血红扇头蜱卵黄原蛋白基因的克隆及生物学特性分析

Cloning and biological characteristics analysis of vitellogenin gene of Rhipicephalus sanguineus

为研究卵黄原蛋白(Vg)基因对蜱虫卵的生理机制作用,参考GenBank中微小扇头蜱(登录号:EU086096)卵黄原蛋白基因的核苷酸序列,设计特异性引物,PCR扩增血红扇头蜱相关基因,将其克隆至pGEM-T Easy载体,并测序。对获得的阳性克隆进行序列分析,选取Vg基因的VWFD功能区设计表达引物,构建重组表达载体pGEX-4T-1-Vg,并进行表达纯化。用Western-blot鉴定表达产物的免疫原性。结果显示,获得的1 218bp的核苷酸序列片段与预期结果一致。系统发生树显示,血红扇头蜱与微小扇头蜱具有较高的同源性,两者之间的同源性高达93%,其次与变异革蜱、希伯来花蜱、肩突硬蜱的同源性分别为85%、78%、62%。序列分析显示,该基因含有1个由221个氨基酸组成的VWFD功能区。该功能区重组融合蛋白的分子质量约为51ku。Western-blot分析表明,该重组蛋白与家兔抗血红扇头蜱全蜱阳性血清、家兔抗长角血蜱全蜱阳性血清、家兔抗残缘璃眼蜱全蜱阳性血清以及家兔抗草原革蜱全蜱阳性血清均出现不同程度的反应活性。结果表明,经表达Vg基因VWFD区域而制备的重组抗原对于不同蜱种血清具有广谱性。

Vitellogenin(Vg)plays an important role in the regulation of the growth and development of animal eggs.In this study,Vg-related genes in Rhipicephalus sanguineus were amplified by PCR with the use of primers designed based on vitellogenin gene in Rhipicephalus microplus from GenBank(accession number:EU086096),cloned into pGEM-T Easy vector and sequenced.Following analysis of the positive clone's sequences,the functional domain Von Willebrand Factor type D(VWFD)was selected for expression and then the recombinant protein of pGEX-4T-1-Vg was purified.In addition,its immunogenicity was identified by Western-blot.It turned out that the nucleotide sequence obtained from this study was 1 218 bp in length,including a function domain consisting of 221 amino acids and VWFD region.The phylogenetic tree showed that R.sanguineus and R.microplus shared a high sequence similarity up to 93%,which indicates they have high homology with each other.In addition,the similarity to Dermacentor variabilis,Amblyomma hebraeum,and Ixodes scapularis were 85%,78% and 62%,respectively.The recombinant protein,of which the molecular mass was about 51 ku,has been proved that it presented the different levels of reactivities with various positive sera such as rabbit anti-R.sanguineus,Haemaphysalis longicornis,Hyalomma detritumand Dermacentor nuttalli by Western-blot assay.The result showed that the recombinant antigen synthesized based on Vg genes VWFD region had a broad immunochemical reaction spectrum with the different tick species sera,which has important implications for the further study of efficient vaccines against ticks.