Kramer Shannon F, Kostov Yordan, Rao Govind, Bentley William E
Center for Biosystems Research, University of Maryland Biotechnology Institute, and Department of Chemical Engineering, University of Maryland, College Park, Maryland 20742, USA.
Biotechnol Bioeng. 2003 Jul 20;83(2):241-7. doi: 10.1002/bit.10668.
Trichoplusia ni larvae were infected with baculoviruses containing genes for the expression of ultraviolet optimized green fluorescent protein (GFPuv) and several product proteins. A GFP-specific optical probe was used to both excite the green fluorescent protein (lambda(ex) = 385 nm), and subsequently monitor fluorescence emission (lambda(em) = 514 nm) from outside the infected larvae. The probe's photodetector was connected to a voltmeter, which was used to quantify the amount of GFPuv expressed in infected larvae. Voltage readings were significantly higher for infected vs. uninfected larvae and, by Western analysis, linear with the amount of GFPuv produced. In addition, the probe sensitivity and range were sufficient to delineate infection efficiency and recombinant protein production for model proteins, chloramphenicol acetyltransferase and human interleukin-2. This work represents a critical step in developing an automated process for the production of recombinant proteins in insect larvae.
将粉纹夜蛾幼虫用含有用于表达紫外线优化绿色荧光蛋白（GFPuv）和几种产物蛋白的基因的杆状病毒进行感染。使用一种GFP特异性光学探针来激发绿色荧光蛋白（激发波长λ(ex)=385nm），随后监测受感染幼虫外部的荧光发射（发射波长λ(em)=514nm）。该探针的光电探测器连接到电压表，用于量化受感染幼虫中表达的GFPuv的量。受感染幼虫的电压读数显著高于未感染幼虫，并且通过蛋白质免疫印迹分析，其与产生的GFPuv的量呈线性关系。此外，该探针的灵敏度和范围足以描绘模型蛋白氯霉素乙酰转移酶和人白细胞介素-2的感染效率和重组蛋白产量。这项工作是开发昆虫幼虫中重组蛋白生产自动化过程的关键一步。
