title: Gene cloning, expression and immunogenicity of the protective antigen subolesin in Dermacentor silvarum.
abstract: Subolesin (4D8), the ortholog of insect akirins, is a highly conserved protective antigen and thus has the potential for development of a broad-spectrum vaccine against ticks and mosquitoes. To date, no protective antigens have been characterized nor tested as candidate vaccines against Dermacentor silvarum bites and transmission of associated pathogens. In this study, we cloned the open reading frame (ORF) of D. silvarum 4D8 cDNA (Ds4D8), which consisted of 498 bp encoding 165 amino acid residues. The results of sequence alignments and phylogenetic analysis demonstrated that D. silvarum 4D8 (Ds4D8) is highly conserved showing more than 81% identity of amino acid sequences with those of other hard ticks. Additionally, Ds4D8 containing restriction sites was ligated into the pET-32(a+) expression vector and the recombinant plasmid was transformed into Escherichia coli rosetta. The recombinant Ds4D8 (rDs4D8) was induced by isopropyl β-D-thiogalactopyranoside (IPTG) and purified using Ni affinity chromatography. The SDS-PAGE results showed that the molecular weight of rDs4D8 was 40 kDa, which was consistent with the expected molecular mass considering 22 kDa histidine-tagged thioredoxin (TRX) protein from the expression vector. Western blot results showed that rabbit anti-D. silvarum serum recognized the expressed rDs4D8, suggesting an immune response against rDs4D8. These results provided the basis for developing a candidate vaccine against D. silvarum ticks and transmission of associated pathogens.
authors.affiliation: Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang, 050024, China.
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang, 050024, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Zeng
authors.affiliation: Shijiazhuang Post and Telecommunication Technical College, Shijiazhuang, 050021, China.
authors.forename: Jincheng
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang, 050024, China.
authors.forename: Duo
authors.initials: D
authors.lastname: Wang
authors.affiliation: Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang, 050024, China.
authors.forename: Dongming
authors.initials: D
authors.lastname: Li
authors.affiliation: Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang, 050024, China.
authors.forename: Tiantian
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang, 050024, China.
authors.forename: Shujie
authors.initials: S
authors.lastname: Yang
authors.affiliation: Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang, 050024, China.
authors.forename: Jingze
authors.initials: J
authors.lastname: Liu
mesh_terms: D000818:Animals; D000941:Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D060829:Arthropod Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D016000:Cluster Analysis; D017124:Conserved Sequence; D003870:Dermacentor / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D000941:Antigens; D060829:Arthropod Proteins; D011994:Recombinant Proteins; C550207:subolesin, tick
keywords: 4D8; Dermacentor silvarum; protective antigen; rDs4D8; subolesin; vaccine