Identification And Functional Of Peroxiredoxin From Haemaphysalis Doenitzi

Ticks are blood-sucking arthropods.The process of sucking blood can spread a variety of pathogens,which poses a serious threat to human health and animal husbandry.In this study,peroxiredoxin in Haemaphysalis doenitiz were studied for their structures and functions,which were used gene clone,prokaryotic expression,fluorescence quantitative PCR,RNAi and other technologies.In present study,two peroxiredoxins were successfully cloned from H.doenitiz,which type are 1-cys Prx and 2-cys Prx.They were named HDPrx1 and HDPrx2respectively.The sizes of open reading frames were 489bp and 582 bp,and the number of amino acids were 162aa and 193aa which the molecular weight of proteins were 17 k Da and 22 k Da.The isoelectric points were 10.85 and 7.60,respectively.The prediction results of signal peptide showed that both of HDPrx1 and HDPrx2 had no signal peptide.HDPrx1 encoded a conserved domain PRX?1cys and a conserved catalytic center.HDPrx2 encoded two conserved domains PRX?Typ2cys and two conserved catalytic centers.Both of them belong to thioredoxin?superfamily super protein family.Their amino acid were mostly predicted as hydrophilic amino acid,which were speculated to be hydrophilic protein.SDS-page electrophoresis showed that the bands of the recombinant proteins were 37k Da and 42k Da,respectively.The optimal expression conditions of r HDPrx1 were as follows:IPTG concentration0.5m M/ml,30?for 6h.And the optimal expression conditions of r HDPrx2 were as follows:IPTG concentration 0.5m M/ml,37?for 6h.Protein was adsorbed by Ni column and eluted with different gradients of imidazole and the optimal concentration of imidazole elution were both 500m M.The results of quantitative PCR showed that HDPrx1 and HDPrx2 were expressed in the four developmental stages with similar distribution patterns,and the relative expression levels were in the order of larva>adult>nymph>egg,and the relative quantity in larva were significantly higher than other stages.HDPrx1 and HDPrx2 were both expressed in different tissues.The relative quantity of HDPrx1 in midgut and ovary were higher than other tissues,and the relative quantity were in the order of midgut>ovary>Malpighian tube>salivary gland.The relative quantity of HDPrx2 in midgut of sated female ticks was higher than that in other tissues,and the relative expression level was in the order of midgut>salivary gland>Malpighian tube>ovary.The expression changes of HDPrx1 and HDPrx2 genes of H.doenitiz were analyzed after low temperature treatment(4?)for different days:HDPrx1 and HDPrx2 were expressed in after different days of low temperature treatment,and the relative quantity varied with different time.At 6 days of low temperature treatment,and the relative expression was the highest at 6 days.RNAi interference experiments showed that knockdown of HDPrx1 and HDPrx2had obvious effects on mortality,engorged weight and spawning capacity of female ticks,and significantly affected the blood sucking time.After interference,ticks became smaller and the abnormal shriveled eggs were appeared.In addition,there are significant differences between treatment group with control group about engorged weight.Antioxidant activity analysis showed that both r HDPrx1 and r HDPrx2 could reduce with H₂O₂,and the reduction ability was dependent on the concentration of recombinant protein.The antibacterial activity of r HDPrx1 and r HDPrx2 against Gram-positive bacteria(Staphylococcus aureus),Gram-negative bacteria(Escherichia coli),fungi(Candida albicans)showed significant inhibitory effect.Furthermore,the inhibition effects of the two recombinant proteins were dose-dependent.