Establishment and Identification of Chinese Hamster Ovary Cell Lines with Stable Expression of Soluble CD40 Ligands

• Main Authors: JIANG Hua-wei, YANG Quan-liang, LIU Yong-ping, LING Yang
• Format: Article
• Language: English
• Published: Third Party Medicine International Publishing Group Co. Limited 2014-09-01
• Series: Journal of International Translational Medicine
• Subjects: Soluble CD40 ligand; Green fluorescent protein; Apoptosis; Fatty acid synthase
• Online Access: http://www.jitm.hk/EN/10.11910/2227-6394.2014.02.03.05

Objective: To establish the Chinese Hamster Ovary (CHO) cell lines with stable expression of soluble CD40 ligands (sCD40L). Methods: Recombinant plasmid pIRES2-EGFP-sCD40L, enzyme digestion and sequencing identification were obtained by cloning sCD40L coding sequences into eukaryotic expression vector pIRES2-EGFP from carrier pDC316-sCD40 containing sCD40L. CHO cells were transfected by electroporation, followed by screening of resistant clones with G418, after which monoclones were obtained by limited dilution assay and multiply cultured. Flow cytometer and reverted fluorescence microscope were applied to observe the expression of green fluorescent protein, while sCD40L expression was detected by polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) from aspects of deoxyribose nucleic acid (DNA), messenger ribonucleic acid (mRNA) and protein, respectively. CHO-sCD40L was cultured together with MDA-MB-231 cells to compare the expression changes of surface molecule fatty acid synthase (Fas) by flow cytometer and observe the apoptosis of MDA-MB-231 cells after Fas activated antibodies (CH-11) were added 24 h later. Results: Plasmid pIRES2-EGFP-sCD40L was successfully established, and cell lines with stable expression of sCD40L were obtained with cloned culture after CHO cell transfection, which was named as B11. Flow cytometer and reverted fluorescence microscope showed ＞90% expression of green fluorescent protein, while PCR, RT-PCR and ELISA suggested integration of sCD40L genes into cell genome DNA, transcription of sCD40L mRNA and sCD40L protein expression being (4.5±2.1) ng/mL in the supernatant of cell culture, respectively. After co-culture of B11 and MDA-MB-231 cells, the surface Fas expression of MDA-MB-231 cells was increased from (3±1.02) % to (34.8±8.75)%, while the apoptosis rate 24 h after addition of CH11 from (5.4±1.32)% to (20.7±5.24)%, and the differences were statistically significant when compared with those in control group (P＜0.01). Conclusion: CHO cell lines with stable expression of sCD40L are successfully obtained and provide useful appliances for the application of CD40/CD40L pathways in tumor immnuotherapy.