[Study of the construction and function of an eukaryotic expression plasmid of immunotoxin DT390 gene].
OBJECTIVE
To construct a new recombinant eukaryotic expression plasmid of murine IFN-gamma-inducible protein 10 (mIP10) gene and a truncated diphtheria toxin (DT390) gene and investigate its biological function.


METHODS
mIP10 cDNA was cloned from murine liver tissue by RT-PCR and was inserted into eukaryotic expression plasmid SRalpha containing DT390 gene to construct plasmid SRalpha-DT390-mIP10. After being identified with restriction endonucleases digestion analysis and DNA sequencing, the recombinant plasmid was transfected into NIH3T3 cells through PolyFect. The expression of recombinant plasmid was detected by immunofluorescence assay and the bioactivity of mIP10-DT390 in cultured supernatant of the transfected NIH3T3 cells was detected by MTT colorimetry.


RESULTS
A new recombinant immunotoxin expression plasmid SRalpha-DT390-mIP10 was constructed successfully and was expressed in NIH3T3 cells. The expressed DT390-mIP10 killed the activated T cells effectively.


CONCLUSIONS
A new recombinant eukaryotic expression plasmid SRalpha-DT390-mIP10 has been constructed and expressed successfully, which is useful in further studying the treatment of cancer and autoimmune diseases.
Chen
Wen-jie
WJ
Department of Immunology, West China Preclinical College and Forensic Medicine College, Sichuan University, Chengdu 610041, China. chenwenjie168@sohu.com
Li
Hong
H
Jia
Yi
Y
Zhang
Lin
L
Lu
Mei-li
ML
Li
Ming-yuan
MY
Jiang
Zhong-hua
ZH
D000818:Animals; D054357:Chemokine CXCL10 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D005057:Eukaryotic Cells / Q000187:drug effects / Q000378:metabolism; D005260:Female; D000922:Immunotoxins / Q000235:genetics / Q000378:metabolism*; D008213:Lymphocyte Activation / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D041681:NIH 3T3 Cells; D011993:Recombinant Fusion Proteins / Q000097:blood / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000187:drug effects; D014162:Transfection
D054357:Chemokine CXCL10; C518733:DT390-RANTES-SRalpha; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins