AEG-1跨膜蛋白结构及其在新型肿瘤疫苗制备中的应用研究

AEG-1 is highly expressed in various tumor cells, playing important roles in the tumorigenesis, development and drug resistance. However, there remains considerable debate about the structure of AEG-1 transmembrane protein, which limits its applications as tumor vaccine antigen. Our previous studies showed the antibodies against C terminal of AEG-1 could combine with extracellular peptide fragment of AEG-1 and inhibited tumor growth. Therefore, we speculate that the AEG-1 belongs to the type Ⅱ topology. In this study, Fusion Tags technology and Immunofluorescence Staining technology will be used to investigate the structure of AEG-1 transmembrane protein, and the functions of extracellular peptide fragment of AEG-1 in tumor growth will also be confirmed in vivo and in vitro. On the basis of these studies, a new chimeric VLPs vaccine will be constructed by using extracellular peptide fragment of AEG-1 and adjuvant Flagellin. BALB/C mice will be immunized with the chimeric VLPs vaccines, and ELISA, ELISPOT, Flow Cytometry will be used to evaluate cellular and humoral immune response levels. Then, anti-tumor effects of the novel vaccines will also be investigated in mice models with breast cancer. In general, our aims are to lay the foundation for application of extracellular peptide fragment of AEG-1 in antitumor vaccine, and offer a new method to design anti-tumor vaccines with other tumor antigens.

AEG-1在多种恶性肿瘤中高表达，并与肿瘤发生、发展密切相关，是一个重要的肿瘤生物治疗新靶点，但因其跨膜蛋白结构分型仍存有较大争议，限制了AEG-1在抗肿瘤疫苗研发中的应用。课题组前期研究表明，抗AEG-1蛋白C末端抗体可结合于AEG-1胞外区且抑制肿瘤生长，推测AEG-1为Ⅱ型跨膜蛋白。本课题拟应用融合标签蛋白及免疫细胞化学等技术进一步验证AEG-1跨膜蛋白结构分型，并结合体内外实验分析AEG-1胞外肽段在肿瘤细胞增殖中的作用机制；进而制备AEG-1胞外肽段携带免疫佐剂Flagellin的嵌合型VLPs疫苗，免疫BALB/c鼠，通过ELISA、ELISPOT及流式细胞技术等方法评价小鼠细胞及体液免疫应答水平；并借助乳腺癌小鼠模型探讨该新型抗肿瘤VLPs疫苗的抑瘤作用，最终为AEG-1胞外段作为肿瘤疫苗治疗靶点提供理论及实验依据，也为其它肿瘤抗原疫苗的设计提供一个全新思路和方法。

AEG-1在多种恶性肿瘤中高表达，并与肿瘤发生、发展密切相关，是一个重要的肿瘤生物治疗新靶点，但因其跨膜蛋白结构分型仍存有较大争议，限制了AEG-1在抗肿瘤疫苗研发中的应用。本课题首先应用融合标签蛋白及免疫细胞化学等技术验证AEG-1跨膜结构分型，证实其C末端位于胞外，并证实AEG-1胞外段单克隆抗体在体外水平能够抑制乳腺癌细胞增殖、促进细胞凋亡，且在体内水平对乳腺癌荷瘤小鼠具有抑瘤作用；接着，制备AEG-1 C末端携带免疫佐剂Flagellin的VLPs疫苗（AEG-1-C-Flic-VLPs）免疫BALB/c小鼠，通过ELISA、细胞因子ELISA等技术评价小鼠细胞及体液免疫应答水平。结果显示，与AEG-1-C-VLPs免疫组相比，AEG-1-C-Flic-VLPs免疫后刺激小鼠产生较高的抗体水平，并刺激脾淋巴细胞分泌较多的细胞因子IL-2与INF-γ；接种乳腺癌细胞后，AEG-1-C-Flic-VLPs免疫组小鼠成瘤率低，生存率较高； VLPs对小鼠的免疫治疗作用研究结果显示：AEG-1-C-FliC VLPs免疫组小鼠生存率高且成瘤后肿瘤生长缓慢。我们进一步表明，VLPS长期接种对小鼠体质量无影响且接种后重要器官未见明显的病理组织学变化。以上结果为AEG-1胞外段作为肿瘤疫苗治疗靶点提供理论及实验依据，也为其它肿瘤抗原疫苗的设计提供一个全新思路和方法。