Shahein Yasser Ezzat
Molecular Biology Department, National Research Centre, Cairo, Egypt.
Vet Immunol Immunopathol. 2008 Feb 15;121(3-4):281-9. doi: 10.1016/j.vetimm.2007.10.008. Epub 2007 Oct 22.
A full-length cDNA of an immunogenic protein was cloned from a cDNA library of the local Egyptian cattle tick Boophilus annulatus. Antibodies raised against B. annulatus larval proteins were used to screen a cDNA expression library. A 936bp cloned fragment was sequenced and showed an open reading frame of 516bp encoding a protein of 171 amino acids. Comparison of the deduced amino acid sequence with protein data bank revealed that the sequence is related to a sequence isolated from the hard tick Haemaphysalis qinghaiensis (Hq05). Southern blot analysis of B. annulatus genomic DNA showed that the cloned cDNA hybridized to double bands per restriction digest, suggesting that the cloned cDNA is a double copy gene. Amino acid analysis of the cloned gene revealed the presence of two casein kinase II phosphorylation sites in the N-terminal domain suggesting that this molecule may be involved in the signal transduction or gene expression pathways. RT-PCR and northern blotting revealed the presence of two isoforms of the Ba05 gene in salivary glands and in the 3-day-old eggs. The cloned gene without the signal peptide, was expressed in Escherichia coli under T7 promotor of pET-30b vector, and purified under denaturation conditions. The purified protein appeared as a single band on 12% SDS-PAGE with a molecular weight around 22.8kDa including the histidine tag of the vector. Antibodies raised against the purified molecule were used to detect the B. annulatus homologue to the Hq05 gene in whole tick, larvae and gut protein extracts. Immunoblotting revealed the presence of this molecule Ba05 only in whole tick and larval protein extracts and not in the gut protein extract. Using the same antibodies, homologues to the Ba05 gene were detected in other tick species as Hyalomma dromedarii and Rhipicephalus sp. but not in Ornithodoros moubata.
从埃及当地牛蜱微小牛蜱的cDNA文库中克隆了一种免疫原性蛋白的全长cDNA。用针对微小牛蜱幼虫蛋白产生的抗体筛选cDNA表达文库。对一个936bp的克隆片段进行测序，发现其开放阅读框为516bp，编码一个由171个氨基酸组成的蛋白质。将推导的氨基酸序列与蛋白质数据库进行比较，发现该序列与从硬蜱青海血蜱分离的一个序列（Hq05）相关。微小牛蜱基因组DNA的Southern印迹分析表明，克隆的cDNA与每个限制性酶切片段的两条带杂交，表明克隆的cDNA是一个双拷贝基因。对克隆基因的氨基酸分析显示，在N端结构域存在两个酪蛋白激酶II磷酸化位点，表明该分子可能参与信号转导或基因表达途径。RT-PCR和Northern印迹分析显示，Ba05基因在唾液腺和3日龄卵中有两种异构体。去除信号肽的克隆基因在pET-30b载体的T7启动子控制下在大肠杆菌中表达，并在变性条件下纯化。纯化后的蛋白在12% SDS-PAGE上呈现为一条单带，分子量约为22.8kDa，包括载体的组氨酸标签。用针对纯化分子产生的抗体检测微小牛蜱中与Hq05基因同源的Ba05基因，在全蜱、幼虫和肠道蛋白提取物中进行检测。免疫印迹显示，这种Ba05分子仅存在于全蜱和幼虫蛋白提取物中，而不存在于肠道蛋白提取物中。使用相同的抗体，在其他蜱种如嗜驼璃眼蜱和扇头蜱属中检测到了与Ba05基因同源的基因，但在波斯锐缘蜱中未检测到。
