不同EGFP-蜘蛛丝融合基因在昆虫细胞的表达及融合丝蛋白形成机制初探(英文)

Insights Into Formation Mechanism of the Silk Fusion Protein From Expression of Different Sizes of EGFP-spider Silk Fusion Protein in Insect Cells

蜘蛛能吐出具有优异机械特性的多种类型的丝,其中管状腺丝用于构建卵囊并具有良好的分子和机械性能,是目前唯一报道的全长cDNA序列的蜘蛛丝。基于为将来利用生物工程方法大量生产高性能的仿蜘蛛丝纤维提供基础信息,通过Bac-to-Bac/AcNPV杆状病毒表达系统,在强启动子驱动下,对2个大小不同的蜘蛛卵囊丝蛋白基因序列(全长cDNA序列和部分cDNA序列)与EGFP报告基因实现了在AcNPV昆虫细胞中的融合表达。绿色荧光和W estern印迹分析表明,被表达出的2个大小不同的卵囊丝融合蛋白在昆虫细胞胞质中呈现出明显不同的溶解性。含有部分卵囊丝基因的短EGFP-蜘蛛丝融合基因的表达产物在胞质里具有可溶性;而含有全长卵囊丝基因的巨大EGFP-蜘蛛丝融合基因的表达产物倾向于自我装配形成沉淀聚合物。研究结果也暗示了蜘蛛丝蛋白的分子大小可能对其装配成高性能的丝纤维有重要影响。

Spiders spin multiple types of silks that are renowned for their superb mechanical properties. Cylindrical silk with novel molecular and mechanical properties, used in the construction of egg case outercover, is only one type of spider silk full-length cDNA reported data to date. In order to provide some valuable messages for large-scale mimicking the outstanding high-performance properties of native spider silk by bioengineering method in the future, two different sizes of cylindrical silk protein genes （full-length and partial cDNA sequences） and EGFP fusion genes were expressed in insect cells under the control of strong promoter by Bacto-Bac/AcNPV baculovirus expression system. EGFP fluorescence and western blotting analysis showed two different sizes of the expressed egg case silk fusion protein presented obviously different solubility within the aqueous cytosol of insect cells. The expression products of partial egg case silk fusion gene were soluble in aqueous cytosol while another larger fusion protein containing almost full-length egg case silk protein tends to form aggregate by self assemblage. Our results also suggested that the molecular size of spider silk protein could be important to assemble them into high-performance silk fiber.