摘要
目的构建抗人表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)人源化单克隆抗体CHO工程细胞株,并进行筛选及表达。方法将抗HER2抗体重链基因Fd及轻链基因VL分别克隆至同一质粒p RH上,将构建正确的重组表达质粒命名为p HER2。通过脂质体法将p HER2转染至二氢叶酸还原酶(DHFR)缺陷型CHO细胞(CHO-DG44)中,筛选稳定表达的工程细胞株,进行ELISA及SDS-PAGE检测。同时对抗HER2抗体高表达细胞株进行batch模拟试验。结果从近10 000株克隆中筛选获得3株备选细胞株,命名为H6、H7和H9。细胞株培养上清表达的抗体仅能与重组HER2发生特异性结合;SDS-PAGE检测结果表明,于相对分子质量50 000和25 000处可见重链和轻链的目的蛋白条带。H6、H7和H9细胞株在简单的batch悬浮培养条件下周期约为1周,抗体水平随培养时间的延长而逐渐升高,H9细胞株表达水平最高,为0.52 mg/ml。结论本研究筛选获得的工程细胞株H9具有较高的抗HER2单克隆抗体水平,为靶向治疗HER2过表达的乳腺癌和胃癌等恶性肿瘤奠定了基础。
Objective To construct, screen and express a recombinant CHO cells for monoclonal antibody（Mc Ab）against human epidermal growth factor receptor 2（HER2）. Methods The heavy chain gene Fd and light chain gene VL of Mc Ab against HER2 were cloned into plasmid p RH. The constructed recombinant plasmid p HER2 was transfected to DHFR defective CHO-DG44 cells in mediation of Lipofectamine 2000, based on which a recombinant CHO cell strain for stable expression was screened, identified by ELISA and SDS-PAGE, and subjected to batch mock test. Results Three recombinant cell lines, H6, H7 and H9, were screened from nearly 10 000 clones. The antibody expressed in supernatant showed only specific binding to recombinant HER2. The bands of heavy and light chains, with relative molecular masses of 50 000 and 25 000 respectively, were shown on SDS-PAGE profile. The cell cycles of H6, H7 and H9 strains in simple batch suspension culture were about one week, which the secreted antibody level increased gradually with the increasing time for culture. The expression level of H9 was the highest, which was 0. 52 mg / ml. Conclusion The screened recombinant cell line H9 secreted high level of Mc Ab against HER2, which laid a foundation of targeting therapy of tumors in which HER2 were overexpressed, such as breast cancer and gastric carcinoma.
出处
《中国生物制品学杂志》
CAS
CSCD
2016年第10期1056-1059,共4页
Chinese Journal of Biologicals
关键词
人表皮生长因子受体2
单克隆抗体
中国仓鼠卵巢细胞
Human epidermal growth factor receptor 2（HER2）
Monoclonal antibody（McAb）
Chinese hamster ovary（CHO）cells
