外源蛋白在CHO细胞染色体上一个新位点的定点整合和稳定表达

Site-specific integration and stable expression of exogenous protein at a novel site on CHO cell chromosome

寻找中国仓鼠卵巢(CHO)细胞染色体上稳定表达位点是解决CHO细胞长期培养表达不稳定问题的有效手段。本课题组前期利用慢病毒转染将示踪基因(Zsgreen1)整合CHO细胞染色体上并发现多个潜在稳定表达位点。本研究验证了其中一个位点稳定表达外源蛋白的能力,该位点位于染色体NW_003614241.1上148052~148157 bp区域。首先观察Zsgreen1基因的表达情况,随后采用CRISPR/Cas9技术将增强绿色荧光蛋白(EGFP)基因定点整合至该位点,获得3株EGFP基因定点整合的细胞,经60代悬浮培养,细胞荧光强度无明显变化,证明此位点可稳定表达EGFP基因。采用同样方法构建了表达人血清白蛋白(HSA)基因的重组CHO细胞株,经Western blot验证,此位点可分泌表达HSA,表明上述位点可定点整合和稳定表达外源蛋白。

Finding stable expression sites on the chromosomes of Chinese hamster ovary(CHO)cells is an effective method to solve the problem of unstable expression of CHO cells in long-term culture.Our group used lentiviral transfection to integrate the tracer gene(Zsgreen1)into the chromosome of CHO cells and found multiple potential stable expression sites.This study verified the ability of one of the sites located in the 148052-148157 bp region on chromosome NW_003614241.1 to stably express exogenous proteins.The expression of Zsgreen1 gene was first observed,and CRISPR/Cas9 technology was then used to integrate the enhanced green fluorescent protein(EGFP)gene into this site.Three strains of EGFP gene integrated cells were obtained.After 60 generations of suspension culture,the fluorescence intensity of the cells had no significant changes,which proved that this site can stably express the EGFP gene.The same method was used to construct recombinant CHO cell lines expressing the human serum albumin(HSA)gene,and was verified by Western blot that this site could express and secrete HSA.It shows that the above-mentioned sites can be integrated and can stably express exogenous proteins.