Institutes of Health Central Plains, Xinxiang Medical University, No. 601 Jinsui Road, Xinxiang, 453003, Henan Province, China.
International Joint Research Laboratory for Recombinant Pharmaceutical Protein Expression System of Henan, Xinxiang Medical University, No. 601 Jinsui Road, Xinxiang, 453003, Henan Province, China.
Sci Rep. 2024 Sep 6;14(1):20856. doi: 10.1038/s41598-024-71915-0.
At present, biopharmaceuticals have received extensive attention from the society, among which recombinant proteins have a good growth trend and a large market share. Chinese hamster ovary (CHO) cells are the preferred mammalian system to produce glycosylated recombinant protein drugs. A highly efficient and stable cell screening method needs to be developed to obtain more and useful recombinant proteins. Limited dilution method, cell sorting, and semi-solid medium screening are currently the commonly used cell cloning methods. These methods are time-consuming and labor-intensive, and they have the disadvantage of low clone survival rate. Here, a method based on semi-solid medium was developed to screen out high-yielding and stable cell line within 3 weeks to improve the screening efficiency. The semi-solid medium was combined with an expression vector containing red fluorescent protein (RFP) for early cell line development. In accordance with the fluorescence intensity of RFP, the expression of upstream target gene could be indicated, and the fluorescence intensity was in direct proportion to the expression of upstream target gene. In conclusion, semi-solid medium combined with bicistronic expression vector provides an efficient method for screening stable and highly expressed cell lines.
目前，生物制药受到了社会的广泛关注，其中重组蛋白具有良好的增长趋势和较大的市场份额。中国仓鼠卵巢（CHO）细胞是生产糖基化重组蛋白药物的首选哺乳动物系统。需要开发一种高效、稳定的细胞筛选方法，以获得更多有用的重组蛋白。有限稀释法、细胞分选和半固体培养基筛选是目前常用的细胞克隆方法。这些方法耗时耗力，克隆存活率低。在这里，开发了一种基于半固体培养基的方法，可在 3 周内筛选出高产、稳定的细胞系，以提高筛选效率。半固体培养基与含有红色荧光蛋白（RFP）的表达载体结合，用于早期细胞系的开发。根据 RFP 的荧光强度，可以指示上游靶基因的表达，荧光强度与上游靶基因的表达成正比。总之，半固体培养基与双顺反子表达载体相结合，为筛选稳定、高表达的细胞系提供了一种有效的方法。
