[Construction and expression of eukaryotic expression vector of human 4-1BB ligand gene in tumor cells and its antitumor activity in vitro].

Abstract

AIM:

To construct eukaryotic expression vector of human 4-1BB ligand (4-1BBL) gene and express it in HT-29 cell line. To explore the effect on activation and cytotoxicity of human cytotoxic T lymphocytes(CTLs) induced by human 4-1BBL gene transfection into tumor cells in vitro.

METHODS:

RT-PCR was applied to amplify the full-length of human 4-1BBL gene from Raji cells. After sequencing, the cDNA was recombinated into the eukaryotic expression vector pcDNA3.1(-) and transfected into HT-29 cells through Lipofect AMINE 2000. Human 4-1BBL mRNA and protein expression of transfected cells was detected by RT-PCR and FACS respectively. Human peripheral blood mononuclear cells were stimulated with anti-CD3 mAb and incubated with non-transfected or transfected HT-29 cells, respectively. The MTT colorimetry was used to detect the proliferation and cytotoxic effect of T lymphocytes. Meanwhile, the expression of intracellular IFN-gamma was detected by FCM.

RESULTS:

The HT-29 cells transfected by pcDNA3.1(-)-h4-1BBL could express human 4-1BBL efficiently. As compared with wild type HT-29 cells, the transfected HT-29 cells had more effect for proliferation, IFN-gamma production and cytotoxic activity of lymphocytes.

CONCLUSION:

The recombinant eukaryotic expression vector of pcDNA3.1(-)-h4-1BBL is successfully constructed. The transfection of human 4-1BBL gene in HT-29 cells is effctive in enhancing its immunogenicity and inducing antitumor immune response in vitro.

• Authors: Kun Lu, Jun-ying Wu, Bai-qing Li
• Journal: Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology (Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi) Vol. 23 Issue 6 Pg. 511-4 (Jun 2007) ISSN: 1007-8738 [Print] China
• PMID: 17553345 (Publication Type: Journal Article)

Chemical References

• 4-1BB Ligand
• Antibodies, Monoclonal
• CD3 Complex
• Interferon-gamma

Topics

• 4-1BB Ligand (genetics, metabolism)
• Antibodies, Monoclonal (pharmacology)
• CD3 Complex (immunology)
• Cell Proliferation (drug effects)
• Cells, Cultured
• Colorimetry
• Flow Cytometry
• Gene Expression Regulation
• Genetic Vectors (genetics)
• HT29 Cells
• Humans
• Interferon-gamma (metabolism)
• Reverse Transcriptase Polymerase Chain Reaction
• T-Lymphocytes, Cytotoxic (cytology, drug effects, immunology, metabolism)
• Transfection