稳定表达sCD40L的CHO细胞系的建立和鉴定

Establishment and identification of a Chinese hamster ovary cell line stably expressing sCD40L

目的:建立稳定表达sCD40L的CHO细胞系。方法:从含sCD40L的载体pDC316-sCD40L中,将sCD40L编码序列克隆入真核表达载体pIRES2-EGFP,获得重组质粒pIRES2-EGFP-sCD40L,酶切及测序鉴定。电穿孔转染CHO细胞,G418筛选抗性克隆,有限稀释法获取单克隆后扩大培养。流式细胞术、倒置荧光显微镜观察绿色荧光蛋白的表达;使用PCR、RT-PCR、ELISA方法分别从DNA、mRNA、蛋白水平对sCD40L的表达进行检测。Westernblotting检测CHO-sCD40L分泌的sCD40L与膜型CD40的结合;将CHO-sCD40L与MDA-MB-231细胞共孵育24h后,流式细胞术检测MDA-MB-231细胞表面分子Fas的表达变化;加入Fas激活性抗体(CH-11)后观察MDA-MB-231细胞的凋亡。结果:成功构建质粒pIRES2-EGFP-sCD40L,转染CHO细胞后经克隆化培养获得稳定表达sCD40L的细胞系CHO-sCD40L。流式细胞术、倒置荧光显微镜检测绿色荧光蛋白表达高达90%以上;PCR检测证实sCD40L基因整合入细胞基因组DNA;RT-PCR检测到sCD40LmRNA的转录;ELISA法检测到细胞培养上清中有sCD40L蛋白表达,高达(4.5±2.1)ng/ml。Western blotting证实CHO-sCD40L分泌的sCD40L可与膜型CD40结合。CHO-sCD40L与MDA-MB-231细胞共培养后,MDA-MB-231细胞表面Fas表达由(3.0±1.02)%升高到(34.8±8.75)%;加入CH-1124h后,凋亡率由(5.4±1.32)%提高到(20.7±5.24)%(P<0.01)。结论:成功获得稳定表达sCD40L的CHO细胞系,为研究CD40/CD40L途径在肿瘤免疫治疗中的应用提供了有用的工具。

Objective： To establish a Chinese hamster ovary cell line stably expressing sCD40L. Methods： sCD40L was obtained from vector pDC316-sCD40L by restricted enzymatic resection and was inserted into eukaryotic expression plasmid pIRES2-EGFP to construct a recombinant expression plasmid plRES2-EGFP-sCD40L. The recombinant plasmid was then digested with Bgl Ⅱ and Sal Ⅰ and was confirmed to contain full length of pIRES2-EGFP-sCD40L cDNA by agarose gel analysis and DNA sequence analysis. The CHO cells were transfected with the recombinant plasmid by using electroporation and screened with antibiotic G418. Single clones expressing sCD40L were obtained by limited-dilution method. EGFP-positive cells wet＇e detected by flow cytometry and inverted fluorescence microscopy. The DNA integration and mRNA expression of sCD40L in positive clones were detected by PCR and RT-PCR; the protein of sCD40L was evaluated by ELISA; and the conjugation between sCD40L and CD40 was detected by Western blotting. MDA-MB-231 cells were cocultured with CHO-sCIMOL for 24 h and the changes of Fas expression on MDA-MB-231 cell surface were detected by FCM; the apoptosis of MDA-MB-231 cells was observed 24 h after cocultured with anti-Fas activating antibody（CH11 ）. Results： A recombinant eukaryotic expression plasmid plRES2-EGFP-sCD40L was successfully constructed. A CHO- sCD40L cell line stably expressing sCD40L was subsequently obtained. FCM and inverted fluorescence microscope showed that 90% of the cells had positive fluorescent signals ; PCR and RT-PCR confirmed that incorporation of sCD40L DNA and expression of sCD40L mRNA ; ELISA revealed an expression of sCD40L protein of （4.5 ± 2.1 ） ng/ml in the supernatant ; and Western blotting displayed CHO-sCD40L cells-secreted sCD40L could conjugate with CD40. The expression of Fas on the surface of MDA-MB-231 cells increased from （ 3.0 ± 1.02 ） % to （ 34.8 ± 8.75 ） % after cocultured with CHO- sCI40L cells ; the apoptotic rates of MDA-MB-231 cells increased from（5.4± 1.32） % to（ 20.7 ± 5.24 ） % after cocultured with CH-11 （P 〈0.01 ）. Conclusion： We have successfully established a CHO cell line stably expressing sCI40L, which may help to investigate the role of CD40/CD40L pathway in the adjuvant immunotherapy of tumor.