[Cloning, eukaryotic expression and function assay of recombinant leukemia inhibitory factor gene LIF].
Leukemia inhibitory factor (LIF), a cytokine belonging to IL-6 family, which was discovered to inhibit the proliferation of murine myeloid leukemic cell line M1, has multiple functions in various biological processes. This factor is highly glycosylated when it binds to receptor to activate the signal transduction. Therefore, expression of LIF through eukaryotic system is the best way to obtain the correct glycosylation. In this study, human LIF cDNA with the sequence of signal peptide was cloned from adult blood cells by RT-PCR, and then subcloned into pcDNA3 for expression in HEK-293T cells. After transfection of the recombinant plasmid pcDNA3/LIF into HEK-293T cells, the conditioned medium containing the secreted LIF was obtained. The activity of the secreted LIF in the conditioned medium was detected through the phosphorylation of STAT3, EMSA and luciferase reporter (pGL2-APRE-luc) assays in Hep3B and 293T cells. Furthermore, to investigate the biological functions of the overexpressed recombinant LIF, a [(3)H]-thymedine incorporation assay was performed, and the results showed that the recombinant LIF inhibited the growth of M1 cells strongly. Taken together, all the function assays suggest that the recombinant LIF has the normal functions, suggesting that the recombinant LIF could be used for further studies.
Ning
Hong-Xiu
HX
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China.
Chen
Yue
Y
Rong
Yu
Y
Zhang
Xiu-Fang
XF
Chang
Zhi-Jie
ZJ
D000328:Adult; D000818:Animals; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D053605:Leukemia Inhibitory Factor; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D050796:STAT3 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D015850:Interleukin-6; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; D011994:Recombinant Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; D015246:Deoxyribonuclease EcoRI; C034510:CTCGAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific