In vitro biological activities of transmembrane superantigen staphylococcal enterotoxin A fusion protein.
The bacterial superantigen staphylococcal enterotoxin A (SEA) stimulates T cells bearing certain TCR Vbeta domains when binding to MHC II molecules, and is a potent inducer of CTL activity and cytokine production. Antibody-targeted SEA such as C215 Fab-SEA and C242 Fab-SEA has been investigated for cancer therapy in recent years. We have previously reported significant tumor inhibition and prolonged survival time in tumor-bearing mice treated with a combination of both C215Fab-SEA and Ad IL-18 (Wang et al., Gene Therapy 8:542-550, 2001). In order to develop SEA as an universal biological preparation in cancer therapy, we first cloned a SEA gene from S. aureus (ATCC 13565) and a transmembrane (TM) sequence from a c- erb-b2 gene derived from human ovarian cancer cell line HO-8910, then generated a TM-SEA fusion gene by using the splice overlap extension method, and constructed the recombinant expression vector pET-28a-TM-SEA. Fusion protein TM-SEA was expressed in E. coli BL21(DE3)pLysS and purified by using the histidine tag in this vector. Purified TM-SEA spontaneously associated with cell membranes as detected by flow cytometry. TM-SEA stimulated the proliferation of both human PBLs and splenocytes derived from C57BL/6 (H-2b) mice in vitro. This study thus demonstrated a novel strategy for anchoring superantigen SEA onto the surfaces of tumor cells without any genetic manipulation.
Ma
Wenxue
W
Cancer Institute, Zhejiang University School of Medicine, 88 Jiefang Road, 310009, Hangzhou, China. wma@unmc.edu
Yu
Hai
H
Wang
Qingqing
Q
Bao
Jianfang
J
Yan
Jie
J
Jin
Hongchuan
H
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004768:Enterotoxins / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D018734:Genes, erbB-2 / Q000235:genetics*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D066298:In Vitro Techniques; D008213:Lymphocyte Activation / Q000187:drug effects*; D008546:Melanoma, Experimental / Q000188:drug therapy* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D013154:Spleen / Q000166:cytology; D013211:Staphylococcus aureus / Q000235:genetics; D018089:Superantigens / Q000235:genetics
D004768:Enterotoxins; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins; D018089:Superantigens; C026066:enterotoxin A, Staphylococcal