基于反义肽简并性策略的靶向多肽探针的设计、筛选与肝癌活细胞成像分析研究

Novel analytical methods capable of highly specific and sensitive detection of critical tumor markers are promising for early cancer diagnosis, metastasis alarming and targeted therapy. Considering the limitation of antibodies in bulky size, poor stability and incapability in cell penetrating, herein a novel strategy to design and screen small peptide probes based on the degeneracy of antisense peptides is proposed for cancer analysis. A novel tumor marker, lysosome-associated protein transmembrane 4-beta (LAPTM4B), is chosen as the target protein. And an important extracellular fragment of LAPTM4B protein is chosen as the "epitope" for designing antisense peptides. High performance affinity screening system based on affinity chromatography or integrated microfluidic chip is constructed. Positional scanning and stepwise affinity screening is developed to obtain an optimal peptide. Providing the targeted recognition towards LAPTM4B in physiological condition, the screened peptide probe will be further applied in specific tumor cell detection and real-time tracking. The peptide probe capable of subcellular imaging and differentiation of the biomarker LAPTM4B in live cancer cells is promising for highly selective and sensitive tumor detection and targeted therapy. Such molecular probes also can provide important information and detailed insights into the biological functions of LAPTM4B protein.

针对由癌细胞产生的、与肿瘤特性密切相关的生物标志物，发展高特异性、高灵敏度的检测新方法，可望为肿瘤的早期发现、转移预警和治疗提供新思路。鉴于抗体等生物大分子探针分子量大、稳定性差、细胞穿透能力弱等缺点，本项目拟选择新型肿瘤标志物溶酶体四次穿膜蛋白LAPTM4B为靶蛋白，以其关键胞外结构域为靶点，发展基于反义肽简并性的逐步扩展-位置扫描肽库设计筛选新方法；构建基于亲和色谱或微流控芯片的高效亲和筛选体系，筛选出生理条件下高亲和力、高选择性的靶向小分子多肽探针；考察多肽探针对LAPTM4B蛋白的专一性识别，进行人肝癌活细胞的靶向成像分析研究，以多肽探针示踪监测LAPTM4B蛋白在亚细胞水平的定位和动态转运信息。为实现体内癌细胞的快速、高特异性、高灵敏度分析检测奠定基础，为阐述关键癌蛋白在恶性肿瘤发生发展中的生物学功能提供新的思路与方法。

针对由癌细胞产生的、与肿瘤特性密切相关的生物标志物，发展高特异性、高灵敏度的检测新方法，必将为肿瘤的早期发现、转移预警和治疗提供新思路。本研究以新型肿瘤标志蛋白溶酶体四次穿膜蛋白LAPTM4B为靶蛋白，选择其胞外结构域EL1片段为靶点；基于遗传密码的简并性，以及正-反义肽相互作用，设计构建了反义肽库；发展了基于聚集诱导发光（AIE）效应的快速、灵敏、高通量靶向多肽探针筛选新方法，筛选出了对LAPTM4B蛋白具有高亲和力和高选择性的多肽探针AP2和AP5。以荧光标记的AP2和AP5多肽探针进行了人肝癌活细胞的靶向成像分析，该探针可以选择性识别癌细胞表面的LAPTM4B 蛋白，并示踪、监测LAPTM4B蛋白在亚细胞水平的定位和动态转运。进一步以靶向多肽为识别元件，构建了三维微纳结构的肿瘤细胞特异性捕获芯片，实现了全血中循环肿瘤细胞的高效捕获与无损释放。所构建的靶向多肽探针为肿瘤的检测、捕获和靶向治疗提供了新手段。本项目在Chem. Sci.、Anal. Chem.、Chem. Commun.等杂志发表论文15篇，申请发明专利3项，获得2016年中国分析测试协会科学技术奖二等奖1项。