| The tick Haemaphysalis doenitzi is mainly distributed in Fujian,Yunnan and Taiwan,which can attack reptiles,birds and mammals,can carry Babesiidae pathogens and lead to many diseases that bring great harm to human health and livestock production.Defensin can resist pathogens,plays an important role in the innate immune system of ticks.In current study,five defensin genes were cloned from H.doenitzi,we performed the bioinformatics analysis(protein sequence,homology,physicochemical properties and three-dimensional structure)of the five defensin genes and the functional studies of recombinant proteins(antibacterial activity,antioxidant activity,hemolytic activity).These genes were named as Hd-defensin-1,Hd-defensin-2,Hd-defensin-3,Hd-defensin-4,and Hd-defensin-5.The open reading frames successively were246 bp,225bp,225 bp,210bp and 225 bp,which encoded amino acids 88,74,74,69 and 74,respectively.The puptive mature peptide is 48,38,38,38and38,and theoretical pIswere7.47,8.73,11.51,7.47 and 9.65,respectively.By comparing with defensins in NCBI,Hd-defensin-1 and Hd-defensin-2 showed a high homology with the defensin(AB028926.1)of H.longicornis,and the similarity was 95% and that of the other three was more than 60%.Hd-defensin-3 and Hd-defensin-5 were further studied by prokaryotic expression.The recombinant proteinrHd-defensin-3 andrHd-defensin-5 were obtained.The rHd-defensin-3andrHd-defensin-5 displayed a potent activity against most of the Gram-positive bacteria,against some of Gram-negative bacteria and fungi.Real-time quantitative results suggested a significant difference in expression of Hd-defensin-3.In different developmental stages,the highest expression was in the nymph.And in various tissues the highest expression was in the salivary glands,followed by midgut,ovary and malpighian.The expression of Hd-defensin-5in different developmental stages showed the highest expression is in the larva and nymph,meanwhile in various tissues the highest expression was in the salivary glands.The recombinant proteins had a strong ability to scavenge ABTS·+ and DPPH· free radicals,no hemolytic activity.This study can provide references for the development of new antibacterial and antioxidant agents. |
