[Construction of eukaryotic expression vector of mouse alpha-fetoprotein cDNA and its expression in dendritic cells and in vitro antitumor effect on hepatoma].
OBJECTIVE
Studying the mechanism of why the abnormal protein coded by mutant gene of hepatoma carcinoma cell possesses potential antigenicity, but can not form effective immunogen, searching for specific antigen of hepatoma, and developing DNA vaccine for hepatoma treatment will have active impact on the immunotherapy of hepatoma. This study was to construct fluorescent eukaryotic expression vector of mouse alpha fetoprotein (AFP) cDNA fragment with or without the secretory signal peptide (termed AFP1 cDNA and AFP2 cDNA), and observe its expression in mouse dendritic cells (DCs) and in vitro antitumor effect on hepatoma.


METHODS
AFP1 cDNA and AFP2 cDNA were amplified from mouse hepatoma cell line H22 by reverse transcription-polymerase chain reaction (RT-PCR), and cloned into eukaryotic expression vector PEGF-N3 to form the recombinant PEGF-N3/AFP1 and PEGF-N3/AFP2. The mouse bone marrow cells were induced with recombinant granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant interleukin-4 (rmIL-4) to form mature DCs. The recombinant PEGF-N3/AFP1 and PEGF-N3/AFP2 were transfected into DCs. The DCs vaccine were co-cultured with isogenous mouse spleen lymphocytes. The expression of gamma-interferon (IFN-gamma) in the spleen lymphocytes stimulated by AFP1/DCs or AFP2/DCs was measured by ELISA assay; the specific killing activity of cytotoxic T lymphocytes (CTLs) was evaluated by 51Cr release assay.


RESULTS
Sequencing analysis verified that AFP1 cDNA and AFP2 cDNA were correctly amplified from H22 cells. The recombinant PEGF-N3/AFP1 and PEGF-N3/AFP2 were stably expressed in mouse DCs. AFP2/DCs obviously stimulated proliferation of T lymphocytes; the stimulation index (SI) was significantly higher in AFP2/DCs group than in AFP1/DCs group, empty vector group, and blank control group (5.12 +/- 1.46 vs. 1.41 +/- 0.65, 1.42 +/- 0.7, and 1.41 +/- 0.55, P < 0.01); the specific killing activity of CTLs was stronger in AFP2/DCs group than in empty vector group [(88.15 +/- 16.4)% vs. (12.72 +/- 5.45)%].


CONCLUSIONS
The recombinant expression vectors PEGF-N3/AFP1 and PEGF-N3/AFP2 with or without signal peptide were constructed successfully. AFP2/DCs can specifically activate T lymphocytes in vitro and effectively induce antitumor immune response.
Department of Gastroenterology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, 410011, PR China.
Bin
B
Zeng
Fang-Gen
FG
Lu
Xiao-Wei
XW
Liu
Dong-Ye
DY
Yang
Wei-Yi
WY
Fang
Jian
J
Wang
Ai-Jun
AJ
Liao
Wei
W
Shi
D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018076:DNA, Complementary / Q000235:genetics; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D005822:Genetic Vectors; D008114:Liver Neoplasms, Experimental / Q000235:genetics / Q000276:immunology / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D014162:Transfection; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology; D000509:alpha-Fetoproteins / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019444:Vaccines, DNA; D000509:alpha-Fetoproteins