表达rhGH-Fc融合蛋白的CHO工程细胞的传代稳定性分析

Stability of engineered CHO cells for expression of rhGH-Fc fusion protein during subculture

目的分析表达重组人生长激素(recombinant human growth hormone,rhGH)-Fc融合蛋白的CHO工程细胞的传代稳定性,确定细胞的生产稳定代次。方法取表达rhGH-fc融合蛋白的CHO细胞(35代),连续传代建立50、70、90代细胞库。复苏各代次细胞并进行培养表达,绘制不同代次的细胞生长曲线,比较不同代次细胞在对数生长期的比生长速率;培养结束,对不同代次细胞表达的蛋白进行亲和层析纯化,比较其表达量,Western blot法对表达蛋白进行鉴定,通过对目的基因mRNA测序比较目的基因的稳定性。结果 50和70代细胞与35代相比,生长曲线基本重合,90代细胞曲线出现差异;50、70、90代细胞在对数生长期的比生长速率与35代相比,差异均无统计学意义(P>0.05),但90代的P值已明显降低;50和70代细胞重组蛋白表达量与35代相比,差异均无统计学意义(P> 0.05),但90代细胞重组蛋白的表达量下降比例达42%;各代次细胞rhGH-Fc基因序列保持稳定,与理论一致。结论表达rhGH-Fc融合蛋白的CHO工程细胞的生产稳定代次为70代,可满足后续研究和生产需要。

Objective To analyze the stability of engineered CHO cells for expression of recombinant human growth hormone(rhGH)-Fc fusion protein,and determine the stable passage of cells for production. Methods CHO cells of passage 35 for expression of rhGH-fc fusion protein were subcultured,based on which the cell banks of passages 50,70 and 90 were established. The cells of various passages were resuscitated and cultured,and the cell growth curves were plotted to compare the specific growth rates of various passages in the logarithmic growth phase. The proteins expressed in the cells of various passages were purified by affinity chromatography and identified by Western blot,of which the expression levels were compared. The stability of the target gene was evaluated by sequencing of the target mRNA.Results The growth curves of passages 50 and 70 were basically coincided with,while that of passage 90 was different from,that of passage 35. However,the specific growth rates in the logarithmic growth phase of passages 50,70 and 90 showed no significant difference with that of passage 35(P > 0. 05),while the P value of passage 90 decreased significantly. The expression levels of rhGH-Fc fusion protein in the cells of passages 50 and 70 showed no significant difference with that of passage 35(P > 0. 05),while that in the cells of passage 90 decreased by 42%. However,the rhGH-Fc gene sequences in the cells of various passages were stable and consistent with that in theory. Conclusion The engineered CHO cells of passage 70 was stable for expression of rhGH-Fc fusion protein,which met the needs for further research and production.