SBIR Phase I: Intracellular delivery of nematicidal proteins

SBIR 第一阶段：杀线虫蛋白的细胞内递送

• 批准号: 0611206
• 负责人: Michelle Hresko
• 金额: $ 10万
• 依托单位: Divergence, Inc.
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0611206&HistoricalAwards=false
• 关键词: SBIR; Phase; Intracellular; delivery; nematicidal

This Small Business Innovation Research (SBIR) Phase I research project will develop a method to use cell-penetrating peptides (CPPs) to carry bioactive proteins across cell membranes in plant parasitic nematodes. Multiple CPPs will be developed which cross nematode cell membranes when attached to a 15 kDa protein scaffold. This scaffold is the starting structure for future novel libraries (called STEM), which will involve the selection of proteins that inhibit essential intracellular targets in the nematode. CPP-STEM fusion proteins will be generated in bacteria and purified. Soybean cyst nematode (SCN) and root knot nematode (RKN) J2 larvae will be soaked in these proteins under conditions that facilitate molecular uptake into the parasite intestine. To confirm that the CPP-STEM scaffolds can be expressed in plants in a form that is accessible to the nematode, transgenic expression of the fusion proteins within hairy roots as well as uptake by SCN and RKN will be used as the bioassay. The CPP-STEM proteins will also be localized in the nematode, which will provide information on what tissues and cellular compartments can be targeted by this approach. Commercially, the application is a transgenic solution for the control of parasitic nematode infections. Parasitic nematodes cost the agricultural industries in excess of $8 billion annually in the United States and $78 billion annually worldwide. The use of nematicides is tightly restricted. They are also cumbersome to apply in a safe manner and are expensive to use. Many chemicals, such as methyl bromide, are hazardous to the environment and are slated for deregistration. The broader impact of this project centers on the potential for novel means of nematode control to lessen the need for the application of toxic and environmentally unfriendly chemicals on major crops. This approach is target based, expressing nematicidal proteins that cross the nematode cell membrane in transgenic plants. This strategy can be used to develop control of a broad array of parasitic nematodes from cyst nematodes on soybean, root knot on vegetables, fruits and cotton, to lesion nematode on corn. Further application to insects and fungal pathogens of plants could broaden the impact of this technology.

这个小企业创新研究（SBIR）第一阶段研究项目将开发一种方法，使用细胞穿透肽（CPP）携带生物活性蛋白穿过植物寄生线虫的细胞膜。将开发多个CPP，其在连接到15 kDa蛋白质支架时穿过线虫细胞膜。这种支架是未来新型文库（称为STEM）的起始结构，这将涉及选择抑制线虫中必需的细胞内靶点的蛋白质。CPP-STEM融合蛋白将在细菌中产生并纯化。大豆胞囊线虫（SCN）和根结线虫（RKN）J2幼虫将在有利于分子吸收进入寄生虫肠道的条件下浸泡在这些蛋白质中。为了证实CPP-STEM支架可以以线虫可接近的形式在植物中表达，将使用毛状根内融合蛋白的转基因表达以及SCN和RKN的摄取作为生物测定。CPP-STEM蛋白也将定位于线虫中，这将提供关于这种方法可以靶向哪些组织和细胞区室的信息。在商业上，该应用是用于控制寄生线虫感染的转基因解决方案。在美国，寄生线虫每年给农业造成的损失超过80亿美元，在全世界每年造成的损失超过780亿美元。杀线虫剂的使用受到严格限制。 它们也难以以安全的方式应用并且使用昂贵。许多化学品（例如甲基溴）对环境有害，因此将被取消注册。该项目更广泛的影响集中在线虫控制新方法的潜力上，以减少对主要作物施用有毒和对环境不利的化学品的需要。这种方法是基于靶标的，在转基因植物中表达穿过线虫细胞膜的杀线虫蛋白。该策略可用于开发从大豆上的孢囊线虫、蔬菜、水果和棉花上的根结线虫到玉米上的病害线虫的广泛的寄生线虫防治。进一步应用于植物的昆虫和真菌病原体可以扩大这项技术的影响。