【作者】 马小兵；

【导师】 畅继武；

【作者基本信息】 天津医科大学 ， 外科学， 2006， 博士

【摘要】 背景和目的 热休克蛋白gp96作为分子伴侣参与了肿瘤抗原向MHC-Ⅰ类分子途径的递呈过程,并与抗原肽形成gp96-肽复合物激活CD8~+T淋巴细胞,产生抗肿瘤的特异性免疫反应。因此,gp96-肽复合物疫苗为肿瘤的免疫治疗提供了新的思路。但是,如此充满前景的疗法需要从有限的肿瘤组织中快速提取高纯度的gp96,传统的方法因为需要使用ConA-Sepharose柱,很容易造成ConA的污染,而ConA本身是一种T细胞丝裂原,可对疫苗接种者的免疫系统产生有害作用。所以,寻找一种快速有效而又高度特异性的方法分离纯化gp96,就成为该疫苗大规模临床应用所要解决的首要问题。噬菌体抗体库技术可有效地解决以上问题。我们利用噬菌体表面展示技术构建人天然Fab段噬菌体抗体库,建立人源抗体制备技术平台,为寻求恶性肿瘤的免疫治疗新方法打下基础。 方法 取4个健康献血员新鲜血液各200ml,分离淋巴细胞,提取总RNA,经逆转录合成cDNA,设计多对引物,以PCR扩增抗体轻链和重链Fd段cDNA。轻链PCR产物和噬粒载体pComb3经Sac Ⅰ和Xba Ⅰ酶切,由T4 DNA连接酶连接,电穿孔转化感受态大肠杆菌XL1-Blue,扩增后提取噬粒,构建轻链库。轻链库和重链Fd段PCR产物经Xho Ⅰ和Spe Ⅰ酶切,以同样方法连接后转化XL1-Blue,加入辅助噬菌体VCSM13,产生噬菌体抗体全库。提取噬粒酶切鉴定轻链库和全库有无插入片段。分别以噬菌体全库和提取的噬粒为模板,以轻、重链不同引物组合进行PCR扩增。构建的抗体库用人血白蛋白、IL-2和gp96等不同蛋白进行3轮筛选。 结果 提取的淋巴细胞总RNA及逆转录合成的cDNA质量好,部分重链Fd片段、部分κ轻链和λ轻链cDNA得到扩增。Fab全库库容达6.6×10~6,酶切和PCR显示有插入片段。用人血白蛋白、IL-2和gp96等不同蛋白进行几轮筛选,抗体库得到了不同程度的富集。更多还原

【Abstract】 Background/Objective: It has long been known that the cellular heat shock protein gp96 derived from cancer cells or tissues can elicit cancer-specific immunity. Recent studies have indicated that gp96 molecules, 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, gp96 can be used for cancer immunotherapy. However, this very promising application will require the rapid purification of gp96 molecules from a limited amount of sample material at a very high purity. The conventional procedure to isolate gp96 molecules requires ConA-Sepharose column purification, which result in contamination of gp96 with ConA due to bleeding from the column. Because ConA is a T-cell mitogen, it might harmfully interfere with the vaccinee’s immune system. Using phage antibody library can avoid from the trouble depicted previously. In addition, the anti-gp96 Fab antibodies speed up the purification process and allow the processing of small tumor samples.Phage surface display technique, which has become an increasingly important tool in biotechnology, provides a new way for antibody development. In this project, we constructed a naive human Fab fragment phage display library, provide a platform for human antibody preparation and a basis to new therapy for the malignant tumors.Methods: With lymphoprep, peripheral blood lymphocytes were isolated from 800 ml blood, which was obtained from four healthy blood donors. The heavy chain Fd and light chain cDNA synthesized from the total RNA of lymphocytes were amplified by PCR with variable regions 5’ and 3’ primers of heavy and light chain, and the amplification products were ligated into the phagemid vector pComb3, then the ligated sample was transformed into competent E. coli XL 1-Blue by Electroporation.The transformed cells were infected with VCSM13 helper phage toyield recombinant phage antibody Fabs. The phagemids abstracted from amplified E.coli were cut with endonucleases such as Sac I , Xba I , Spe I and Xho I and bother the phage antibody Fabs or phagemids abstracted from amplified E.coli were amplified by PCR to monitor the insertion of the genes of light chain or heavy chain Fd fragment. Human albumin, interleukin-2 and gp96 were utilized as antigens to conduct respectively three rounds of panning to the original Fab antibody library.Results: The quantity of total RNA and cDNA were qualified. By combination of light chain and heavy chain genes, an antibody library containing 6.6x 106 clones was obtained, and both the cutting of enzymes and PCR showed that there were the genes of light chain or heavy chain Fd fragment in the phagemids. After having been panned respectively by three kinds of antigen proteins, the original antibody library gained enrichment in different degree.Conclusion: Utilizing the technology of phage surface display, special antibody can be gained from the human naive Fab phage display library, which can be used as a new therapy for tumors.更多还原