Using DIA Method To Study The Effect Of Protein Abundances And Phosphorylation Of Rabbit Skin Bitten By Haemaphysalis Longicornis

Ticks are blood-sucking arthropods in vitro carrying and spreading pathogens.They are detrimental to not only human health,but also animal husbandry.In this study,different stages of Haemaphysalis longicornis were used to bite the New Zealand white rabbit.Dynamic changes of proteins expression and phosphorylation in rabbit skin tissue bitten by different stages ticks were analyzed.Systematic analysis of differentially expressed proteins and proteins with modifications was made to identify key immune response proteins in mammals that are resistant to ticks,and to explore the role these proteins play in tick bites.In this study,the novel Data Independent Acquisition(DIA)quantitative proteomics technique was used to analyze the changes in protein expression and phosphorylation in rabbit skin tissue bitten by larva,nymph and adult ticks.The results showed that 636 differentially expressed proteins were co-identified in rabbits bitten by larva at 24 h,48 h,and 72 h;317 differentially expressed proteins were co-identified in rabbits bitten by nymph at 24 h,48 h,and 72 h;375 differentially expressed proteins were co-identified in rabbits bitten by adult at 24 h,48 h and 72 h.Finally,a total of 202 proteins were differentially expressed at 9 time points in these three periods.Among them,89 proteins were up-regulated with the time of blood-sucking,and 113 proteins were down-regulated.Bioinformatics analysis showed that the phagocytic pathway plays an important role in the host's defense against H.longicornis.Proteins that play important roles include coronin,NADPH oxidase,calnexin,calreticulin and two lectins(Mannose receptor C-type 1 and DC-SIGN).These proteins combine to initiate an immune defense mechanism,excreting foreign substances introduced by H.longicornis and the metabolites produced by the rabbit itself by phagocytosis.Proteins such as ezrin,integrin alpha,integrin beta,and cadherin play important roles in Blood coagulation and wound healing.These proteins regulate growth of vascular endothelial cells,promote the formation and development of blood vessels and blood coagulation by promoting cell proliferation,cell migration,and platelet aggregation.In addition,the two proteins,trichohyalin and peptidyl arginine deiminase 3,play an important role in the formation of hair follicles and hair.The bite of H.longicornis causes a decrease in hair on the surface of rabbit skin,which may be inseparable from the down-regulation of these two proteins in rabbit skin.The bite of the H.longicornis caused a significant change in the phosphorylation of the protein in the rabbit skin.The results showed that 1478 differentially expressed phosphorylation sites were co-identified at 24 h,48 h,and 72 h in rabbits bitten by larva,involving 590 phosphorylated proteins;931 differentially expressed phosphorylation sites were co-identified at 24 h,48 h and 72 h in rabbits bitten by nymph,435 phosphorylated proteins were involved,and 1013 differentially expressed phosphorylation sites were co-identified at 24 h,48 h,and 72 h in the rabbits bitten by adult,involving 449 phosphorylated proteins.Finally,a total of 435 differentially expressed phosphorylation sites were co-identified at 9 time points in these three periods,of which 126 were up-regulated with the time of blood-sucking and 276 were down-regulated,involving 215 phosphorylated proteins.Bioinformatics analysis of phosphorylated proteins involved in differentially expressed phosphorylation sites revealed that the bacterial invasion of epithelial cells pathway,endocytic pathway,tight junction pathway,and T cell receptor signaling pathway play important roles in the process that the host resists H.longicornis.Phosphorylated proteins play a key role.The interleukin 4 receptor is involved in a series of immunoregulatory mechanisms to regulate the inflammatory response.Nck adaptor 1 and Pak2 are closely related to vascular lumen formation.There are also some phosphorylated proteins,such as myosin VA,epidermal growth factor receptor pathway substrate 15,protocadherin 1 and so on,relating to immune function and cell migration.These phosphorylated proteins may play an important role in the host's defense against H.longicornis.The protein screened in this experiment is likely to become a drug target for prevention and control of H.longicornis in the future.This study not only provides theoretical support for the early diagnosis of tick-borne diseases,but also provides new ideas for the development of anti-tick vaccines and related drugs.This study also provides new methods for further research and exploration of integrated prevention and control of ticks.