Transcriptomic And Proteomic Analysis Of Parabronema Skrjabini And Identification Of Immune-related Genes

Parabronema skrjabini is blood-feeding nematode which resides in abomasum of ruminants,and camels are considered as the best definitive host.Diarrhea,anemia and even death may occur if hosts are infected with P.skrjabini seriously.This parasite poses a threat to camel's health,which can be a disaster to the local economy.Unluckily,only few research associated with basic theories,omics,immunological diagnosis and immune prophylaxis of P.skrjabini can be found until now.In our study,P.skrjabini at four specific stages,which including the egg,the third larva,adult female and adult male were identified by RNA-Seq and iTRAQ,and the results of sequencing were tested by qPCR.The integrated analysis of transcriptomic and proteomic data fully indicated the multiple biological characteristics and the differences in mRNA and protein expression of P.skrjabini at different developmental stages,and explore some secretory genes or proteins which were involved in immune response.Then,we verified immunogenicity of three special secretory genes by prokaryotic expression systerm,and developed the iELISA diagnostic methods of Parabronemiasis.This study provides the foundation for further investigation of prophylaxis and treatment on Parabronemosis.The main resluts were as following:1.More than 10GB data were obtained through the transcriptome sequencing of four stages.54102 Unigenes were identified by qulity control and de novo assembly method,of these,33816 Unigenes were differentially expressed genes,32006 Unigenes were found functional annotation.The gene temporal expression analysis indicated that expression pattern characteristics of P.skrjabini at four specific stages.In egg stage,most of fuctional genes highly enriched in protein synthesis.In the third larva stage,genes were mainly involved in larvae development and autoimmune protection processes.In male stage and feamle stage,genes were respectivly involved in processes of spermiogenesis and energy metabolism,and fertilization and egg activation in response to sperm entry.12 genes were selected to detect the validation rate using qPCR,11 genes expression tendency were consistent with RNA-Seq.Comparative transcriptomics elucidated the number,functional classifications and KEGG pathway of differentially expressed genes(DEGs)to improve understand the characteristics of expression profiles changes in pairwise comparation of egg and the third larva,the third larva and adult(coFemale/Male),and fenale and male.Our study predicted 1975 secretory genes which were likely to play roles in immunoregulation,as well as identified some significant functional secretory genes.2.The proteome of the third larva,adult female and adult male of P.skrjabini were analysed using iTRAQ technology.A total of 2072 proteins were generated,of which 2009 proteins were functionally annotated.The differentially expressed proteins(DEPs)in pairwise comparation analysis illuminated differentially expressed proteins in comparation of adult and the third larva were mainly associated with larval development,the positive of growth and determination of adult lifespan,and involved in ribosome,oxidative phosphorylation and Pyruvate metabolism pathways.In female and male comparation,differentially expressed proteins were not only highly enriched in reproduction-related function,but also involved in regulation of actin cytoskeleton,vascular smooth muscle contraction and PPAR signaling pathways.Moreover,we predicted 197 secreted proteins,and further identified their expression3.A joint analysis of differentially expressed genes and differentially expressed proteins verified that there exist high correlation in terms of number,expression quantity,functional classification and metabolic pathways between mRNA transcripts and generated protein4.Screening and cloning nine genes act as diagnostic antigen candidates based on analysis of secretory genes proteins,and successfully constructed BL21(pETSTPK),BL21(pETCPI)and BL21(pETCPR)three recombinant expression system.Western blotting analysis illustrated that three recombinant proteins exclusively reacted with camels' serums which were infected by P.skrjiabini,suggested three recombinant proteins were considered as high specificity and good immunogenicity.5.Three indirect ELISA approaches for detecting the antibodies against P.skrjabini recombinant antigens rSTPK?rCPI and rCPR were established.Serum from 81 camels with Parabronemiasis and 9 normal camels without Parabronemiasis were tested to assess three indirect ELISA diagnosis value.The specificity of three recombinant antigens were all 100%,the sensibility showed that rSTPK was 97.5%,rCPI was 98.8%and rCPR was 96.3%,respectively,and the repeatability of three recombinant antigens were high.Furthermore,the field test of 140 camels' serums through three indirect ELISA approaches found that positive rates of three antigens were not significant differences(P>0.05),therefore,the established indirect ELISA approaches using recombinant antigens rSTPK,rCPI and rCPR would be applied to serological diagnosis of Parabronemiasis.