[Construction and functional analysis of a novel eukaryotic expression plasmid of recombinant immunotoxin DT390-Rantes].
OBJECTIVE
To construct a novel eukaryotic expression plasmid for recombinant immunotoxin DT390-Rantes and perform preliminary analysis of its function.


METHODS
The gene fragment coding for Rantes was obtained from the liver tissues of C57BL/6 mice using RT-PCR, and inserted into the eukaryotic expression plasmid SRalpha containing DT390 gene to construct the recombinant plasmid DT390-Rantes-SRalpha, which was transformed into E. coli JM109, followed by selection of the positive clones containing the target inserts. The eukaryotic expression plasmid was analysed by PCR, restriction endonuclease digestion and DNA sequencing. The recombinance plasmid DT390-Rantes-SRalpha was transfected into NIH3T3 cells and its expression was observed by immunofluorescence detection. The activity of the expressed DT390-Rantes in vitro was evaluated by MTT assay.


RESULTS
The gene fragment of Rantes was correctly inserted into the eukaryotic expression plasmid SRalpha as verified by restriction endonuclease digestion and DNA sequencing, and could be expressed in NIH3T3 cells. MTT assay confirmed that the expression product DT390-Rantes could kill activated T cells in vitro.


CONCLUSIONS
The recombinant eukaryotic expression plasmid DT390-Rantes-SRalpha is successfully constructed and expressed in eukaryotic cells. The expressed product can specifically kill activated T cells in vitro.
Jia
Yi
Y
College of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. yijia781021@tom.com
Li
Hong
H
Cheng
Wen-jie
WJ
Lü
Mei-li
ML
Li
Ming-yuan
MY
Jiang
Zhong-hua
ZH
Zhang
Lin
L
D000818:Animals; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013601:T-Lymphocytes / Q000166:cytology; D014162:Transfection
C518733:DT390-RANTES-SRalpha; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins