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Published on: October 6, 2017
[Construction and functional analysis of a novel eukaryotic expression plasmid of recombinant immunotoxin
Yi Jia1, Hong Li, Wen-jie Cheng
1College of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. yijia781021@tom.com
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.
