencing.The correlation analysis of DEGs and DEPs verified that transcriptome and proteome had a good correlation in quantity,expression abundance,functional classification and metabolic pathways between m RNA and protein lever.3.A total of 392 excreted and secreted proteins(ESPs)were identified by mass spectrometric analysis of M.orientalis,all of which were associated with transcriptome and protein database.Based on the results of ESPs combined with transcriptome and proteome,7candidate genes related to immunity were screened out,and the prokaryotic expression vectors of candidate genes were successfully constructed,and the immunogenicity was identified bywestern blot.r Mo ENO1 and r Mo CTSF were selected as diagnostic antigens for serological detection of M.orientalis.The sensitivity of indirect ELISA for r Mo ENO1 and r Mo CTSF were1:1600 and 1:800,respectively.There was no cross reaction with duck coccidia,Amphimerus anatis,Echinochasmus japonicus,Tracheophilus cymbius and Echinostoma revolutum.Clinical samples were detection by the indirect ELISA methods,the resluts showed that r Mo ENO1 and r Mo CTSF had no significant difference in positive detection rate(P>0.05),which could be used for the serological diagnosis of metorchiasis.In conclusion,we performed the transcriptome and proteome sequencing on three developmental stages,and established the indirect ELISA diagnostic method for M.orientalis.The results can be insight into the biological characteristics and regulation mechanism for three development stage of M.orientalis,provides important data for pathogenic mechanism and immune prevention of M.orientalis.