Karbanowicz Thomas P, Lew-Tabor Ala, Rodriguez Valle Manuel
Queensland Alliance for Agriculture & Food Innovation, The University of Queensland.
Queensland Alliance for Agriculture & Food Innovation, The University of Queensland; Centre for Comparative Genomics, Murdoch University.
J Vis Exp. 2017 Jul 23(125):55747. doi: 10.3791/55747.
Rhipicephalus microplus - the cattle tick - is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.
微小牛蜱——牛蜱——作为多种病原体的传播媒介，是对家畜经济影响最大的体外寄生虫。人们致力于控制牛蜱以减轻其有害影响，重点是发现候选疫苗，如位于蜱肠道上皮细胞表面的BM86。当前的研究集中在利用cDNA和基因组文库来筛选其他候选疫苗。分离蜱肠道细胞在研究蜱肠道细胞膜表面蛋白组成方面具有重要优势。本文介绍了一种从半饱血微小牛蜱的蜱肠道内容物中分离上皮细胞的新颖可行方法。该方案利用三（2-羧乙基）膦（TCEP）和乙二胺四乙酸（EDTA）从上皮下支持组织中释放上皮细胞，并使用不连续密度离心梯度从其他细胞类型中分离上皮细胞。利用链霉亲和素连接的磁珠对蜱肠道上皮细胞的细胞表面蛋白进行生物素化和分离，以便在荧光激活细胞分选（FACS）或液相色谱-串联质谱（LC-MS/MS）分析中进行下游应用。
