Title: Establishment and identification of a Chinese hamster ovary cell line stably expressing sCD40L
Abstract: 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 pIRES2-EGFP-sCD40L. The recombinant plasmid was then digested with BglⅡand SalⅠand 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 were 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-sCD40L 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 pIRES2-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-sCD40L 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 (P0.01). Conclusion: We have successfully established a CHO cell line stably expressing sCD40L, which may help to investigate the role of CD40/CD40L pathway in the adjuvant immunotherapy of tumor.
Publication Year: 2007
Publication Date: 2007-01-01
Language: en
Type: article
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