[Construction and expression of the fusion protein DT389-hIL-13 and its cytotoxicity to glioma cell lines].
OBJECTIVE
To construct a fusion protein toxin DT389-hIL-13 which comprises the N-terminal 389 amino acids of diphtheria toxin (DT389) and human interlukin 13 (hIL-13), and to explore its cytotoxicity on U251 glioma cells.


METHODS
The cDNA of hIL-13 gene was amplified by PCR and linked with the 3'-terminus of the gene encoding the N-terminal 389 amino acids, which correspond to the enzyme domain and transmembrane domain of diphtheria toxin. The tandem constructed gene was then inserted into an E. coli expression vector pET30a. The resulted expression vector was transformed into E. coli BL21 and induced by IPTG. The expressed protein was analyzed by SDS-PAGE and Western blot analysis. U(251) glioma cells were cultured DT389-hIl-13 was added into the culture. The cytotoxicity was determined using colorimetric MTS proliferation assay.


RESULTS
The expression plasmid pET30a/DT389-hIL13 was constructed with correct sequence. The recombinant protein was successfully expressed in E. coli in manner of inclusion body and with a relative molecular weight of about 55 000, which reacted well with both anti-diphtheria toxin and anti-hIL-13 polyclonal antibody in Western blot assay. The purified recombinant chimeric toxin was found to effectively inhibit the prolifieration of glioblastoma multiforme cells bearing high affinity hIL-13 receptors, and resulted in dose-dependent relationship with 50% inhibition concentration (IC(50)) of 5 x 10(-)11mol/L.


CONCLUSIONS
Prokaryotic expression system can be recruited to produce recombinant chimeric toxin DT389-hIL-13. The results may lay a foundation for preparing specific the agent targets for tumors overexpressing IL-13 receptor.
Tuo
Hou-zhen
HZ
Department of Neurology, Beijing Friendship Hospital, Capital University of Medical Sciences, Beijing 100050, China.
Wang
Jian-wei
JW
Wang
De-xin
DX
Li
Ji-mei
JM
Ouyang
Jing
J
Hong
Tao
T
D001932:Brain Neoplasms / Q000473:pathology*; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic; D004274:DNA, Recombinant / Q000235:genetics; D004167:Diphtheria Toxin / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005910:Glioma / Q000473:pathology*; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D018793:Interleukin-13 / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011387:Prokaryotic Cells / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D004274:DNA, Recombinant; D004167:Diphtheria Toxin; D000922:Immunotoxins; D018793:Interleukin-13; D011993:Recombinant Fusion Proteins