Tsuji N, Morales T H, Ozols V V, Carmody A B, Chandrashekar R
Heska Corporation, Fort Collins, CO 80525, USA.
Mol Biochem Parasitol. 1998 Nov 30;97(1-2):69-79. doi: 10.1016/s0166-6851(98)00131-5.
A full length D. immitis cDNA (nDiCal) encoding a protein with significant similarity to the calreticulin protein family was isolated from a 6-day fourth-stage larval cDNA expression library by immunoscreening, using serum from a rabbit immunized by repeated injection of small numbers of third-stage larvae. nDiCal is 1538 bp long and contains the 21 bp nematode splice leader sequence SL1 at the 5' end. nDiCal encodes for a protein (pDiCal) with a predicted molecular mass of 46 kDa. pDiCal sequence analysis revealed similarities with calreticulin, a protein that typically resides in the endoplasmic reticulum. pDiCal possesses three consensus sequences of the calreticulin family of proteins: a neutral N-terminal region with a putative signal sequence; a proline- and tryptophan-rich P region; and a highly acidic C-terminal region. A 45Ca2+-overlay assay showed that recombinant pDiCal (rDiCal) is a Ca2+-binding protein. Antibodies to rDiCal identified a 56 kDa native antigen in all developmental stages including the excretory-secretory products derived from larvae and adult worms. Localization studies demonstrated the ubiquitous presence of pDiCal with intense expression in the hypodermis and syncitial muscle cells in both male and female adult worms. Labeling was also seen in the developing embryos within the uterus of the female worms. Sera from immune as well as chronically-infected microfilaremic dogs contained antibodies that bind rDiCal. In addition, immunoblot analysis showed that serum from a rabbit immunized with L3 cuticles reacted with rDiCal.
通过免疫筛选，从六日龄第四期幼虫的cDNA表达文库中分离出了一个全长的犬恶丝虫cDNA（nDiCal），该cDNA编码一种与钙网蛋白家族具有显著相似性的蛋白质，使用的血清来自一只经多次注射少量三期幼虫免疫的兔子。nDiCal长1538 bp，在5'端含有21 bp的线虫剪接前导序列SL1。nDiCal编码一种预测分子量为46 kDa的蛋白质（pDiCal）。pDiCal序列分析显示与通常位于内质网的钙网蛋白相似。pDiCal具有钙网蛋白家族蛋白质的三个共有序列：一个带有假定信号序列的中性N端区域；一个富含脯氨酸和色氨酸的P区域；以及一个高度酸性的C端区域。一项45Ca²⁺覆盖试验表明重组pDiCal（rDiCal）是一种Ca²⁺结合蛋白。针对rDiCal的抗体在所有发育阶段都鉴定出一种56 kDa的天然抗原，包括幼虫和成虫的排泄-分泌产物。定位研究表明pDiCal普遍存在，在雌雄成虫的皮下组织和合体肌细胞中强烈表达。在雌虫子宫内的发育胚胎中也可见标记。免疫犬以及慢性感染微丝蚴血症犬的血清中都含有能与rDiCal结合的抗体。此外，免疫印迹分析表明，用L3角质层免疫的兔子血清与rDiCal发生反应。
