Gene Cloning, Prokaryotic Expression And Functional Analysis Of Defensins From The Parthenogenetic Population Of Haemaphysalis Longicornis

The tick Haemaphysalis longicornis is obligate blood-sucking ectoparasites,and consists two reproductive populations in nature, bisexual and parthenogenetic population. It can carry a great variety of pathogens and lead to many diseases which bring great harm to human health and livestock production. As antimicrobial peptide,defensin is commonly found in animals and plants, and especially in arthropods which lacks adaptive immune response, defensin is particularly important which can effectively combat invading pathogens during innate immunity response. As obligate blood feeding arthorpods, ticks also contain many defensins which help to resist microbes or pathogens.In current study, three defensins were cloned from the cDNA library of the parthenogenetic population of H. longicornis using construction kit and the 3’RACE method, and they were named as Hp-defensin-1, Hp-defensin-2 and Hp-defensin-3.The open reading frame successively is 225 bp, 210 bp and 237 bp, which encodes amino acids 74, 69 and 78, respectively. The puptive mature peptide is 38, 37 and 46,and theoretical pI is 9.69, 8.33 and 9.50, respectively.Hp-defensin-1 was further studied for prokaryotic expression and function analysis, and results indicated that, the amino acid sequence showed a high homology with defensin of Dermacentor marginatus(ACJ04433.1), and the similarity was75.68%. A closest genetic relationship was revealed between Hp-defensin-1 and defensin of D. silvarum after construction of the phylogenetic tree. Real-time quantitative results suggested a significant difference in expression of Hp-defensin-1in different developmental stages and tissues. Hp-defensin-1 was ligated into pET-32(a+) and then transformed into Escherichia coli BL21 for expression.SDS-PAGE results showed that the molecular weight was 23 kDa, which is consistent with prediction. The recombinant protein displayed a potent activity against most of the Gram-positive bacteria, some of Gram-negative bacteria and fungi, meanwhile,distinct ABTS·+and DPPH·free radical scavenging capacity with no hemolytic activity was observed. The above results provided the basis for the futuredevelopment of new antibacterial drugs.