单细胞PCR技术获取人源抗体平台的建立

Therapeutic monoclonal antibody (mAb) has been an effective strategy for cancer and autoimmune disease, etc. Therapeutic mAb underwent development from murine antibody, chimeric antibody, humanized antibody to fully human antibody. Fully human antibody has become the comparatively ideal choice and because of the small side effects and becomes the hot spot in Bio/Pharmaceutical Industry. Most of the therapeutic human mAbs were derived from either transgenic mouse or phage display technique. But the broad use of these techniques was limited due to the technique monopolization and intellectual property rights reserved by the top pharmaceutical company. Research & development of the therapeutic mAbs in China stay at the level of chimeric antibody and humanized antibody. The development of fully human mAb needs to be improved. While, fully human mAb can be derived from human who live in the conditions with complicated antigens. Many kinds of neutralizing mAbs are generated in human body to protect the human from the attack of different pathogens. And it's enormously valuable to explore these neutralizing mAbs from human. . In this study, the platform for fully human monoclonal antibody using single cell PCR technique is going to be constructed. The B cell from peripheral blood with the expression of the specific antibody will be marked with specific antigen and sorted by FACS. Single cell PCR technique will be used to obtain human mAb which comes through antigen stimulation and affinity maturation in human body. More than three neutralizing mAbs against hepatitis B virus is going to be obtained. It might be a potential technique to get the therapeutic human mAb and make contribution to the research & development of the therapeutic mAbs in China.

抗体药物是治疗癌症、自身免疫病等较理想的药物，其发展经历了鼠源抗体、人-鼠嵌合抗体、人源化抗体、人源抗体的阶段。人源抗体不含异源蛋白序列，是较理想的抗体药物，成为当今生物制药领域中的研发热点。目前，转基因小鼠和噬菌体抗体库技术是人源抗体研发的主要途径，但由于大制药公司的技术垄断和知识产权的限制，阻碍了这些技术的广泛应用。我国抗体药物的研发基本处于嵌合抗体、人源化抗体阶段，人源抗体的研发水平亟待提高。人源抗体的一个重要来源是人体，人生活在纷繁复杂的抗原环境中，体内存在着针对多种病原的优质中和性抗体，这些抗体历经抗原刺激和体内亲和力成熟，极具开发价值。.本研究要建立单细胞PCR技术获取人源抗体的技术平台。从人外周血中，用抗原直接分选能表达目标抗体的B细胞，利用单细胞PCR技术，克隆抗体基因，获得抗体。本研究拟获得3株以上具有中和活性的乙肝抗体，这项技术将为我国人源抗体药物的研发提供技术支持。

治疗性抗体药物中,人源抗体是较理想的抗体药物。人生活在纷繁复杂的抗原环境中，体内存在着针对多种病原的优质中和性抗体，这些抗体历经抗原刺激和体内亲和力成熟，极具开发价值。本研究旨在建立从人外周血B细胞中获得人源抗体的技术平台。通过研究，完成以下工作：利用流式细胞仪分选，建立了高效分选表达特异性抗体B细胞的技术；优化了高通量操作，完善了克隆人源抗体基因各环节的反应体系和条件；建立了高效经济的抗体瞬时真核表达体系。 .研究以乙型肝炎抗体为具体目标，通过乙肝表面抗原标记、流式细胞仪分选，获得表达乙肝抗体的B细胞。利用单细胞PCR技术，克隆得到抗体VH基因、VL基因，并构建抗体IgG真核表达载体，获得针对乙肝S抗原的抗体基因35个。经真核细胞表达，获得32个针对S抗原的抗体蛋白。.通过Elisa抗原结合实验，筛选到1个针对S抗原的抗体HSA。采用HBV感染的NTCP（牛磺胆酸钠共转运蛋白）过表达的HepG2细胞系，进行抑制乙肝病毒感染实验。结果显示，4μM乙肝抗体HSA能够抑制HBV感染体系中表面抗原HBsAg和核心抗原HBeAg的表达，显示抑制活性。同时用qPCR检测感染细胞内HBV的共价闭合环状DNA(cccDNA)含量，这部分实验还在继续，数据还在整理中。.通过本研究，以乙肝抗体为具体内容，建立了从人外周血B细胞中获得人源抗体的技术平台。基于这个平台，利用不同的抗原，经过流式细胞筛选、抗体基因克隆和表达，可以获得针对特异抗原的人源抗体。