Construction of expression vector of hTERT- hIL18 fusion gene and induction of cytotoxic T lymphocyte response against hTERT.
OBJECTIVE
Recombiant human telomerase reverse transcriptase (hTERT)-human IL-18(hIL18) was constructed to investigate its expression and biological function in eukaryotic cells. DCs transfected with hTERT-IL18 acquired stronger telomerase activity and were able to elicit an hTERT-specific cytotoxic T lymphocyte CTL response in vitro.


METHODS
hIL-18 gene fragment was amplified by polymerase chain reaction (PCR) and TA cloned. The hIL-18 gene was then subcloned into eukaryotic expression vector pcDNA3.1(+) containing human TERT via a linker. The sequence of gene fusion was confirmed using both restrictive enzyme digestion and DNA sequencer. The expression vector with gene fusion was transfected into 3T3 cell line with Lipofectamine 2000. ELISA, Western blot, immunofluorescence stain were performed to determine the expression properties of hTERT-hIL18 in 3T3 cells. Its biological effect on the anti-apoptosis was measured by Flow cytometry and its effect on INF-gamma expression was determined using ELISA. After preparation of dendritic cells, hTERT-hIL18, hTERT, hIL-18 expression vectors were transfected into DCs respectively by electroporation to generate hTERT-specific DCs lines. The peripheral blood mononuclear cells PBMCs were stimulated with different DCs lines to create specific CTL. The response of target cell (leukemia cell line-K562 cell) to hTERT-specific CTL was evaluated by LDH release assay.


RESULTS
The human IL-18 gene fragment was amplified from the human mononuclear cells and was inserted into pcDNA3.1(+)/hTERT vector successfully. The correct sequence was proved by both restrictive enzyme digestion and sequencing. The correct open reading frame was also verified. Fusion protein of hTERT-hIL18 was effectively expressed in eukaryotic cells, which was detected by both Western-blotting and immunofluorescence stain. The expressed recombinant fusion protein induced similar levels of INF-gamma to that of native IL-18 protein. FCM assay showed that the transfected fusion protein inhibited the apoptosis, which was consistent with the effects of hTERT as a universal tumor associated antigen. CTL assay shows that hTERT- hIL18 and hTERT gene-transfected DCs stimulated T-cell responses that recognized and lysed tumor target cells of high hTERT expression, whereas DCs transfected with hIL-18 gene didn't induced the response of tumor targets lyses.


CONCLUSIONS
The Recombinant hTERT- hIL18 fusion protein had both biological activity of hTERT and hIL-18, indicating that this rationally designed protein can be further developed as novel tumor therapeutics. DCs transfected with hTERT-IL18 gene were capable of eliciting a stronger hTERT-specific CTL response in vitro.
Tong
X M
XM
The First Affiliated Hospital, School of Medicine, Zhejiang University, Qinchun Road, Hangzhou 310003, Zhejiang, China.
Zheng
S E
SE
Bader
A
A
Yao
H P
HP
Wu
N P
NP
Altmeyer
P
P
Brockmeyer
N H
NH
Jin
Jie
J
D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D019496:Cancer Vaccines / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000494:pharmacology; D003601:Cytotoxicity Tests, Immunologic; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D020382:Interleukin-18 / Q000235:genetics* / Q000378:metabolism; D020014:K562 Cells; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D019098:Telomerase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D014162:Transfection
D019496:Cancer Vaccines; D017077:Culture Media, Conditioned; D020382:Interleukin-18; D011993:Recombinant Fusion Proteins; D007371:Interferon-gamma; C509186:TERT protein, human; D019098:Telomerase; D008727:Methotrexate