[The construction and expression of superantigen SEA and antimelanoma ScFv fusion gene].
Two strategies, direct ligation after enzyme digestion and over-lap PCR technology, were adopted to construct a fusion gene which was composed of the antimelanoma single chain antibody gene and the staphylococcal enterotoxin A gene without N-terminal signal sequence. The fusion gene was subcloned into pET28-a vector and transformed into E. coli BL21(DE3). Ni-NTA system was selected to separate and purify the expresstd products. The inhibition ratio of the fusion protein was tested by MTT method. It is shown that the 6His-ScFv-SEA fusion protein can be expressed stably in E. coli BL21 (DE3). The quantity of the fusion protein was shown up to 30% of the total protein of the bacteria and mainly in inclusion body. By activation the effective cells, the fution protein can inhibit the melanoma cell whith expressed corresponding antigen.
Sun
Jing
J
Shenyang Institute of Applied Ecology of Chinese Academy of Science, Shenyang 110015, China.
Lü
An-Guo
AG
Wu
Wen-Fang
WF
Bai
Xiang-Yang
XY
Ren
Xiu-Bao
XB
Liu
Hong
H
D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D008545:Melanoma / Q000188:drug therapy / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology* / Q000627:therapeutic use; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism*
D004768:Enterotoxins; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C026066:enterotoxin A, Staphylococcal