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[Retroviral vector-mediated in vitro expression of human soluble fas]
Liang-Li Wang1, Ping Zou, Zhong-Bo Hu
1Institute of Hematology, The Affiliated Union Hospital of Tongji Medical College, Huazhong University of Science and Techology, Wuhan 430022, China. liaixiang302@sohu.com
Abstract:
Leukemic cells from patients expressed high level FasL cause apoptosis of autologous activated T cells via the Fas/FasL pathway. To investigate the role of soluble Fas (sFas) in reversing this process, a retroviral-mediated expression vector pLXIN-sFas was established. A retroviral-mediated expression system of human sFas was established in vitro and the biological activity of the expression product sFas was observed. To obtain the soluble Fas cDNA, the specific part of the full-length Fas cDNA was deleted by multiple PCR. After pLXIN-sFas packaged by PA317 cells, it was transferred into the target cell COS-7. The quantity of the sFas was (2.2 +/- 0.7) micro g/ml in supernatant of cultured COS-7 cells, and it could greatly inhibit apoptosis of Jurket cells induced by anti-Fas antibody. Our results suggested that the recombinant is able to express the target proteins in vitro and it has the perfect biological activity.
Insights
Soluble Fas (sFas) can inhibit leukemic cells from inducing T cell death. This study developed a method to produce biologically active sFas in vitro, offering potential therapeutic strategies.
Area of Science:
- Immunology
- Molecular Biology
- Biotechnology
Background:
- Leukemic cells express high levels of Fas Ligand (FasL), inducing apoptosis in autologous activated T cells through the Fas/FasL pathway.
- The role of soluble Fas (sFas) in reversing this T cell apoptosis remains an area of investigation.
Purpose of the Study:
- To establish a retroviral-mediated expression system for human soluble Fas (sFas) in vitro.
- To assess the biological activity of the produced sFas and its potential to inhibit Fas-mediated apoptosis.
Main Methods:
- A retroviral expression vector (pLXIN-sFas) was constructed by deleting a specific portion of the full-length Fas cDNA using multiple PCR.
- The vector was packaged by PA317 cells and transferred into COS-7 target cells.
- Quantification of sFas in the supernatant and assessment of its inhibitory effect on anti-Fas antibody-induced Jurket cell apoptosis.
Main Results:
- A functional retroviral expression system for human sFas was successfully established in vitro.
- The recombinant sFas was produced at a concentration of (2.2 +/- 0.7) micro g/ml in the supernatant of cultured COS-7 cells.
- The expressed sFas significantly inhibited apoptosis of Jurket cells induced by anti-Fas antibody, demonstrating potent biological activity.
Conclusions:
- The developed retroviral system effectively produces biologically active soluble Fas (sFas) in vitro.
- Recombinant sFas exhibits significant anti-apoptotic activity, suggesting its potential therapeutic application in Fas/FasL-mediated diseases.
- This study provides a foundation for further research into sFas as a modulator of immune cell apoptosis.