Regulation Of Immune Function Of Recombinant Arginine Kinases In Sarcoptes Scabiei On Host Peripheral Blood Mononuclear Cells(PBMSs)

Scabies is a widely distributed ectoparasitic disease.Its geographical distribution and the number of parasitic host species show a continuous growth trend.The disease affects the growth and development of economic animals,and leads to the death of animals.At the same time,scabies is also an important zoonotic parasitic disease,and was included in the list of neglected tropical diseases in 2017.Globally,approximately300 million people are affected by scabies each year.Participating in parasite-host interaction and causing host immune responses,arginine kinases are considered to be immune regulators of pro-inflammatory cytokines and effector.At present,the pathogenic mechanism of S.scabiei is incompletely clear,and the function of arginine kinase in the interaction between S.scabiei and host is still lacking.In this study,bioinformatics analysis and prokaryotic expression of two different arginine kinases from the S.scabiei arginine kinase family were carried out.The immunoreactivity,enzyme activity and localization in S.scabiei and host skin were analyzed.The two recombinant proteins at four different concentration gradients were used to stimulate the host’s peripheral blood mononuclear cells(PBMCs);the effects of the recombinant proteins on cell proliferation,apoptosis,and migration were observed,and the m RNAs of apoptosis-related factors and NF-κB Transcript level and secretion level of Th1/Th2,Treg/Th17 related cytokines were detected.The main research findings are as follows:1.Prokaryotic expression and protein characteristics analysis of arginine kinases from S.scabiei.Ss AK-1 and Ss AK-2 genes were successfully cloned,and had typical ADP binding sites,arginine binding sites and substrate specificity loops.r Ss AK-1 and r Ss AK-2 were successfully obtained through prokaryotic expression,and had good reactogenicity and certain catalytic activity by WB and enzyme activity assay.The Ss AK-1 was widely distributed in the whole body of scabies mite by immunofluorescence localization,especially in the stomach and exoskeleton area;and the Ss AK-2 was widely distributed in the mouthparts,stomach and exoskeleton of S.scabiei.The natural Ss AK-1 and Ss AK-2 can be secreted or excreted into the pool around the parasitic site of S.scabiei(the clear liquid secreted around the body of S.scabiei parasitizing on the skin)and the epidermis of the skin,which may indicate that they are involved in the host-S.scabiei interaction to modulate host immune function.2.Effects of proliferation,apoptosis and migration of host PBMCs by r Ss AK-1and r Ss AK-2.Healthy New Zealand rabbit’s blood were collected from ear veins to isolate PBMCs,and the cells were stimulated with 10 μg/ml,20 μg/ml,40 μg/ml,and 80 μg/ml of r Ss AK-1 and r Ss AK-2,respectively.The effect of cell proliferation was detected by CCK-8.Flow cytometry and q RT-PCR were used to detect the effect of cell apoptosis and the m RNA transcription level of apoptosis factors(Fas,Bax,Bcl-2,Bcl-xl)and NF-κB(p65).The traswell chamber was used to detect the effect of cell migration.The results showed that r Ss AK-1 and r Ss AK-2 significantly promoted the proliferation of PBMCs,inhibited cell apoptosis,and induced cell migration in a concentrationdependent manner.The effect of r Ss AK-1 on promoting proliferation and apoptosis was more obvious than that of r Ss AK-2,while the effect of r Ss AK-2 on promoting migration was more obvious than that of r Ss AK-1.Both r Ss AK-1 and r Ss AK-2increased the transcription levels of anti-apoptotic factors(Bcl-2,Bcl-xl)and NF-κB(P65)in a concentration-dependent manner.3.Effects of the balance of Th1/Th2 and Treg/Th17 of host PBMCs by r Ss AK-1and r Ss AK-2.10 μg/ml,20 μg/ml,40 μg/ml,and 80 μg/ml of r Ss AK-1 and r Ss AK-2 were used to stimulate cells.Th1 cytokines(IL-2,IFN-γ),Th2 cytokines(IL-4),Treg cytokines(IL-10,TGF-β1)and Th17 cytokines(IL-17)in PBMCs were detected by ELISA.With the increase of the concentration of r Ss AK-1 and r Ss AK-2,the secretion of Th1cytokines(IL-2,IFN-γ)gradually decreased,and the secretion of Th2 cytokines(IL-4)gradually increased;the secretion of IL-10 decreased,and the secretion of IL-17 increased,which indicated that the Th1/Th2 balance shifted towards Th2 and changed the Th17/Treg balance.