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首页> 外文期刊>BMC Biotechnology >Evaluation of Gaussia luciferase and foot-and-mouth disease virus 2A translational interrupter chimeras as polycistronic reporters for transgene expression
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Evaluation of Gaussia luciferase and foot-and-mouth disease virus 2A translational interrupter chimeras as polycistronic reporters for transgene expression

机译：高斯荧光素酶和口蹄疫病毒2A翻译中断嵌合体作为多顺反子报道转基因表达的评价。

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BackgroundThe 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. ResultsWe 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. ConclusionsWe 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.
机译：背景技术由于快速简便地监测荧光素酶活性，高斯普林斯荧光素酶被用作体外和体内表达系统的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princeps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。结果我们产生了6个质粒，每个质粒编码一个开放阅读框，口蹄疫病毒2A序列位于高斯princeps荧光素酶基因的N端或C端。两个质粒包括新的高斯普林斯萤光素酶变体，其中删除了1位甲硫氨酸。将口蹄疫病毒2A翻译中断序列置于高斯princeps荧光素酶基因的N或C端不能阻止所得嵌合荧光素酶蛋白的分泌或发光。我们还测量了另一种具有2A荧光素酶序列的多顺反子质粒载体的能力，该序列位于口蹄疫病毒P1和3C蛋白酶基因的下游，可从转染的病毒中产生口蹄疫病毒样颗粒和萤光素酶活性细胞。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，保留了荧光素酶活性和形成病毒样颗粒的能力。结论我们证明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物中转基因表达的有价值的方法。

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