胃肠道肿瘤相关抗原表位嵌合病毒样颗粒疫苗构建及其免疫原性研究

Gastrointestinal cancer is a common disease and ranked first in all kinds of malignant tumors in our country. Nowadays, the vaccine is considered as the fundamental way to cancer prophylaxis and prevention,and molecular vaccine which based on epitopes has shown its good application.However, the disadvantages of epitope vaccines on weak immunogenicity and natural conformation need to further study.The L1 structural protein of the human papillomavirus(HPV) expressed in eukaryotic expression system in vitro can self-assemble into virus-like particles(VLPs) and be used to carry heterologous epitopes with strong immunogenicity and stable epitope conformation.In this study MAGE-3, HER-2/neu and CEA tumor antigen which highly expressed in various gastrointestinal cancer will be selected as the targets of potential vaccine. After prediction,screening and identification of their CTL and B cell epitopes,the sequence containing these epitopes will be inserted into the carboxyl terminus of HPV16 L1 protein to produce chimeric VLPs. The immune responses and protective effects of the chimeric VLPs contained MAGE-3 / HER-2/neu/CEA multi-epitopes will further be evaluated. The aim of this study is to generate specific antibodies and CTL reaction after immunized by chimeric VLPs and to prevent gastrointestinal tumor.

胃肠道肿瘤是我国的多发病，居恶性肿瘤首位，疫苗则是其防治的根本途径。以抗原表位为基础的分子疫苗已显示了其防治肿瘤的良好前景，但表位疫苗的免疫原性弱和其天然构象问题仍急待解决。鉴于人乳头瘤病毒（HPV）结构蛋白可在体外真核表达系统自组装形成病毒样颗粒（VLPs），利用其携带异源表位疫苗，具有免疫原性强且保持其异源表位空间构象不变等优点。本研究拟选择胃肠肿瘤组织中高表达的肿瘤抗原MAGE-3、HER-2/neu和CEA，通过对其CTL和B细胞表位的预测、筛选和鉴定，并将胃肠肿瘤相关抗原表位基因插入HPV16 L1 蛋白的羧基端，制备以HPV16 L1为载体含MAGE-3/ HER-2/neu/CEA多表位的嵌合VLPs，并进一步探讨其刺激机体产生的免疫保护效应。目的是使嵌合VLPs能刺激机体免疫系统同时产生肿瘤抗原特异性的抗体反应和CTL 反应，从而达到用特异性免疫方法防治胃肠肿瘤。

胃癌、结肠癌均是发病率排名前五的恶性肿瘤，且以手术和放疗、化疗为主的胃肠肿瘤的治疗缺乏肿瘤特异性，毒副作用大，医疗费用巨大。而针对肿瘤细胞特异性表达的肿瘤相关抗原（TAA）或肿瘤特异性抗原（TSA）发展起来的肿瘤治疗性疫苗，能有效激活免疫系统、杀伤肿瘤细胞，具特异、安全、有效的特点。本研究选择精子相关抗原9 （sperm associated antigen 9，SPAG-9）和黑色素瘤抗原A3（Melanoma antigen A3, MAGE-A3），采用免疫信息学方法预测SPAG-9蛋白的CTL 和B细胞表位并筛选出了富含HLA-A2限制的CTL表位和B细胞表位的短肽。根据表位筛选验证结果，选择具可形成病毒样颗粒（VLPs）的人乳头瘤病毒（HPV16）L1 为载体，将富含CTL表位和B细胞表位的AA 551-573片段插入到HPV16-L1 蛋白的羧基端，利用毕赤酵母表达系统成功表达并纯化了HPV16 L1/SPAG-9多表位嵌合VLPs。另外，通过基因克隆技术，构建含MAGE-A3全长基因的重组质粒 pET21a(+)/MAGE-A3(FULL-LENGTH)，并进行原核蛋白表达、纯化及鉴定。进一步免疫小鼠，免疫效应分析显示，HPV16 L1/SPAG-9多表位嵌合VLPs和MAGE-A3 重组蛋白均能有效诱导特异性体液免疫和细胞免疫。本研究拟从肿瘤细胞特异性表达的肿瘤相关抗原（TAA）或肿瘤特异性抗原（TSA）发展起来的肿瘤治疗性疫苗这一全新角度探讨胃肠肿瘤的新治疗方法，为特异性免疫防治胃肠肿瘤的研究提供理论和实验依据，具广阔的应用前景。