Engineering transgenic silkworms to produce spider silk fibers

工程转基因蚕生产蜘蛛丝纤维

• 批准号: 7364972
• 负责人: Donald L. Jarvis
• 金额: $ 21.52万
• 依托单位: UNIVERSITY OF WYOMING
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7364972
• 关键词: Adopted; Area; Bandage; Biochemical Genetics; Bombyx; Burn injury; Condition; Cosmetic surgery; Elasticity; Engineering; Ensure; Fiber; Fibroins; Future; Genes; Genetic Transformation; Gland; Human Resources; Individual; Insecta; Knowledge; Ligaments; Light; Measures; Mechanics; Moths; Nature; Neurologic; Organ; Patients; Peptide Leader Sequences; Peptides; Production; Property; Proteins; Purpose; Recombinant Proteins; Recombinants; Recording of previous events; Research; Research Personnel; Silk; Skin graft; Spiders; Staging; Surgical sutures; Synthetic Genes; System; Tendon structure; Tensile Strength; Time; Transgenic Animals; Transgenic Organisms; base; design; experience; member; mutant; physical property; programs; promoter; protein structure function; scaffold; success; tissue regeneration; vector

DESCRIPTION (provided by applicant): Silk fibers have many current and future biomedical applications. They are widely used as fine suture materials and even thinner fibers are needed for ocular, neurological, and cosmetic surgeries. Silk fibers also hold promise as materials for artificial ligaments and tendons and they have many other potential biomedical applications. Many different recombinant protein production systems have been used to try to meet current needs and to develop additional biomedical applications of silks. Each has yielded silk proteins, but none has consistently yielded useful silk fibers. Thus, the overall purpose of this R21 exploratory proposal is to develop a system that can produce spider silk fibers. Our basic approach will be to adopt the silkworm as a surrogate host for spider silk protein production. The highly efficient piggybac system will be used to genetically transform mutant silkworms, which produce no native silk, with a synthetic gene encoding an unusually large spider silk protein. The genetic and biochemical properties of the resulting transgenic silkworms, particularly their ability to produce spider silk fibers, will then be critically assessed. A key feature of our plan is that it includes specific measures that will optimize our ability to obtain fibers. These include: (i) using a synthetic gene encoding an unusually large spider silk protein, (ii) using a promoter that targets expression of the heterologous silk proteins to the silk gland, which is naturally equipped to spin silk fibers, and (iii) using an appropriate leader peptide for secretion from the silk gland, and (iv) using a surrogate host, the silk moth, which is highly amenable to genetic transformation and naturally equipped to spin silk fibers. The successful isolation of a transgenic silkmoth that can produce spider silk fibers in this exploratory project will set the stage for future projects designed to further develop this system to produce spider silk fibers with pre-determined physical properties optimized for specific biomedical applications. This will exploit current knowledge of specific peptide motifs contributing tensile strength or elasticity to spider silks. Theoretically, these motifs can be combined in various ways to design silk fibers differing in strength and elasticity. The overall likelihood that the current project can be successfully completed is enhanced by the fact that it will be undertaken by a team of three researchers with established, complementary programs in three areas key to the project: spider silks (Lewis), insect expression systems (Jarvis), and insect transformation (Fraser). Silk fibers have many current and future biomedical applications. They are widely used as fine suture materials and even thinner fibers are needed for ocular, neurological, and cosmetic surgeries. Silk fibers also hold promise as materials for artificial ligaments and tendons and they have many other potential biomedical applications. Many different recombinant protein production systems have been used to try to meet current needs and to develop additional biomedical applications of silks. Each has yielded silk proteins, but none has consistently yielded useful silk fibers. Thus, the overall purpose of this R21 exploratory proposal is to develop a system that can produce spider silk fibers for these current and future biomedical applications. Our plan for this exploratory project is to isolate a transgenic silkworm that can produce spider silk fibers. If this project is successful, it will set the stage for future projects designed to further develop this system to produce spider silk fibers with pre-determined physical properties optimized for specific biomedical applications. These projects will exploit current knowledge of specific peptide motifs contributing tensile strength or elasticity to spider silks.

描述（由申请人提供）：丝纤维在当前和未来有许多生物医学应用。它们被广泛用作精细缝合材料，眼科、神经科和美容手术需要更细的纤维。丝纤维也有望作为人工韧带和肌腱的材料，并且它们还有许多其他潜在的生物医学应用。许多不同的重组蛋白生产系统已被用来尝试满足当前的需求并开发丝的其他生物医学应用。每一种都产生了丝蛋白，但没有一种能够持续产生有用的丝纤维。因此，这个R21探索性提案的总体目的是开发一种可以生产蜘蛛丝纤维的系统。我们的基本方法是采用蚕作为蜘蛛丝蛋白生产的替代宿主。高效的piggybac系统将用于对不产生天然丝的突变蚕进行基因改造，并用编码异常大的蜘蛛丝蛋白的合成基因进行改造。然后将严格评估由此产生的转基因蚕的遗传和生化特性，特别是它们产生蜘蛛丝纤维的能力。我们计划的一个关键特点是它包括优化我们获取纤维能力的具体措施。这些包括：（i）使用编码异常大的蜘蛛丝蛋白的合成基因，（ii）使用将异源丝蛋白表达靶向到丝腺的启动子，丝腺天然具备旋转丝纤维的能力，以及（iii）使用适当的前导肽从丝腺分泌，以及（iv）使用替代宿主，即蚕蛾，它非常适合遗传转化和 天然具备纺丝纤维的能力。在这个探索性项目中成功分离出能够产生蜘蛛丝纤维的转基因蚕蛾，将为未来的项目奠定基础，这些项目旨在进一步开发该系统，以生产具有针对特定生物医学应用而优化的预定物理特性的蜘蛛丝纤维。这将利用当前对特定肽基序的了解，这些肽基序有助于蜘蛛丝的拉伸强度或弹性。理论上，这些图案可以通过多种方式组合来设计强度和弹性不同的丝纤维。当前项目成功完成的总体可能性因以下事实而增强：该项目将由三名研究人员组成的团队承担，并在该项目的三个关键领域建立了互补的计划：蜘蛛丝（刘易斯）、昆虫表达系统（贾维斯）和昆虫转化（弗雷泽）。丝纤维在当前和未来有许多生物医学应用。它们被广泛用作精细缝合材料，眼科、神经科和美容手术需要更细的纤维。丝纤维也有望作为人工韧带和肌腱的材料，并且它们还有许多其他潜在的生物医学应用。许多不同的重组蛋白生产系统已被用来尝试满足当前的需求并开发丝的其他生物医学应用。每一种都产生了丝蛋白，但没有一种能够持续产生有用的丝纤维。因此，R21 探索性提案的总体目的是开发一种可以为当前和未来的生物医学应用生产蜘蛛丝纤维的系统。我们这个探索性项目的计划是分离出一种可以产生蜘蛛丝纤维的转基因蚕。如果该项目成功，将为未来旨在进一步开发该系统的项目奠定基础，以生产具有针对特定生物医学应用而优化的预定物理特性的蜘蛛丝纤维。这些项目将利用当前对特定肽基序的了解，这些肽基序有助于蜘蛛丝的拉伸强度或弹性。