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[Construction of eukaryotic expression vector of hdll1(ext)-Fc and its expression in COS-7 cells].

AIM
To construct an eukaryotic expression vector pEF-BOSneo-hdll1(ext)-Fc, and to express the fusion protein consisting of the extracellular region of delta-like1 and Fc fragment of human IgG1 in COS-7 cells.


METHODS
The extracellular region of human delta-like1 was amplified from a human brain cDNA library by PCR. The expression vector was constructed by DNA recombination. The recombinant plasmid was transfected into COS-7 cells via liposome mediation. The expression of the fusion protein was detected by RT-PCR, immunofluorescence assay and sandwich ELISA.


RESULTS
DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic expression vector pEF-BOSneo-hdll1(ext)-Fc had been constructed successfully. After recombinant plamid had been transfected into COS-7 cells, RT-PCR and DNA sequencing verified that the dll1(ext) gene and IgG1Fc gene were fused correctly. The results of immunofluorescence assay were positive and the fusion protein could be detected by sandwich ELISA in culture supernatant of transfected COS-7 cells.


CONCLUSION
hdll1(ext) was successfully cloned and expressed in the form of Fc fusion protein, which is helpful for further study of the function of Notch pathway.