[Construction, expression and identification of recombinant plasmid encoding bifunctional protein sflk1-IFN-gamma].
To construct, express and identify the recombinant plasmid pcDNA3.1(+)/sflk1-IFN-gamma encoding bifunctional protein sflk1-IFN-gamma (soluble fetal liver kinase 1 and interferon-gamma).



sflk1 and IFN-gamma gene fragments were cloned by RT-PCR, and then inserted into pcDNA3.1(+) plasmid between BamHI-EcoRI and XhoI-XbaI restriction sites to form the recombinant plasmid pcDNA3.1(+)/sflk1-IFN-gamma. The recombinant sflk1-IFN-gamma transiently expressed in COS-7 cells was detected by ELISA and Western blotting. Bioactivities of sflk1-IFN-gamma fusion protein were identified by proliferation inhibition assay with H5V cells and NK activity assay.



pcDNA3.1(+)/sflk1-IFN-gamma can be effectively expressed in COS-7 cells. Concentrations of sflk1 and IFN-gamma in culture supernatants of pcDNA3.1(+)/sflk1-IFN-gamma transfected COS-7 cells were (20.85+/-2.48) ng/ml and (1.08+/-0.09) ng/ml, respectively. Western blotting showed that the molecular weight of sflk1-IFN-gamma fusion protein was about 130 kDa, while that of sflk1 was 115 kDa. The supernatants of transfected cells significantly inhibited the proliferation of H5V cells stimulated by mouse VEGF 164 and enhanced the NK activity of splenocytes, demonstrating that sflk1-IFN-gamma fusion protein possessed the bioactivities of both sflk1 and IFN-gamma.



The constructed plasmid pcDNA3.1(+)/sflk1-IFN-gamma can be effectively expressed in eukaryotes. The expressed sflk1-IFN-gamma fusion protein has the biological activities of both sflk1 and IFN-gamma.
Wu
Qian-qian
QQ
Department of Pathogen Biology, Zhejiang University School of Medicine, Hangzhou, China.
Chen
Hong-hu
HH
Guo
Jia
J
Wang
Sheng-chao
SC
Pan
Jian-ping
JP
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005260:Female; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; D007371:Interferon-gamma; D040301:Vascular Endothelial Growth Factor Receptor-2