Maccarrone M, Veldink G A, Finazzi Agrò A, Vliegenthart J F
Bijvoet Center for Biomolecular Research, Department of Bio-Organic Chemistry, Utrecht University, The Netherlands.
Biochim Biophys Acta. 1995 Jan 18;1243(1):136-42. doi: 10.1016/0304-4165(94)00124-g.
Protoplasts were isolated from lentil (Lens culinaris) roots and their suitability as a transient expression system was investigated. After transfecting the protoplasts with the beta-glucuronidase (GUS) gene by either electroporation or polyethylene glycol (PEG), the specific activity of the reporter enzyme and the cell viability were determined. Electroporation was more effective than PEG treatment as transfection procedure and its efficiency was affected by the plasmid length. The feasibility of electro-transferring at the same time (coelectroporation) inhibitory anti-lipoxygenase monoclonal antibodies and the GUS-carrying plasmid pBI 221 was investigated as well. The amount of transferred immunoglobulins was quantitated by ELISA and the inhibitory ability of monoclonal antibodies on the intracellular target enzyme was determined. Evidence is presented for the successful coelectroporation of immunoglobulins and plasmid DNA into lentil protoplasts, the two types of macromolecules acting independently of each other in the recipient cells.
从扁豆（Lens culinaris）根中分离出原生质体，并研究了其作为瞬时表达系统的适用性。通过电穿孔或聚乙二醇（PEG）用β-葡萄糖醛酸酶（GUS）基因转染原生质体后，测定报告酶的比活性和细胞活力。作为转染程序，电穿孔比PEG处理更有效，其效率受质粒长度影响。同时还研究了电转移（共电穿孔）抑制性抗脂氧合酶单克隆抗体和携带GUS的质粒pBI 221的可行性。通过酶联免疫吸附测定（ELISA）对转移的免疫球蛋白量进行定量，并测定单克隆抗体对细胞内靶酶的抑制能力。本文提供了证据，证明免疫球蛋白和质粒DNA成功共电穿孔进入扁豆原生质体，这两种大分子在受体细胞中彼此独立发挥作用。
