【作者】 李常颖；

【导师】 畅继武；

【作者基本信息】 天津医科大学 ， 外科学， 2010， 博士

【摘要】 目的热休克蛋白gp96作为分子伴侣,能够与肿瘤抗原肽形成gp96—肽复合物,参与肿瘤抗原向MHC-I类分子途径的递呈过程,从而激活CD8+T淋巴细胞,产生抗肿瘤特异性免疫应答。因此,gp96—肽复合物疫苗为肿瘤的免疫治疗提供了新的思路。传统制备gp96的方法虽然已经成熟,但是由于组织来源的限制以及纯化工序过于繁杂,难以进行工业化生产。因此寻找一种快速有效而又高度特异性的方法纯化gp96,成为该疫苗大规模临床应用所要解决的首要问题。研究进一步证实,在gp96—肽复合物中起肿瘤免疫作用的是抗原肽,不是gp96自身,gp96结合的多肽才是疫苗制备和诱导特异性免疫应答的分子基础。因此,对gp96结合肽库的鉴定和分析,成为构建gp96—肽复合物疫苗、探讨相关作用机理及疫苗人工化生产等急待解决的问题。方法用固相化的gp96蛋白从人天然噬菌体抗体库中筛选出表达抗gp96蛋白Fab抗体的阳性克隆,酶切及连接反应构建其可溶性表达噬菌粒,转化至大肠杆菌BL21(DE3)pLysS中,IPTG诱导特异性抗体的高效表达,并用SDS-PAGE鉴定抗体表达情况、ELISA鉴定其抗原结合活性及特异性；所得抗体经纯化后作为偶联配体,吸附纯化膀胱癌及其癌旁组织中的gp96—肽复合物；通过酸洗脱法得到gp96所结合的多肽片段,经基质辅助激光解析电离飞行时间质谱测定其分子量及氨基酸序列；应用生物信息学技术分析各多肽片段的生物学特性。结果1通过对人天然Fab段噬菌体抗体库的四轮淘选,特异性抗gp96蛋白Fab噬菌体抗体得到了富集；2构建的抗gp96可溶性Fab抗体表达载体,在IPTG的诱导下得到了抗体的高效表达；进一步应用亲和层析技术得到了纯化的抗gp96可溶性Fab抗体；3利用基因工程抗体技术结合免疫亲和层析技术,可以从少量膀胱组织中纯化出实验用量的gp96—肽复合物,每克组织可获得约20μg-50μ.g的gp96—肽复合物；4在膀胱癌及其癌旁组织来源的gp96结合的多肽中,14条多肽的氨基酸序列被鉴定,其中8条来源于膀胱癌,6条来源于癌旁组织,分子量在997.5281Da-2211.33Da之间：5条多肽具有明确的蛋白来源,5条多肽经鉴定具有抗原性。结论1利用基因工程抗体技术结合免疫亲和层析技术,可成功的从有限的组织中分离纯化出实验用量的gp96—肽复合物；2gp96结合肽库中的肽为一些小分子肽片段,长短不一,部分蛋白来源明确,部分多肽具有抗原性,具有潜在的临床应用价值。更多还原

【Abstract】 Objective It has been known heat shock protein gp96derived from cancer cells or tissues can elicit cancer-specific immunity. Recent studies have indicated that gp96molecules, as a chaperon, participate in the presentation of the associated peptides to T cells through histocompatibility complex class I-restricted antigen presentation pathway. On the basis of these features, gp96can be used for cancer immunotherapy. Though the traditional preparation method of gp96had been developed, it had many limitations and shortage including a limited amount of sample material and the complicated purification procedures. The construction of a new rapid and high effective preparation method is necessary. Further Researches had confirmed that the antigenicity derived from not gp96but gp96associated peptides. The whole peptide pool associated with gp96is the molecular basis of the vaccine preparation and the induction of specific immune reaction. So, to further identify and analyze the gp96-associated peptide pool is very necessary for developing effective cancer vaccine and investigating related mechanism of action.Methods The specific gp-96clones were screened from a’naive’human Fab fragment phage diasplay library against the immobilized gp-96antigens, then the clones were transformed to the BL21(DE3)pLysS to give high-performance soluble expression of Fab antibodies by using IPTG as inducing agent. Soluble Fab antibodies were purified and acted as couple ligand to adsorb gp96peptide complexes from tissue protein suspensions of bladder cancer and paratumor tissue, gp-96associated peptides were eluted and identified by mass spectrometry. Using bioinformatics technology was used to obtain bionomics of polypeptidesResults1. Specific anti-gp96Fab antibodies were enriched by screening from a human naive Fab fragment phage antibody library.2. Soluble expressions of specific anti-gp96Fab antibodies were performed.3. gp-96peptide complexes can be obtained from small amounts of bladder tissue by antibody library technology and immuno affinity chromatography technology. The preparation was identified by Western blot. One gram tissue can obtain20～50μg gp-96peptide complexes.4.14peptides were identified,8peptides were from bladder cancer tissue, and others were from paracancerous tissue of bladder. The molecular weight range is997.5281Da～2211.33Da. Protein source and antigenicity were identified.Conclusions1. gp-96peptide complexes can be obtained from small amounts of bladder tissue by antibody library technology and immunoaffinity chromatography technology.2. Peptides in gp-96associated peptide pool are some small molecular peptides, their length is different. Protein sources of some peptides are known, some are unknown. Some peptides have antigenicity. Further analysis must be accomplished before the application in peptide vaccine.更多还原