Oker-Blom C, Suomalainen A M, Akerman K, Qi Z, Lindqvist C, Kuusisto A, Karp M
Abo Akademi University, Turku, Finland.
Biotechniques. 1993 May;14(5):800-9.
A fusion construct encoding two antibody-binding sites of protein A from Staphylococcus aureus and click beetle, Pyrophorus plagiophthalamus, luciferase (LucGR) was designed and expressed using the baculovirus system. The construct was inserted under the transcriptional regulation of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed in the insect Spodoperta frugiperda cell line during viral infection. The properties of the resultant chimeric protein product, protA-LucGR, were studied both in vivo and in vitro by using i) luminometry, ii) immunoblot analysis, iii) immunoprecipitation, iv) metabolic labeling procedures and v) luminescent immunoassays. Together, the results clearly demonstrate that the light-emitting properties of the fused luciferase construct remain intact. Further, the antibody-binding domain of protein A retains its activity as it binds to both rabbit and goat as well as human immunoglobulins. Due to the dual biological function of this fusion protein, it should provide a potential reagent within the field of molecular biology and diagnostics.
设计并使用杆状病毒系统表达了一种融合构建体，该构建体编码来自金黄色葡萄球菌的蛋白A的两个抗体结合位点和叩甲（Pyrophorus plagiophthalamus）荧光素酶（LucGR）。该构建体被插入苜蓿银纹夜蛾核型多角体病毒（AcNPV）多角体蛋白基因启动子的转录调控之下，并在病毒感染期间在昆虫草地贪夜蛾细胞系中表达。通过以下方法在体内和体外研究了所得嵌合蛋白产物protA-LucGR的特性：i）发光测定法，ii）免疫印迹分析，iii）免疫沉淀，iv）代谢标记程序和v）发光免疫测定法。总之，结果清楚地表明融合荧光素酶构建体的发光特性保持完整。此外，蛋白A的抗体结合结构域在与兔、山羊以及人免疫球蛋白结合时保留其活性。由于这种融合蛋白的双重生物学功能，它应该在分子生物学和诊断领域提供一种潜在的试剂。
