CHO细胞中靶向CYLD的miRNA筛选及其对抗体表达的影响

The demand for the therapeutic antibody in medical market is booming. Increasing antibody yield in Chinese Hamster Ovary (CHO) cell lines has become a research focus. Previously we found CYLD is up-regulated during anti-EGFR antibody production in CHO-K1 engineering cells. Furthermore, inhibition of CYLD by CRISPR-Cas9 could effectively increase antibody yield. However, the role of CYLD and the mechanism regulating CYLD in CHO cells remain unclear. So we would like to further study the function of CYLD in antibody production by gene knockout and overexpression; Screen cgr-miRNA targeting CYLD by bioinformatics, luciferase reporter gene detection system, fluorescence quantitative PCR, Western Blot; Furthermore explore the potential transcriptional factor/pathway regulating cgr-miRNA by bioinformatics, EMSA；Exam the function of miRNA in antibody production. We believe our investigation of miRNA-CYLD axis in antibody production will not only help us to further understand the regulating mechanism of CYLD in CHO cells, but will also provide novel basis for increasing antibody yield in CHO cell engineering.

医疗市场对治疗性抗体药物的需求与日俱增，提高中国仓鼠卵巢（CHO）细胞株的抗体表达能力成为研究热点。我们前期研究发现：CHO-K1工程细胞株生产抗EGFR抗体过程中，去泛素化酶CYLD被显著上调，通过CRISPR-Cas9抑制CYLD能有效提高抗体产量。而CYLD在CHO细胞工程中的作用和调控CYLD的机制尚不清楚。本项目拟进一步以miRNA-CYLD为对象，通过基因敲除、过表达等手段研究CYLD在抗体表达中的作用和功能，并综合利用生物信息学、荧光素酶报告基因检测系统、荧光定量PCR和免疫印迹等技术筛选靶向CYLD的仓鼠源miRNA，进而通过生物信息学分析、EMSA等技术探究调控特异性miRNA的转录因子/信号通路，并检测特异性miRNA对抗体表达的影响。从而阐明“转录因子/信号通路-特异性miRNA-CYLD”参与抗体表达的分子机制，同时为提高CHO细胞表达抗体提供依据。

提高治疗性抗体的产量一直是抗体生产领域的核心科学问题之一。而通过改造单一基因可以优化多种生物学功能，并显著提升高产细胞株的构建速度和抗体表达能力。因此，筛选影响宿主细胞产抗能力的功能性基因十分重要。课题组首次发现了去泛素化酶CYLD参与影响中国仓鼠卵巢（CHO）细胞表达治疗性抗体，而失活CYLD可以显著提高CHO细胞的增殖能力、抗凋亡能力以及抗体表达水平。进一步通过生物信息学筛选并鉴定了仓鼠源miR-106b可以直接靶向CYLD，过表达miR-106b可通过调控CYLD、及其下游NF-κB和Wnt/β-catenin信号通路增强CHO细胞的培养表现和相应的抗体表达水平。该研究不仅阐明了CHO细胞中调控CYLD的分子机制，并为提高治疗性抗体的产量提供了重要的应用基础。