鼠抗人CD52单克隆抗体制备

发布时间：2018-05-19 17:45

本文选题：CD52 + 真核表达载体　；参考：《中国协和医科大学》2009年硕士论文

【摘要】： 人类CD52抗原属于一个只有十二个氨基酸残基(GQNDTSQTSSPS)的GPI锚定糖蛋白,N末端在3位天冬酰氨上连接有一个具有负电荷唾液酸残基的复杂的糖基,C末端通过与GPI锚连接而使整个分子固定在细胞膜上。CD52抗原广泛分布于淋巴细胞、单核细胞、嗜酸性粒细胞等造血细胞上以及雄性生殖上皮细胞表面。CD52在细胞上表达密度高,就淋巴细胞而言每个淋巴细胞上约有450 000个分子,约占到细胞表面成分的5%,因此成为了抗体攻击的理想的靶抗原。在1980年剑桥大学就发现了CD52抗体能在体外通过加入人的补体后杀伤带有CD52抗原的靶细胞。因此寻找到有杀伤抑制作用的CD52抗体具有重要的应用价值。至今最成功的抗CD52抗体药物是Alemtuzumab (campath-1H),是一个经过人源化改造的抗CD52单克隆抗体,被成功用于一些慢性淋巴细胞白血病和自身免疫疾病的治疗和研究。本课题主要是采用杂交瘤技术制备功能性的抗人CD52单克隆抗体,同时建立稳定CD52真核转染细胞系,以便于快速准确筛选抗人CD52单克隆抗体。实验方法如下：首先,应用HUT-78细胞提取细胞总RNA,运用RT-PCR方法扩增CD52基因,定向克隆入真核表达载体pcDNA3.1 (+),用脂质体转染法转染CHO细胞,建立稳定转染的CHO细胞系,用RT-PCR、FACS、免疫组织化学技术检测转染细胞系中目的基因和蛋白的表达。其次,根据NCBI提供的CD52抗原的结构特点及campath-1H作用的抗原表位特点,设计合成了靠近CD52抗原C基末端的八个氨基酸的短肽,用于免疫和筛选抗人CD52单克隆抗体。最后,采用合成肽CD52-KLH免疫Balb/c鼠,进行细胞融合与培养,用合成肽CD52-BSA进行间接ELISA实验初步筛选,通过FACS和Western blot鉴定抗体,MTS实验筛选鉴定有细胞抑制作用的抗体。结果显示建立了稳定转染的CHO细胞系,初步筛选鉴定出两株抗人CD52单克隆抗体,为进一步研究单克隆抗体的功能和鼠抗体人源化改造奠定了基础。
[Abstract]:Human CD52 antigen belongs to a GPI anchored glycoprotein N terminal with only 12 amino acid residues (GQNDTSQTS SPS), which is connected to a complex glycosylated C terminal with a negatively charged sialic acid residue on the 3 position aspartate by connecting with the GPI anchor. The whole molecule is immobilized on the cell membrane. CD52 antigen is widely distributed in lymphocytes. In monocytes, eosinophils and other hematopoietic cells, and on the surface of male reproductive epithelial cells, CD52 is highly expressed in cells, with about 450,000 molecules per lymphocyte, as far as lymphocytes are concerned. It accounts for about 5% of the cell surface components, so it is an ideal target antigen for antibody attack. In 1980, the University of Cambridge discovered that CD52 antibodies can kill target cells with CD52 antigen by adding human complement in vitro. Therefore, it has important application value to find CD52 antibody which can kill and inhibit it. The most successful anti CD52 antibody is Alemtuzumab campath-1HG, a humanized anti CD52 monoclonal antibody, which has been successfully used in the treatment and research of chronic lymphoblastic leukemia and autoimmune diseases. In this study, functional anti-human CD52 monoclonal antibodies were prepared by hybridoma technique, and stable CD52 eukaryotic transfection cell lines were established for rapid and accurate screening of anti-human CD52 monoclonal antibodies. The experimental methods are as follows: Firstly, total RNAs were extracted from HUT-78 cells, CD52 gene was amplified by RT-PCR method, and cloned into eukaryotic expression vector pcDNA3.1 (pcDNA3.1). CHO cells were transfected with liposome, and stable CHO cell lines were established. The expression of target gene and protein in transfected cell line was detected by RT-PCR- FACS and immunohistochemistry. Secondly, according to the structural characteristics of CD52 antigen provided by NCBI and the antigenic epitope characteristics of campath-1H, a short peptide of eight amino acids near the C-terminal of CD52 antigen was designed and synthesized, which was used to immunize and screen monoclonal antibodies against human CD52. Finally, Balb/c mice were immunized with synthetic peptide CD52-KLH. The cells were fused and cultured. The indirect ELISA assay was performed with synthetic peptide CD52-BSA. The antibodies were identified by FACS and Western blot. The results showed that a stable transfected CHO cell line was established and two monoclonal antibodies against human CD52 were identified by preliminary screening, which laid a foundation for further study on the function of monoclonal antibody and humanization of mouse antibody.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.1

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