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Published on: May 21, 2018
[Expression of human granulysin in murine macrophages and its effects on cell damage and apoptosis]
Zheng-jun Yi1, Dao-yin Zhu, Jun-ming Li
1Department of Microbiology and Immunology, Chongqing University of Medical Sciences, Chongqing 400016, China. yizhengjun@sohu.com
Aim:
To construct an eukaryotic expression vector pBudCE4.1/GLS encoding human granulysin(GLS) derived from activated CTLs and observe its effects on cell damage and apoptosis when expressed in macrophages of various murine germ lines.
Methods:
The gene sequence coding GLS carried on pEGFP-C1/GLS was subcloned into pBudCE4.1 plasmid and then transfected into different murine macrophages. The expression of GLS was detected by RT-PCR and immunocytochemical staining. The impairment and apoptosis of the host cells was assesed by measuring LDH in culture media and fluorescent staining of nuclear DNA, respectively.
Results:
The pBudCE4.1/GLS recombinant containing accurate ORF of GLS was obtained and successfully expressed in the target cells at 48 h after transfection. No obvious effects on cell damage and apoptosis was detected during this course.
Conclusion:
The gene coding human GLS can be expressed in murine macrophages. The expressed product has no obvious effects on cell damage and apoptosis.
Insights
Human granulysin (GLS) was successfully expressed in murine macrophages using the pBudCE4.1/GLS vector. The study found no significant cell damage or apoptosis in host cells following GLS expression.
Area of Science:
- Molecular biology
- Immunology
- Cell biology
Context:
- Granulysin (GLS) is a key cytotoxic protein found in activated cytotoxic T lymphocytes (CTLs).
- Understanding GLS expression and its effects in different cellular contexts is crucial for immunotherapy research.
- Murine macrophages are key immune cells involved in host defense and inflammation.
Purpose:
- To construct an eukaryotic expression vector, pBudCE4.1/GLS, for human granulysin (GLS).
- To investigate the expression of human GLS in murine macrophages.
- To evaluate the impact of GLS expression on macrophage cell damage and apoptosis.
Summary:
- The pBudCE4.1/GLS vector was successfully constructed and transfected into murine macrophages.
- Human GLS was confirmed to be expressed in the target cells 48 hours post-transfection via RT-PCR and immunocytochemistry.
- No significant alterations in cell damage (LDH levels) or apoptosis (nuclear DNA staining) were observed.
Impact:
- Demonstrates the feasibility of expressing human granulysin in murine macrophages.
- Provides foundational data on the non-cytotoxic effects of human GLS in macrophages.
- Suggests potential for further investigation into GLS function and therapeutic applications in immune modulation.
