[Construction of an anti-tumor immunogene vaccine pVAX-IL-12-GB and its expression in vivo and in vitro].
OBJECTIVE
To construct a novel immunogene therapeutic plasmid that expresses human interleukin-12 (IL-12), granulocyte-macrophage colony stimulating factor (GM-CSF) and B7.1 and observe its expression in vivo and in vitro.


METHODS
Human IL-12 gene fragment was cloned into the upper stream of IRES gene in the previously constructed plasmid pVAX-IRES-GM-CSF-B7.1, and the positive recombinant plasmid pVAX-IL-12-GB was transfected into 293T cells via Lipofectamine 2000. The expressions of IL-12 and GM-CSF-B7.1 mRNA and proteins in the transfected cells were assayed by RT-PCR and ELISA, and B7.1 expression was tested by fluorescence-activated cell sorting and immunofluorescence assay. The plasmid pVAX-IL-12-GB was delivered into mouse muscle by electroporation, and the expression of IL-12 in the muscle tissue was identified by immunohistochemistry.


RESULTS
Enzyme digestion, PCR and sequence analysis all confirmed successful construction of the recombinant plasmid pVAX-IL-12-GB. IL-12, GM-CSF and B7.1 expressions were all detected in transfected 293T cells, and the expression of IL-12 was also detected in the transfected mouse muscular tissues.


CONCLUSIONS
A novel anti-tumor immunogene vaccine constructed can be expressed both in vivo and in vitro, which facilitates further studies of tumor immunogene therapy.
Department of Urology, General Hospital of PLA, Beijing, China. jk_dong@163.com
Jinkai
J
Dong
Jiangping
J
Gao
Jinqi
J
Yan
Liang
L
Zhang
Yi
Y
Xiao
Wei
W
Wang
Xiaoxiong
X
Wang
Jiyun
J
Yu
D000818:Animals; D018122:B7-1 Antigen / Q000235:genetics* / Q000276:immunology; D019496:Cancer Vaccines / Q000235:genetics* / Q000276:immunology; D018274:Electroporation; D015316:Genetic Therapy; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics* / Q000276:immunology; D006801:Humans; D018664:Interleukin-12 / Q000235:genetics* / Q000276:immunology; D051379:Mice; D010957:Plasmids; D014162:Transfection
D018122:B7-1 Antigen; D019496:Cancer Vaccines; D018664:Interleukin-12; D016178:Granulocyte-Macrophage Colony-Stimulating Factor