[The research on construction and expression of eukaryotic expression plasmid for a recombinant immunotoxin mMIP-1alpha-DT390].
OBJECTIVE
To construct a novel recombinant immunotoxin (IT) expression vector by fusing mouse macrophage inflammatory protein-1alpha gene (mMIP-1alpha) into a truncated diphtheria toxin gene (DT390), and examine the expression of mMIP-1alpha-DT390 fusion protein in NIH3T3 cells.


METHODS
mMIP-1alpha cDNA was cloned from mouse liver tissue through reverse transcription-polymerase chain reaction (RT-PCR), and inserted in the expression plasmid SRalpha, that includes the DT390 gene, to form the recombinant vector SRa-mMIP-1alpha-DT390. The positive recombinant plasmid was identified by PCR, the restriction endonucleases digestion and DNA sequencing, and then by liposome protocol, the identified positive plasmid was transferred into NIH3T3 cells for observing the fusion protein expression by immunofluorescence, with detecting the activities of the immunotoxin in vitro through MTT.


RESULTS
We have successfully constructed a recombinant immunotoxin expression vector named as SRalpha-mMIP-1alpha-DT390. The immunofluorescence photograph sourced from fluorescence immunocytochemical method showed that the fusion gene could express in the cytomembrane and cytoplasm of NIH3T3 cells. In the bioactivity detection assay, the supernatant of the transfected cell culture was observed to have the obvious cytotoxic activity to activated T-lymphocyte.


CONCLUSIONS
The SRalpha-mMIP-1alpha-DT390 recombinant immunotoxin expression vector will provide the basis of studying the targeted cytotoxic activity to inflammatory cells, and may have some potential value for clinical application.
Lü
Mei-li
ML
Department of Forensic Biology, West China School of Preclinical and Forensic Medcine, Sichuan University, Chengdu 610041, China.
Li
Hong
H
Liang
Wei-Bo
WB
Chen
Wen-Jie
WJ
Jia
Yi
Y
Jiang
Zhong-hua
ZH
Zhang
Wei-dong
WD
Zhang
Lin
L
D000818:Animals; D054405:Chemokine CCL3 / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000633:toxicity; D004167:Diphtheria Toxin / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000633:toxicity; D005057:Eukaryotic Cells / Q000166:cytology; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D000922:Immunotoxins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000633:toxicity; D051379:Mice; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000633:toxicity; D017422:Sequence Analysis, DNA; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection
D054405:Chemokine CCL3; D004167:Diphtheria Toxin; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins