HSV-IgM人-鼠嵌合抗体制备及稳定细胞株构建工艺开发

Preparation of HSV-IgM human-mouse chimeric antibody and development of stable recombinant cell line

摘要:

为实现体外大规模制备单纯疱疹病毒HSV-IgM (HSV1, HSV2)人鼠嵌合抗体，本研究通过RNA连接酶介导的cDNA末端快速扩增(RNA ligase-mediated rapid amplification of cDNA ends, RLM-RACE)技术获取其对应杂交瘤细胞基因序列，构建嵌合抗体至真核表达载体，在CHO-S细胞中稳定表达所需目的蛋白。同时优化稳定细胞株筛选工艺，对细胞池构建阶段和单克隆筛选阶段的加压条件进行摸索与探究，最后目的抗体采用蛋白L亲和纯化法进行纯化并进行生物活性检测；最终成功制备899 kDa和909 kDa的稳定高表达重组IgM抗体(HSV1, HSV2)细胞株。结果表明，最适筛选压力为20P200M (一轮加压)和50P1000M (二轮加压)；使用加压培养基进行单克隆筛选抗体表达量较高，HSV1-IgM和HSV2-IgM单克隆最终表达量分别为1 620 mg/L和623 mg/L。本研究为HSV1和HSV2的IgM系列重组抗体质控品开发以及体外高表达分泌IgM亚型抗体提供理论与实践基础。

关键词:人鼠嵌合IgM抗体;CHO-S细胞;构建工艺;表达

Abstract:

In order to achieve large-scale production of HSV-IgM (HSV1, HSV2) human-mouse chimeric antibody in vitro, the gene sequence of the corresponding hybridoma cell was harvested by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) technique to clone the chimeric antibody into eukaryotic expression vectors, and express the target proteins in CHO-S cells. At the same time, the screening process of stable cell lines was optimized, and the pressure conditions of pool construction stage and monoclonal screening stage were explored. Finally, the target protein was purified by protein L affinity purification method and the biological activity was detected. The recombinant IgM antibodies, HSV1 and HSV2, weighted at 899 kDa and 909 kDa respectively, were prepared. The optimal screening pressure was 20P200M (the first phase of pressure) and 50P1000M (the second phase of pressure). The final titer for the monoclonal expression of HSV1-IgM and HSV2-IgM was 1 620 mg/L and 623 mg/L, respectively. This study may facilitate the development of quality control products of HSV1 and HSV2 IgM series recombinant antibodies as well as efficient expression of IgM subtype antibodies in vitro.

Key words:human-mouse chimeric IgM antibody;CHO-S cell;construction technology;expression