【作者】 韦晓明；

【导师】 郝晓柯；

【作者基本信息】 第四军医大学 ， 临床检验诊断学， 2004， 硕士

【摘要】 自1975年单克隆抗体问世以来，MAb已广泛应用于临床疾病的诊断、防治及基础理论研究等领域，取得了令人鼓舞的结果。但鼠源性抗体用于人体防治会产生人抗鼠抗体(human antimouse antibody,HAMA)，它不仅使治疗用MAb迅速失去效用，而且会引起过敏反应。为此这些年来，人们一直致力于鼠源MAb的人源化的工作。噬菌体抗体库是近十几年来发展的一种新的技术，它使单克隆抗体的应用得到了进一步的推广，而且被广泛应用于单克隆抗体人源化，抗体亲和力提高等技术中。1994年Jespers等人提出了一种新的将单克隆抗体人源化的方法，称为抗原表位定向选择法(epitope guided selection,EGS)，它利用鼠源单克隆抗体与人抗体库中的抗体轻重链自由配对，用特异抗原进行筛选从而得到目的抗体片段。该方法体外模拟了抗体亲和力成熟的过程，使得到的抗体不但可以完全的人源化，而且可以具有较高的亲和力。 近年来本室一直进行抗体导向酶一前体药物疗法(antibodydirected enzyme prodrug therapy,ADEPT)对前列腺癌治疗第四军医大学硕士学位论文的的研究，设想将抗人精浆蛋白单克隆抗体与梭肤酶A融合，可靶向到达肿瘤部位，从而在肿瘤部位活化前体药物氨甲喋吟，达到治疗前列腺癌的效果。而将鼠源性的抗人精浆蛋白单克隆抗体人源化则是提高疗效的关键之一。 本研究旨在建立一个大容量的天然人抗体库，从而利用抗原表位定向选择法将本室制备的抗人精浆蛋白单克隆抗体人源化。本研究共收集了2300名志愿者的全血标本1 150ml，分离淋巴细胞，从中共提取总 RNA 1 .3mg，并将总RNA纯化，提取mRNA 36 pg。据文献报道和本室的实践经验选用了扩增及鉴定引物共14条，通过RT一PCR的方法，成功扩增出Fd段40.3 pg，轻链38.3 pg。经鉴定，进一步证实所得到的PCR产物是正确人抗体基因片段，为后继的建立抗体库的工作打下了良好的基础。为了防止EB以及紫外光对DNA片段的破坏，研究过程中选用了Li ghtBlue染液进行电泳，然后胶回收。将所得到的片段全部酶切 shrs，连入载体pC0mb3，共进行了一百多次酶切，十几次连接以及数百次电转化过程，最终建立重链库1.21 x10，，轻链库4.26x10‘。理论上总的库容量可以达到5.巧X10”。建库过程中，对电转化条件进行了一系列探索，优化了对大肠杆菌菌株XLI一Blue菌的电转化条件。实验结果证明，电容25拱F，电阻200。，电压2.SKV，O偏二0.3~0.4，0.Zcm电转化杯，DNA终浓度0.1 pg/m1，感受态菌细胞密度2.5 X10”，氨节青霉素浓度50 pg/ml，可使质粒的转化效率达到10，，基本上可满足建立一个库容量为IJ一1扩抗体库的需要。 对于一个库的筛选来说，纯化的抗原是得到可靠高亲和力抗体的必要前提。本研究利用高效亲和层析柱的方法，直接从增生前列腺组织中提取精浆蛋白，所得到的蛋白纯度高，亲和力好，第四军医大学硕士学位论文前列腺组织中提取精浆蛋白，所得到的蛋白纯度高，亲和力好，并且操作也比较简便。 一般来讲，筛选抗体的亲和力和抗体库容量成正比，而且文献报道使用抗原表位定向选择技术更易得到高亲和力的抗体。本研究建立的库为筛选高亲和力的抗体提供了良好的条件，也为本室后继对ADEPT的研究工作奠定了坚实的基础。更多还原

【Abstract】 Since the advent of monoclonal antibodies in 1975,they have been widely used in many fields, such as diagnoses .prevention and therapeutics for clinical disease and basic research. Although these applications have obtained inspiring achievements, there were still problems remained. Once for therapeutic use in humans, murine monoclonal antibodies often give rise to human anti-mouse antibody(HAMA). The HAMA response not only makes MAb no use in therapy but also causes hypersusceptibility. So during these years, researchers have been working on making murine MAb humanization. Antibody library displayed on phage surface was a new technique developed in the last ten years. It was widely used to humanize monoclonal antibodies and obtain antibodies with higher affinity. A new method called epitope guided selection(EGS) to humanize MAb was put forward by Jespers in 1994. People can obtain target antibody fragments by screen with specific antigens due to the match of murine monoclonal antibodies with the heavy chains and light chains in human antibody library. Because this method was to simulate the process of antibody maturation in vivo, the obtained antibodies was not only fully humanized but also with relatively high affinity.Recent years, research work in our lab was mainly focused on the antibody-directed enzyme prodrug theray(ADEPT) which crosslinks the monoclonal antibody to human r -seminoprotein with carboxypeptidase A active center and thentissue.The mechanism underlining this method is that the fusion protein holds the ability of reaching prostatic tumour tissue specifically due to the recognition of y ?seminoprotein in prostate with its antibody and prodrug ultimately kill tumour cells after activated by carboxypeptidase. The humanization of murine monoclonal antibody to human Y 梥eminoprotein is one of the critical steps for improving therapy effects.The main purpose of this research work is to construct a naive antibody library to human antigens with large repertoire and then to further humanize MAb to our prepared human Y 梥eminoprotein by EGS. 1150 mL peripheral blood from 2300 volunteers was used to isolate lymphocytes and 1. 3 mg total RNA was further obtained. 36ng mRNA was finally purified. We designed and synthesized 14 primers for ampilication and identification according to the reference and our experience. 40. 3 u g Fd fragments and 38.3 u g fragments of light chain were obtained by RT-PCR. And the identification further confirmed that the obtained PCR products encode human antibody fragments. These results permit further work to construct antibody library. In order to advoid the destruction of DNA fragments caused by EB and UV, the LightBlue was choosen to display the corresponding bands after electrophoresis. All the fragments were cloned into pComb3 vector after enzyme digestion for 8hrs. During the construction process, we carried out enzyme digestion for over 100 times, ligation and transformation more than ten times and several hundred times respectively. The ultimate repertoire of heavy chain library was able to reach 1.21X107, the light chain one was. 4. 26X104 . So the total repertoire was estimated to be 5. 15X10". We also did many experiments to obtain optimal condition for transformation with the highest efficiency. The optimized condition for electranformation with XLl-Blue E. coli was: capacitance 25 uF, resistance 200 .voltage 2.5 KV, 0D600=0.3~0.4,the diameter of the container was 0.2 cm, the ultimate concentration of DNA and competent cells were 0.1 ug/mL and 2. 5X 1012 respectively, the working concentration of Amp was2.5X1012respectively,the working concentration of Amp was 50 ug/mL, these conditons which make the efficency of transformation with plasmids 109 meet the demand of constructing a library with 107108 repertoire.For the screen of a library, purified antigens were necessary in order to obtain a reliable library with high affinity.The r-seminoprotein we used in this experiment was extracted by affinity chromatography from proliferating prostate tissues. So th更多还原