Royer Corinne, Jalabert Audrey, Da Rocha Martine, Grenier Anne-Marie, Mauchamp Bernard, Couble Pierre, Chavancy Gérard
Unité Nationale Séricicole, INRA, 25 quai J-J Rousseau 69350 La Mulatière, France.
Transgenic Res. 2005 Aug;14(4):463-72. doi: 10.1007/s11248-005-4351-4.
A gene construct was made by fusing the coding sequence of the red fluorescent protein (DsRed) to the exon 2 of the fibrohexamerin gene (fhx), that encodes a subunit of fibroin, the major silk protein of the silkworm Bombyx mori. The fusion gene was inserted into a piggyBac vector to establish a series of transgenic lines. The expression of the transgene was monitored during the course of larval life and was found restricted to the posterior silk gland cells as the endogenous fhx gene, in all the selected transgenic lines. The exogenous polypeptide was secreted into the lumen of the posterior silk gland together with fibroin, and further exported with the silk proteins as a foreign constituent of the cocoon fiber. The capacity of DsRed to emit fluorescence in the air-dried silk thread led to show that the recombinant protein was distributed over the whole length of the fiber. A remarkable property of the system lies in the localization of the globular protein at the periphery of the silk thread, allowing its rapid and easy recovery in aqueous solutions, without dissolving fibroin. The procedure represents a novel and promising strategy for the production of massive recombinant proteins of biomedical and pharmaceutical interest, with reduced cost.
通过将红色荧光蛋白（DsRed）的编码序列与丝心蛋白六聚体基因（fhx）的外显子2融合，构建了一个基因构建体，该基因编码家蚕主要丝蛋白丝素的一个亚基。将融合基因插入piggyBac载体中，建立了一系列转基因品系。在幼虫期监测转基因的表达，发现在所有选定的转基因品系中，转基因的表达与内源性fhx基因一样，局限于后部丝腺细胞。外源多肽与丝素一起分泌到后部丝腺的管腔中，并作为茧纤维的外来成分与丝蛋白一起进一步输出。DsRed在风干丝线中发出荧光的能力表明重组蛋白分布在纤维的全长上。该系统的一个显著特性在于球状蛋白定位于丝线的外围，使得在不溶解丝素的情况下，能够在水溶液中快速轻松地回收该蛋白。该方法代表了一种生产具有生物医学和药学意义的大量重组蛋白的新颖且有前景的策略，且成本降低。
