| Psoroptes ovis is widespread in the world.P.ovis survives on the epidermis of domestic and wild animals,for example,sheep,buffaloes,yellow cattle,horses,rabbits,deer,wild camels,foxes,minks,etc.P.ovis eats hosts’ body fluids,lymph and exudates,which causes hosts intense pruritus,extensive dermatitis,depilation,emaciation,even death.P.ovis can cause great economic losses to the animal husbandry.In this paper,Psoroptes ovis var.cuniculi transcriptome has been analyzed.Besides,fatty acid-binding proteins and other two functional genes has been studied.The results of the study will provide a reference for the diagnosis and prevention of psoroptic mange.1.Preliminary analysis of P.ovis var.cuniculi transcriptome in different developmental stagesThe transcriptome of P.ovis var.cuniculi was assembled and analyzed using next-generation sequencing technology(Illumina),efficient Trinity software and bioinformatic tools.Their samples were derived from P.ovis var.cuniculi larvae(i.e.the Pso_L group)and nymphs and adults(i.e.the Pso_N_A group).We obtained 59.4 million and 55.4 million raw reads from the Pso_L and Pso_N_A groups,respectively,by RNA-Seq.After a rigorous screening process,56.6 million clean reads from the Pso_L group with a Q20 of 96.91%and a GC of 35.85%were retained.There were 52.9 million clean reads from the Pso_N_A group with a Q20 of 96.54%and a GC of 34.90%.Obtained clean reads were assembled into 38,836 unigenes.The mean length and N50 were 825 bp and 1,607 bp,respectively.On the basis of sequence similarity with seven databases(NR,NT,KO,SwissProt,PFAM,GO and KOG),a total of 17,366 unigenes were annotated.We identified 17,731 CDS from the 38,836 unigenes in the P.ovis transcriptome.Of these,12,950 CDS were identified using the Blastx algorithmic program;4,781 CDS were identified by the ESTScan software.Through analyzing differentially expressed genes in the Pso_L(larval)and Pso_N_A(nymph/adult)groups,a total of 1,316 DEGs were identified,including 496 upregulated unigenes and 820 downregulated unigenes in the Pso_L group compared with the Pso_N_A group.The unigenes of P.ovis from eachdevelopmental stage and the unigenes differentially between developmental stages were compared with allergen protein sequences contained in the allergen database website to predict potential allergens.We predicted 205 allergens genes in the two developmental stages similar to genes from other mites and ticks,of these,14 were among the upregulated DEGs and 26 among the downregulated DEGs.The analysis of DEGs and putative allergen genes may lay the foundation for studies of functional genomics,immunity and gene expression profiles of this parasitic mite species.2.Prokaryotic expression of P.ovis var.cuniculi FABP and Tnl genes and diagnostic value assessment of their recombinant proteinFatty acid-binding proteins(FABPs),multigenic cytosolic proteins are found in most animal groups.They are involved in the uptake and transport of hydrophobic ligands to different cellular fates.Troponin I(Tnl)is a protein that belongs to the troponin protein family,which consists of three separate proteins that each play an important role in regulating the interaction of actin and myosin filaments.Tnl is a molecular switch of muscles’ excitation-contraction,and it can inhibit myosin atpase activity.In this study,FABP and TnI genes were amplified from the total RNA of P.ovis.The FABP and TnI cDNA sequences consisted of an open reading frame of 399 bp and 603bp,encoding putative proteins with 132 and 200 amino acid residues,repectively.The FABP and TnI proteins were predicted to have molecular weights of 15 kDa and 24kDa,pI of 5.79 and 9.78,respectively.C656H1027N183O208S7 and C1052H1754N314O305S10 were the deduced chemical formula of FABP and TnI genes.Molecular mass of recombinant FABP and TnI expressed in E.coli were respectively about 19Kda and 28Kda.The two protein could react with positive sera from rabbit infected P.ovis.When recombinant FABP antigen was used to detect anti-FABP antibody in the ELISA,the specificity and sensitivity were respectively 84.21%(32/38)and 89.66%(26/29);When recombinant TnI antigen was used to detect anti-TnI antibody in the ELISA,the specificity and sensitivity were respectively 92.11%(35/38)and 96.55%(28/29).Preliminary results indicated that TnI was better suitable as a diagnostic antigen and has a greater diagnostic potential.3.Characteristic analysis of Pso o 7 from P.ovis var.cuniculiBased on P.ovis transcriptome data,the gene-specific primers for lipopeptide binding protein Pso o 7 were designed.Pso o 7 gene was amplified from the total RNA of P.ovis.The Pso o 7 cDNA sequence consisted of an open reading frame of 645bp.The analysis of SignalP V4.1 software revealed that the gene had a signal peptide,after removing the signal peptide,the expression sequence length was 597bp,encoding putative proteins with 198 amino acid residues.The Pso o 7 protein was predicted to have a molecular weight of 22kDa,a pI of 5.24.C1003H1615N265O295S4 was the deduced chemical formula of Pso o 7 gene.Molecular mass of recombinant Pso o 7 expressed in E.coli was about 42Kda.The protein could react with positive sera from rabbit infected P.ovis.Immunofluorescence staining showed that Pso o 7 were highly localized to chewing mouthpart,leg and within the epidermal integument.The result of intradermal skin tests showed recombinant protein Pso o 7 could cause an intense immediate hypersensitivity,which promoted the self-grooming behavior of animals,and Pso o 7 could not cause late phase reaction that may promote the development of scabies lesions.Meanwhile,histopathological observation found that the degree of the dermal tissue lesions and inflammatory response at 24h was more light than that at 1h.In this study,it was suggested that Pso o 7 could be used as vaccine candidate molecules of psoroptic mange and lay the foundation for the future development of vaccine research. |
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