Hancock Kathy, Narang Someet, Pattabhi Sowmya, Yushak Melinda L, Khan Azra, Lin Seh-Ching, Plemons Robert, Betenbaugh Michael J, Tsang Victor C W
Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA.
J Immunol Methods. 2008 Jan 31;330(1-2):130-6. doi: 10.1016/j.jim.2007.08.002. Epub 2007 Aug 28.
Baculovirus-mediated expression of recombinant proteins for use in diagnostic assays is commonplace. We expressed a diagnostic antigen for cysticercosis, GP50, caused by the larval stage of Taenia solium, in both High Five and Sf9 insect cells. Upon evaluation of the specificity of recombinant GP50 (rGP50) in a western blot assay, we observed that 12.5% (21/168) of the serum samples from persons with a variety of parasitic infections other than cysticercosis reacted positive when rGP50 was produced in High Five cells. The same samples reacted negative when rGP50 was produced in Sf9 cells. The false positive reactivities of these other parasitic infection sera were abolished when rGP50, expressed in High Five cells, was deglycosylated. In addition, the same sera that reacted with rGP50 from High Five cells also reacted with recombinant human transferrin (rhTf) when expressed in High Five cells, but not Sf9 cells. High Five cells, but not Sf9 cells, modify many glycoproteins with a core alpha(1,3)-fucose. This same modification is found in the glycoproteins of several parasitic worms and is known to be immunogenic. Since the distribution of these worms is widespread and millions of people are infected, the use of recombinant proteins with N-linked glycosylation produced in High Five cells for diagnostic antigens is likely to result in a number of false positive reactions and a decrease in assay specificity.
杆状病毒介导的重组蛋白表达用于诊断检测已很常见。我们在High Five和Sf9昆虫细胞中表达了一种由猪带绦虫幼虫阶段引起的囊尾蚴病诊断抗原GP50。在蛋白质印迹分析中评估重组GP50（rGP50）的特异性时，我们观察到，在High Five细胞中产生rGP50时，来自患有除囊尾蚴病之外各种寄生虫感染的人的血清样本中有12.5%（21/168）呈阳性反应。当rGP50在Sf9细胞中产生时，相同的样本呈阴性反应。当在High Five细胞中表达的rGP50去糖基化后，这些其他寄生虫感染血清的假阳性反应消失。此外，与来自High Five细胞的rGP50反应的相同血清，当在High Five细胞而非Sf9细胞中表达时，也与重组人转铁蛋白（rhTf）反应。High Five细胞而非Sf9细胞会用核心α(1,3)-岩藻糖修饰许多糖蛋白。这种相同的修饰也存在于几种寄生虫的糖蛋白中，并且已知具有免疫原性。由于这些寄生虫分布广泛且数百万人受到感染，使用在High Five细胞中产生的具有N-连接糖基化的重组蛋白作为诊断抗原可能会导致许多假阳性反应并降低检测特异性。
