Gene cloning, expression and immunogenicity of the protective antigen subolesin in Dermacentor silvarum.
Gene cloning, expression and immunogenicity of the protective antigen subolesin in Dermacentor silvarum.
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DOI：
10.3347/kjp.2014.52.1.93
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发表时间：
2014-02
期刊：
影响因子：
--
通讯作者：
Liu J
中科院分区：
文献类型：
--
作者：
Hu Y;Zeng H;Zhang J;Wang D;Li D;Zhang T;Yang S;Liu J
Subolesin（4D 8）是昆虫Akirins的直系同源物，是一种高度保守的保护性抗原，因此具有开发针对蜱和蚊子的广谱疫苗的潜力。迄今为止，没有保护性抗原被表征或测试作为针对森林革蜱叮咬和相关病原体传播的候选疫苗。本研究克隆了D. silvarum 4D 8 cDNA（Ds 4D 8）全长498 bp，编码165个氨基酸残基。序列比对和系统发育分析结果表明，D. silvarum 4D 8（Ds 4D 8）与其它硬蜱的氨基酸序列高度保守，同源性在81%以上。将含有限制性酶切位点的Ds 4D 8连接到pET-32（a+）表达载体上，转化大肠杆菌Rosetta。重组Ds 4D 8（rDs 4D 8）经异丙基β-D-硫代半乳糖苷（IPTG）诱导表达，Ni亲和层析纯化。SDS-PAGE结果显示rDs 4D 8的分子量为40 kDa，这与考虑来自表达载体的22 kDa组氨酸标记的硫氧还蛋白（TRX）蛋白的预期分子量一致。Westernblot结果显示，兔抗D. silvarum血清识别表达的rDs 4D 8，表明针对rDs 4D 8的免疫应答。这些结果为研制抗D.森林蜱和相关病原体的传播。
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.
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