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人源化EGFR抗体轻链拷贝数对CHO-K1bak-/bax-细胞NW_003626341.1位点定点整合的表达影响
发表时间：2021-09-30 浏览量：871 下载量：145
| 全部作者： | 董心怡，陈蕴，吴浩，杨蕾，金坚 |
| 作者单位： | 江南大学药学院 |
| 摘 要： | 目的：采用CRISPR/Cas9基因编辑技术，将表皮生长因子受体（epidermal growth factor receptor，EGFR）人源化抗体基因以不同轻链拷贝数定点整合至抗凋亡CHO-K1bak-/bax-（CHO-Ie3）细胞的LOC103162981基因内NW_003626341.1位点处，比较轻链拷贝数对抗体表达的影响。方法：将编码EGFR抗体轻、重链基因，绿色荧光蛋白基因，嘌呤霉素抗性基因的供体质粒pEF1α-LH、pEF1α-LLH，分别与单导向RNA（sgRNA）、Cas9质粒用转染试剂盒共转染进CHO-Ie3细胞，经药筛及流式分选后在96孔板中获得单克隆细胞株。将这些细胞的培养上清进行Dlot-blotting、Western blotting实验，提基因组后PCR扩增鉴定外源蛋白是否准确整合至目标位点并表达。悬浮驯化成功整合外源基因的细胞株，批次培养分析EGFR抗体的表达情况。结果：获得1株CHO-Ie3-LH细胞，阳性单克隆率约为1.89%，2株CHO-Ie3-LLH细胞，阳性单克隆率约为3.51%，其中轻链双拷贝的细胞株所表达外源蛋白的产量达115.40 mg/L，相较于轻链单拷贝的细胞株表达量提升35.32 %. 结论：在CHO-Ie3细胞中采用CRISPR/Cas9定点整合技术可以成功表达EGFR抗体，且抗体轻链的拷贝数的增加对蛋白表达起到一定的提高作用。 |
| 关 键 词： | 分子生物学；抗表皮生长因子受体；CHO细胞；单克隆抗体 |
| Title： | Influence on expression of humanized anti-EGFR with different copy numbers of light chain which site-specific integrated into CHO-K1bak-/bax-cells NW_003626341.1 |
| Author： | DONG Xinyi, CHEN Yun, WU Hao, YANG Lei, JIN Jian |
| Organization： | School of Pharmaceutical Sciences, Jiangnan University |
| Abstract： | Objective: In order to compare the influence of copy numbers of the light chain on the antibody expression, the genes of humanized anti-epidermal growth factor receptor (anti-EGFR) were site-specific integrated at NW_003626341.1 site of the LOC103162981 gene in anti-apoptotic CHO-K1bak-/bax- (CHO-Ie3) cells with different copy numbers of the light chain through CRISPR/Cas9 gene-editing technology. Methods: Donor plasmids of pEF1α-LH and pEF1α-LLH contained genes encoding the light and heavy chains of anti-EGFR, the enhanced green fluorescent protein (EGFP) and puromycin-resistance were respectively co-transfected with the single guide RNA (sgRNA) plasmid and Cas9 plasmid to CHO-Ie3 cells with mediation of transfection kit. After selecting and sorting, monoclonal cell lines were generated in a 96-well plate. Dolt-blotting and Western blotting were used to verify whether genes of interests were integrated and expressed accurately in the expect locus. The cell lines that successfully targeted exogenous genes were acclimated by suspension. The expression of anti-EGFR was analyzed through batch culture. Results: Two CHO-Ie3-LLH cell lines and one CHO-Ie3-LH cell line that correctly expressed anti-EGFR were observed via screening and verification. The probability of the light chain with double-copy undergoing site-specific integration was about 3.51%, while the light chain with single-copy was about 1.89%. The antibody produced by CHO-Ie3-LLH cell lines were 115.40 mg/L and the expression level was increased by 35.32% compared with CHO-Ie3-LH cell lines. Conclusion: Humanized anti-EGFR was accurately expressed in CHO-Ie3 cells through CRISPR/Cas9 gene-editing technology. The increase in the copy numbers of the antibody light chain showed a significant effect on the expression of exogenous antibody. |
| Key words： | molecular biology; anti-epidermal growth factor receptor; CHO cells; monoclonal antibodies |
| 发表期数： | 2021年9月第3期 |
| 引用格式： | 董心怡，陈蕴，吴浩，等. 人源化EGFR抗体轻链拷贝数对CHO-K1bak-/bax-细胞NW_003626341.1位点定点整合的表达影响[J]. 中国科技论文在线精品论文，2021，14（3）：294-303. |
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