Effect Of Recombinant Tropomyosin Of Psoroptes Ovis On The Biological Function In Keractinocyte And Evaluation Of Its Allergenicity

Psoroptes ovis is a common ectoparasite on the skin surface of domestic and wild animals,and causes allergic skin diseases characterized by loss of weight,pruritus,crusting and skin thickening,resulting in serious animal welfare issue and huge economic losses in the animal industry.The P.ovis transcriptomic data showed that there were a large number of potential allergen genes in P.ovis^[1],and that both P.ovis infestation and its antigen intradermal injection induced the skin allergic inflammation with eosinophil（EOS）infiltration in the host,and the severity of this skin allergic inflammation was closely related to the severity of psoroptic mange^[2].Additionally,P.ovis antigens could regulate the biological function of host keratinocytes^[3],and this process mighte influence the development of the skin allergic inflammation.However,the P.ovis antigen components could affect keractinocyte function and cause allergic skin inflammation were still uncharacterized.Researchers proved that Tropomyosin（TM）,an allergen in many invertebrates,has been shown to cause allergic inflammation in the airway and gastrointestinal tract.In our previous study,we found the presence of TM（Pso TM）in P.ovis excretory secretory proteins by mass spectrometry,so we hypothesized that Psoroptes mites might secrete native Pso TM protein into the skin lesion,and regulat host keratinocyte function and trigger allergic skin inflammation.Therefore,in the present study,we would confirm this hypothesis both in vitro and in vivo,and our results would provide the theoretical basis for further understanding of the pathogenesis of P.ovis.1 Prokaryotic expression,transcriptional levels and localization analysis of TMs in P.ovisIn this study,Pso TM1 and Pso TM2 were successfully obtained by amplification,which contained complete ORFs of 749 bp and 855 bp,respectively,and both were mainly composed ofα-helices with conserved structural domains of TMs and showed typical rod-like structures in tertiary structure,which were consistent with the structural features of TM family.The recombinant proteins（r Pso TM1 and r Pso TM2）obtained by prokaryotic expression were approximately 49 k Da and 55 k Da,respectively,and they were both recognized by the sera of P.ovis-infected rabbits.Pso TM1 and Pso TM2 were transcribed at significantly higher levels in both adult female and male than in nymph and lavar,and Pso TM2 was transcribed at higher levels in both adult female and male than Pso TM1.The natural Pso TM1 and Pso TM2 proteins were mainly distributed in the epidermal spine and granular layer of the P.ovis-infested rabbit ear lesions,indicating that the proteins could be secreted by the Psoroptes mites.In conclusion,Pso TM1 and Pso TM2 were the excretory secretory proteins of P.ovis,and these proteins were be excreted and secreted by the Psoroptes mites into the host skin surface.These results might lay the foundation for the following functional studies of Pso TM1 and Pso TM2.2 Effects of recombinant TMs of P.ovis on biological functions of keratinocyteThe above chapert one showed that P.ovis secreted Pso TM1 and Pso TM2 into the epidermal spine and granular layer of the host skin lesion,and the main constituent cells of these positions were keratinocytes,so in this section the human immortalized keratinocyte（Ha Ca T）was used as the in vitro model to investigate the effect of two recombinant TMs of P.ovis（r Pso TM1 and r Pso TM2）on the biological functions in cells.The results showed that both high concentrations（10μg/m L）of r Pso TM1 and r Pso TM2 significantly inhibited cell proliferation and induced cell apoptosis（P<0.05）,while no-proliferative concentrations（5μg/m L）of r Pso TM1 and r Pso TM2 had no significant effects on cell migration and ROS production（P>0.05）.r Pso TM2 was able to significantly promote Ha Ca T secretion of IL-6 and TNF-α（P<0.05）,while r Pso TM1 had no effect.In conclusion,both r Pso TM1 and r Pso TM2inhibited the proliferation and induced the apoptosis of keratinocytes,but had no significant effect on cell migration and ROS production.3.Evaluation of the sensitization of recombinant TMs in P.ovisIn this section,rabbits naturally infected with P.ovis were used as in vivo model.r Pso TM1and r Pso TM2 were injected intradermally in these rabbits,and the wheal reaction and redness reaction reaction of rabbit skin at different time points were observed,then collected the injected skin for histopathology and fluorescence quantitative PCR assay at the time of the strongest skin reaction to clarify the skin allergic inflammatory cell infiltration and the level of chemokines in the skin.The results showed that r Pso TM1 and r Pso TM2 caused local skin redness and swelling in rabbits at 1 h and 2 h after injection,respectively,and both peaked at10 h after injection and gradually subsided thereafter.r Pso TM1 and r Pso TM2 significantly induced the infiltration of inflammatory cells and eosinophils in the local skin（P<0.05）.Further,the mouse permeation assay revealed that r Pso TM1 significantly promoted vascular permeability in mice（P<0.05）.In conclusion,both r Pso TM1 and r Pso TM2 could induced skin allergic inflammation,and the allergenicity of r Pso TM1 was less than that of r Pso TM2.