Puckette Michael, Burrage Thomas, Neilan John G, Rasmussen Max
U.S. Department of Homeland Security Science and Technology Directorate, Plum Island Animal Disease Center, P.O. Box 848, Greenport, NY, 11944, USA.
Oak Ridge Institute for Science and Education, Plum Island Animal Disease Center Research Participation Program (PIADC), P.O. Box 117, Oak Ridge, 37831, TN, USA.
BMC Biotechnol. 2017 Jun 12;17(1):52. doi: 10.1186/s12896-017-0367-0.
The Gaussia princeps luciferase is used as a stand-alone reporter of transgene expression for in vitro and in vivo expression systems due to the rapid and easy monitoring of luciferase activity. We sought to simultaneously quantitate production of other recombinant proteins by transcriptionally linking the Gaussia princeps luciferase gene to other genes of interest through the foot-and-mouth disease virus 2A translational interrupter sequence.
We produced six plasmids, each encoding a single open reading frame, with the foot-and-mouth disease virus 2A sequence placed either N-terminal or C-terminal to the Gaussia princeps luciferase gene. Two plasmids included novel Gaussia princeps luciferase variants with the position 1 methionine deleted. Placing a foot-and-mouth disease virus 2A translational interrupter sequence on either the N- or C-terminus of the Gaussia princeps luciferase gene did not prevent the secretion or luminescence of resulting chimeric luciferase proteins. We also measured the ability of another polycistronic plasmid vector with a 2A-luciferase sequence placed downstream of the foot-and-mouth disease virus P1 and 3C protease genes to produce of foot-and-mouth disease virus-like particles and luciferase activity from transfected cells. Incorporation of the 2A-luciferase sequence into a transgene encoding foot-and-mouth disease virus structural proteins retained luciferase activity and the ability to form virus-like particles.
We demonstrated a mechanism for the near real-time, sequential, non-destructive quantitative monitoring of transcriptionally-linked recombinant proteins and a valuable method for monitoring transgene expression in recombinant vaccine constructs.
由于可快速简便地监测荧光素酶活性，高斯王子荧光素酶被用作体外和体内表达系统中转基因表达的独立报告基因。我们试图通过口蹄疫病毒2A翻译中断序列将高斯王子荧光素酶基因与其他感兴趣的基因转录连接，从而同时定量其他重组蛋白的产生。
我们构建了六个质粒，每个质粒编码一个单一的开放阅读框，口蹄疫病毒2A序列位于高斯王子荧光素酶基因的N端或C端。两个质粒包含缺失第1位甲硫氨酸的新型高斯王子荧光素酶变体。在高斯王子荧光素酶基因的N端或C端放置口蹄疫病毒2A翻译中断序列并不妨碍所得嵌合荧光素酶蛋白的分泌或发光。我们还检测了另一种多顺反子质粒载体的能力，该载体在口蹄疫病毒P1和3C蛋白酶基因下游放置了2A - 荧光素酶序列，可从转染细胞中产生口蹄疫病毒样颗粒并具有荧光素酶活性。将2A - 荧光素酶序列整合到编码口蹄疫病毒结构蛋白的转基因中可保留荧光素酶活性以及形成病毒样颗粒的能力。
我们展示了一种对转录连接的重组蛋白进行近实时、连续、无损定量监测的机制，以及一种监测重组疫苗构建体中转基因表达的有价值方法。
