Gene Cloning,Prokaryotic Expression And Functional Analysis Of Defensins From Hyalomma Asiaticum

Hyalomma asiaticum is located in the desert and semi-desert areas,distributed in the northwest of China,habitat mainly in Populus euphratica and weed shrub,parasitizes cattle,sheep,horses,wild boar and bactrian camel,also invades people.The ticks H.asiaticum can spread a variety of natural foci disease,such as Xinjiang hemorrhagic fever,Q fever and tropical theileriosis.Defensin is a class of antimicrobial peptides that play an important role in biological innate immunity.The Studies on tick defensins have important significance for exploration of the ralationship beween ticks and pathogens,development of acaricide and control of tick born disease.In this study,five denfensin genes were cloned from a constructed cDNA library H.asiaticum,and named as Ha-defensin-1,Ha-defensin-2,Ha-defensin-3,Ha-defensin-4and Ha-defensin-5.The length of the open reading frame was 219 bp,222bp,222 bp,207bp,and 222 bp respectively.The number of amino acids was 73,74,74,69 and 74,with the theoretical PI 9.08,9.54,9.89,5.53 and 9.99,respectively.Amino acid sequence alignment of Ha-defensin-1-5 with other tick defensins showed the similarity was 53%~82%.The prokaryotic expression and functional analysis of Ha-defensin-1 and Ha-defensin-2 were carried out.Ha-defensin-1 and Ha-defensin-2 were respectively inserted into the vector pET-32(a+)and transferred into Transetta(DE3)competent cells.The expression conditions were explored and optimized.SDS-PAGE showed that the target protein size was about 25 kDa,which was consistent with the predicted size.The expression of Ha-defensin-1 and Ha-defensin-2 were analyzed by Real-time PCR.The results showed that Ha-defensin-1 and Ha-defensin-2 were expressed with various expression levels,in various tissues,at different decelopmental stages,or under the induction of different bacteria.The results of the antibacterial functional analysis showed that the recombinant proteins pET-32(a+)-Ha-defensin-1 and pET-32(a+)-Ha-defensin-2 had antibacterial activity against Gram-positive bacteria,Gram-negative bacteria and fungi,but there are differences.The antioxidantexperiments of recombinant protein pET-32(a+)-Ha-defensin-1 and pET-32(a+)-Ha-defensin-2 showed certain scavenging ability to ABTS·+ and DPPH· free radicals.The hemolytic activity test indected that the recombinant protein pET-32(a+)-Ha-defensin-1 and pET-32(a+)-Ha-defensin-2 hemolysis in 86.8 ?M and93.8 ?M concentration respectively 5.78%~6.39%.The above results lay the foundation for revealing the innate immune mechanism of ticks and developing new bioloping new biological agents.