蜘蛛捕获丝蛋白质成丝机理研究

Spider capture silk is known for its outstanding strength and elasticity, resistance to radiation and heat, fatigue resistance, and superior biocompatibility. Spider capture silk is therefore considered as a strategic resource to be developed urgently, for many potential applications in high-technology fields including military, aerospace and biomedical areas. In our previous work, we have produced the first complete gene sequence of spider capture silk protein, namely the minor ampullate silk protein (MiSp). We have also produced structures of the N terminal, C-terminal domain and non-repetitive Spacer domain of MiSp. In this proposed project, we continue to study the structures change under different physiological and biochemical conditions, how these domains interact with each other, and how they affect the spider silk protein assemblage and silk formation process. Additionally, this project will study the relationship between MiSp repetitive domains and the mechanical properties of silks spun from recombinant MiSp proteins. A mechanism model of spider silk formation will then be established for optimizing the preparation of biomimetic silk fibers with native silk-like performances. Research works in this project can be basis for the production of biomimetic native spider silk-like artificial fibers, and they can also be very helpful for high-performance manmade spider silk industrialization and biomedical applications.

蜘蛛捕获丝具有强度高、弹性大、抗辐射、耐热、耐疲劳、生物相容性好等特性，是一种优质的、难得的天然生物材料，在军工、航空航天以及生物医学工程领域有着巨大的潜在应用价值，是一种亟待开发的战略资源。项目在本课题组已经筛选鉴定的国内外首条大腹园蛛捕获丝蛋白（MiSp）全长序列，和已经解析出MiSp蛋白NT、CT以及Spacer非重复模块的高级结构的基础上，从分子水平上研究NT、CT、Spacer以及重复区功能模块在不同生理和化学条件下的结构变化、相互作用以及对丝蛋白组装、聚集和成丝性能的影响，破解蜘蛛捕获丝蛋白成丝机理，揭示MiSp丝蛋白重复区模块与成丝性能的关系，建立蜘蛛捕获丝蛋白成丝机理模型。研究成果将为制备与天然蛛丝性能相媲美的人造蜘蛛捕获丝奠定理论基础，为高品质重组蛛丝纤维的产业化提供科学依据，为人类创伤修复和治疗开辟新的优质可靠生物资源。

项目表达了多种重组蛛丝蛋白，最高产量可达238mg/L，大多数表达的重组蛛丝蛋白可溶；CD光谱分析多种重组蛛丝蛋白的二级结构显示，在蛋白溶液pH由7.0降至5.5的过程中，α-螺旋结构的含量逐渐降低，β-折叠结构含量逐渐上升，重组蛛丝蛋白NT模块的二聚化和CT模块结构的转变促进了Rep模块的结构转变；α-螺旋结构的含量随重复模块数量的增加略有上升，β-折叠结构含量随重复模块数量增加略有下降；多种重组蛛丝蛋白均可在低浓度通过手工牵拉的方式形成丝纤维，添加NT和CT模块后，只需一个重复区单元即可成丝，所成的丝纤维表面光滑，且无明显褶皱或刻痕；重复模块数量增加，拉伸强度(Stress at break)、伸长量(Strain at break)、杨氏模量(Modulus)和韧性(Toughness)等材料学性能指标都在逐渐上升，揭示NT和CT有助于改善重组蛛丝蛋白纤维的表面形态和材料学性能；高浓度的重组蜘蛛丝蛋白可以通过湿纺纺成丝纤维，丝纤维的材料学性能随重复模块数量的增加，呈现出增加的趋势；高浓度重组蜘蛛丝蛋白溶液可获得光滑的蛋白膜；通过静电纺技术可将不同组合重组蜘蛛丝蛋白质制备成小血管和神经导管支架，并进行了表征。在项目资助下，已经在国际期刊发表论文16篇，国内核心期刊发表5篇，申请专利8个，培养博士生10名，培养硕士生4名。