McDermott M J, Weber E, Hunter S, Stedman K E, Best E, Frank G R, Wang R, Escudero J, Kuner J, McCall C
Heska Corporation, 1613 Prospect Parkway, Fort Collins, CO 80525, USA.
Mol Immunol. 2000 May;37(7):361-75. doi: 10.1016/s0161-5890(00)00061-4.
An 18 kDa protein isolated from saliva of the cat flea, Ctenocephalides felis, elicits a positive intradermal skin test (IDST) in 100 and 80% of experimental and clinical flea allergic dogs, respectively. Using solid-phase enzyme-linked immuno assay (ELISA), this protein detected IgE in 100 and 80% of experimental and clinical flea allergic dogs, respectively. A cDNA (pFSI) encoding a full-length Cte f 1 protein was isolated from a C. felis salivary gland cDNA library, using a combination of PCR and hybridization screening. This cDNA is 658 bp in length, and contains an open reading frame of 528 bp. The open reading frame encodes a protein of 176 amino acids, consisting of an 18 amino acid signal sequence and a 158 amino acid mature protein. The calculated molecular weight and pI of the mature protein are 18106 Da and 9.3, respectively. The protein, named Cte f 1, is the first novel major allergen described for canine flea allergy. Recombinant Cte f 1 (rCte f 1) was expressed in Escherichia coli, Pichia pastoris and baculovirus infected Trichoplusia ni cells. Approximately, 90% of the rCte f 1 expressed in E. coli accumulated in insoluble inclusion bodies, which could be refolded to a soluble mixture of disulfide isomers with partial IgE binding activity. Small quantities of an apparently correctly refolded form of rCte f 1, which had IgE binding activity equal to the native antigen, was isolated from the soluble fraction of E. coli cells. However, P. pastoris and baculovirus infected insect cells expressed and secreted a fully processed, correctly refolded and fully active form of rCte f 1. Mass spectrometry analysis of the active forms of rCte f 1confirmed that eight intact disulfide bonds were present, matching the number observed in the native allergen. The relative ability of rCte f 1 to bind IgE in the serum of flea allergic animals, produced in these three expression systems, matched that of the native allergen. Competition ELISA demonstrated that approximately 90% of the specific IgE binding to native Cte f 1 could be blocked by the different forms of rCte f 1.
从猫栉首蚤（Ctenocephalides felis）唾液中分离出的一种18 kDa蛋白质，在100%的实验性和80%的临床跳蚤过敏性犬中引发了阳性皮内皮肤试验（IDST）。使用固相酶联免疫吸附测定（ELISA），这种蛋白质分别在100%的实验性和80%的临床跳蚤过敏性犬中检测到IgE。利用聚合酶链反应（PCR）和杂交筛选相结合的方法，从猫栉首蚤唾液腺cDNA文库中分离出编码全长Cte f 1蛋白的cDNA（pFSI）。这个cDNA长度为658 bp，包含一个528 bp的开放阅读框。该开放阅读框编码一个由176个氨基酸组成的蛋白质，包括一个18个氨基酸的信号序列和一个158个氨基酸的成熟蛋白。成熟蛋白的计算分子量和pI分别为18106 Da和9.3。这种名为Cte f 1的蛋白质是首个被描述的犬类跳蚤过敏的新型主要过敏原。重组Cte f 1（rCte f 1）在大肠杆菌、巴斯德毕赤酵母和杆状病毒感染的粉纹夜蛾（Trichoplusia ni）细胞中表达。在大肠杆菌中表达的rCte f 1约90%积累在不溶性包涵体中，这些包涵体可以重折叠成具有部分IgE结合活性的二硫键异构体的可溶性混合物。从大肠杆菌细胞的可溶性部分中分离出少量明显正确重折叠形式的rCte f 1，其IgE结合活性与天然抗原相当。然而，巴斯德毕赤酵母和杆状病毒感染的昆虫细胞表达并分泌了一种经过完全加工、正确重折叠且具有完全活性的rCte f 1形式。对rCte f 1活性形式的质谱分析证实存在八个完整的二硫键，与天然过敏原中观察到的数量一致。在这三种表达系统中产生的rCte f 1与跳蚤过敏性动物血清中IgE结合的相对能力与天然过敏原相当。竞争ELISA表明，与天然Cte f 1结合的特异性IgE中约90%可被不同形式的rCte f 1阻断。
