Mauch L, Seissler J, Haubruck H, Cook N J, Abney C C, Berthold H, Wirbelauer C, Liedvogel B, Scherbaum W A, Northemann W
Department of Molecular Biology, ELIAS Entwicklungslabor, Freiburg, Germany.
J Biochem. 1993 Jun;113(6):699-704. doi: 10.1093/oxfordjournals.jbchem.a124106.
cDNAs coding for the full-length human 65 and 67 kDa glutamic acid decarboxylases (GAD65 and GAD67) were amplified from pancreas and hippocampus cDNA libraries by polymerase chain reaction, respectively. Both cDNAs were inserted into a baculovirus vector which mediated highly efficient expression of the human GAD65 and GAD67 with histidine-hexapeptides as affinity ligands at their C-termini in Spodoptera frugiperda (Sf9) cells. The recombinant GAD proteins were purified to homogeneity by affinity chromatography using a metal-chelating matrix. The infected Sf9 insect cells expressed the recombinant human GAD65 and GAD67 with natural-like conformations, as confirmed by measurement of their enzyme activities as well as their fully restored autoantigenicities. Immunoprecipitation of metabolically labeled infected Sf9 cells demonstrated the autoantigenic potential of the recombinant GAD proteins. The practicability of using recombinant GAD65 and GAD67 derived from the baculovirus expression system for the development of an immunoassay for the diagnosis of insulin-dependent diabetes mellitus is discussed.
分别通过聚合酶链反应从胰腺和海马cDNA文库中扩增出编码全长人65 kDa和67 kDa谷氨酸脱羧酶（GAD65和GAD67）的cDNA。将这两种cDNA插入杆状病毒载体，该载体介导人GAD65和GAD67在其C末端带有组氨酸六肽作为亲和配体，在草地贪夜蛾（Sf9）细胞中高效表达。通过使用金属螯合基质的亲和层析将重组GAD蛋白纯化至同质。经测量其酶活性以及完全恢复的自身抗原性证实，被感染的Sf9昆虫细胞表达具有天然样构象的重组人GAD65和GAD67。对代谢标记的被感染Sf9细胞进行免疫沉淀证明了重组GAD蛋白的自身抗原潜力。讨论了使用杆状病毒表达系统衍生的重组GAD65和GAD67开发用于诊断胰岛素依赖型糖尿病的免疫测定法的实用性。
