稳定表达膜结合型CD40配体CHO细胞系的建立和鉴定  

Establishment and identification of CHO cell line stably expressing membrane-bound CD40L

作　　者：陈彦[1] 于津浦[1] 李慧[1] 曹水[1] 任秀宝[1] 

机构地区：[1]天津医科大学附属肿瘤医院免疫室/天津市肿瘤防治重点实验室,300060

出　　处：《中国医药生物技术》2008年第1期40-44,共5页Chinese Medicinal Biotechnology

摘　　要：目的建立稳定表达膜结合型CD40配体(CD40L)的CHO细胞系。方法将编码膜结合型CD40L的重组质粒pcDNA3.1-fCD40L用BgIⅡ和XhoⅠ酶进行双酶切鉴定。电穿孔法向CHO细胞转染该重组质粒,G418筛选抗性克隆,有限稀释法获取单克隆阳性细胞后扩大培养,获得2个整合了fCD40L基因的CHO细胞(CHO-fCD40L)克隆。用RT-PCR、流式细胞仪分别从RNA和蛋白质水平对2个CHO-fCD40L克隆细胞中膜结合型CD40L的表达进行检测;ELISA法检测2个CHO-fCD40L克隆细胞培养上清液中可溶性CD40L(sCD40L)的含量。结果重组质粒pcDNA3.1-fCD40L经双酶切和电泳检测到符合预期大小的基因片段。该重组质粒转染CHO细胞后经克隆化培养获得2个CHO-fCD40L克隆,分别命名为C10、E11。RT-PCR检测显示C10、E11细胞中有膜结合型CD40LmRNA的转录;流式细胞仪检测显示C10、E11细胞表达CD40L蛋白达90%以上;ELISA法检测到显示C10、E11细胞培养液上清中有sCD40L蛋白表达。结论成功获得稳定表达膜结合型CD40L的CHO细胞系,为比较膜结合型CD40L与sCD40L的生物学功能奠定了良好基础。Objective To establish a Chinese hamster ovary （CHO） line stably expressing membrane-bound CD40L. Methods The CD40L gene was inserted into a eukaryotic expression plasmid pcDNA3.1 to construct a recombinant expression plasmid pcDNA3.1-fCD40L. The recombinant plasmid was identified with Bg1 Ⅱ and Xho I enzyme. Then the CHO cells were transfected with the plasmid via electroporation method and screened with antibiotic G418. Single clones expressing CD40L were obtained by limited-dilution method. Expressions of CD40L mRNA and protein in positive clones were detected by RT-PCR and flow cytometry. The protein of soluble CD40L （sCD40L） secreted by the positive clones was measured by ELISA. Results A gene segment with an expected size was detected in the recombinant eukaryotic expression plasmid pcDNA3.1-fCD40L by double digestion and electrophoresis. The recombinant CHO cells stably expression membrane-bound CD40L cell lines named C10, Ell. The CD40L mRNA transcription was detected by RT-PCR in the C10, Ell. As identified by flow cytometry, more than 90% of C10 and Ell cell lines expressed high level of CD40L mRNA. Soluble CD40L protein was detected in the supernatant of C10, Ell by ELISA. Conclusions A CHO cell line stably expressing membrane-bound CD40L protein was established. It provided a useful tool to investigate immunological difference between membrane-bound CD40L and soluble CD40L.

关 键 词：CD40配体 CHO细胞 质粒 转染 

分 类 号：R392[医药卫生—免疫学]