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


CONCLUSIONS
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.
Lu
Kun
K
Department of Immunology, Bengbu Medical College, Bengbu 233030, China.
Wu
Jun-ying
JY
Li
Bai-qing
BQ
D053330:4-1BB Ligand / Q000235:genetics* / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017252:CD3 Complex / Q000276:immunology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003124:Colorimetry; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics*; D019073:HT29 Cells; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D014162:Transfection
D053330:4-1BB Ligand; D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D007371:Interferon-gamma