Superantigen-SEA gene modified tumor vaccine for hepatocellular carcinoma: an in vitro study.
OBJECTIVE
To construct an eukaryotic superantigen gene expression vector containing the recombinant gene of SEA and CD80 molecule transmembrane region (CD80TM), and to express staphylococcus enterotoxin A (SEA) on the membrane of hepatocellular carcinoma (HCC) cell to form a superantigen gene modified tumor vaccine for HCC.


METHODS
SEA and linker-CD80TM gene were amplified through PCR from plasmid containing cDNA of SEA and CD80. Gene fragments were then subcloned into the multiple cloning sites of retroviral vector pLXSN. Recombinant plasmid was transferred into HepG2 cells mediated with lipofectamine, positive clones were selected in culture medium containing G418. RT-PCR and indirect immunofluorescence studies confirmed that SEA was expressed specifically on HCC cell membrane. INFgamma-ELISPOT study demonstrated that SEA protein was expressed on the membrane of HCC cells. Cytotoxicity of HepG2-SEA primed CTLs (SEA-T) was analyzed by (51)Cr release assay. T cells cultured with rhIL-2 (IL-2-T) were used as control.


RESULTS
Restriction digestion and sequence analyses confirmed the correctness of length, position and orientation of inserted fusion genes. SEA was expressed on the surface of HepG2 cells, HepG2-SEA had strong stimulating effect on production of HepG2 specific CTL (P<0.001). SEA-T had enhanced cytotoxicity to HepG2 cells (P<0.05).


CONCLUSIONS
Tumor cell membrane expressed superantigen can be used to reinforce the immune effect of tumor cell vaccine for HCC, which provides a new method of the enhanced active immunotherapy for HCC.
Lu
Shao-Ying
SY
Department of Pathology, Fourth Military Medical University, Xi'an 710032, China. suiyanf@fmmu.edu.cn
Sui
Yan-Fang
YF
Li
Zeng-Shan
ZS
Ye
Jing
J
Dong
Hai-Long
HL
Qu
Ping
P
Zhang
Xiu-Min
XM
Wang
Wen-Yong
WY
Li
Yu-Song
YS
D018122:B7-1 Antigen / Q000235:genetics; D019496:Cancer Vaccines / Q000235:genetics*; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000502:physiology; D004768:Enterotoxins / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D008113:Liver Neoplasms; D018089:Superantigens / Q000235:genetics*; D014162:Transfection
D018122:B7-1 Antigen; D019496:Cancer Vaccines; D004768:Enterotoxins; D018089:Superantigens; C026066:enterotoxin A, Staphylococcal