Gene Cloning, Eukaryotic Expression And Functional Characterization Of Defensin Gene From Dermacentor Silvarum

Ticks are obligate blood-sucking ectoparasites which can attack reptiles, birds andmammals, and are among the most important vectors of pathogens affecting livestock. Thetick Dermacentor silvarum is widely distributed in northeast of China, including InnerMongolia, Hebei, Shanxi and Xinjiang, and usually habitats in the forest-steppe area. Theycan transmit a wide variety of pathogens, causing great damages to human health andlivestock production. Researches about D. silvarum are few and mainly concentrated on itsbiological and ecological features.Four defensin-like DNA sequences were obtained by screening the cDNA library of D.silvarum, and were named as Ds-defensin-1, Ds-defensin-2, Ds-defensin-3and Ds-defensin-4.Then the parameters, including the open reading frame (ORF), theoretical pI, signal peptide,mature peptide and the3D structure of these four DNA sequences were caculated andpredicted.Ds-defensin-1: sequence length is382bp, ORF length is213bp, molecular weight (MW)is7384.6Da, PI is6.07, encoding71amino acids, with a22amino acid residues’ signalpeptide, a15amino acid spacer peptide and a34amino acid residues’ mature peptide.Ds-defensin-2: sequence length is280bp, ORF length is147bp, MW is5163.8Da, PI is4.39, encoding49amino acids, with a23amino acid residues’ signal peptide, a14amino acidspacer peptide and a12amino acid residues’ mature peptide.Ds-defensin-3: sequence length is280bp, ORF length is216bp, MW is7771.0Da, PI is4.85, encoding72amino acids, with a22amino acid residues’ signal peptide, a16amino acidspacer peptide and a34amino acid residues’ mature peptide.Ds-defensin-4: sequence length is278bp, ORF length is216bp, MW is7500.7Da, PI is4.61, encoding72amino acids, with a23amino acid residues’ signal peptide, a14amino acidspacer peptide and a35amino acid residues’ mature peptide.Ds-defenisn-1was selected as the major one for further analysis, including the biologicalfunctions of synthesized peptide and the eukaryotic expression of the Ds-defenisn-1gene. Thesynthesized peptide was evaluated on its antimicrobial activity, antioxidant capacity and hemolytic capacity, and results showed that the synthesized peptide displayed a potent activityagainst most of the tested Gram-positive bacteria and exhibited an obvious inhibitory effect tosome of the Gram-negative bacteria (the growth of Psychrobacter faecalis was inhibited whenthe concentration of Ds-defenisn-1reaches above25μg/mL). SEM observation indicated thatafter30-minutes-treatment by Ds-defensin-1, the bacteria morphology changed obviously.Tiny granules appeared on the surface of the cell wall, and meanwhile the microbial contentwas released. The microbial became twisted and wrinkled, and eventually disrupted. Thesynthesized Ds-defensin-1also showed a significant antioxidant capacity. At the concentrationof200μM, Ds-defensin-1was able to scavenge43.40%of DPPH and53.45%of ABTSradicals in30min. However, no hemolytic ability was detected. The tissue expression patternof Ds-defensin-1in feeding female and male adults of D. silvarum was determined usingReal-time PCR, and results showed that the Ds-defensin-1had a tissue-specific expressionwith high abundance detected in salivary gland and carcass of female and male adults.The recombinant baculovirus of the plasmid of D. silvarum was constructed, andDs-defensin-1was expressed in the current study. Fragments of Ds-defensin-1ORF werecloned into the shuttle vector pFastBac HT C, and then the plasmid was cotransfected intobaculovirus expression vector. The baculovirus P1and P2with infection capability werepicked up after the insect cell of Sf9was infected by the recombinant baculovirus. The vibrantinsect cell of Sf9was infected by P2baculovirus, and Ds-defensin-1was expressed. The MWof Ds-defensin-1is approximate10.7kDa, including the target peptide7.4kDa, which isconsistent with that prediction. However, the peptide of Ds-defensin-1still need furtherpurification and function analysis.