Expression and functional study of human recombinant chemokine-like factor I in Drosophila S2 cells / 中华微生物学和免疫学杂志

Objective To express human chemokine-like factor 1 (CKLF1) in Drosophila S2 cells and study its function. Methods The pMT/V5-His-CKLF1 expression plasmid was constructed and transfected into Drosophila S2 cells. The positive clones were selected through PCR and RT-PCR. The culture medium was analyzed by Western blot with anti-CKLF1 polyclonal antibody. Chemotaxis and MTT assays on human peripheral blood and C2C12 cells, respectively, were then carried out with the medium. Results CKLF1 was transcribed efficiently in S2 cells. The expressed CKLF1 protein could be detected in the culture supernatant by Western blot, which showed weak chemotactic activity on both human peripheral blood neutrophils and lymphocytes as well as enhancing effect on the proliferation of C2C12 cells. Conclusion CKLF1 was expressed successfully in Drosophila S2 cells and secreted into the culture medium. The recombinant CKLF1 expressed in Drosophila cells can chemoattract leucocytes and promote the proliferation of C2C12 cells.

Expression and purification of truncated human recombinant nuclear apoptosis-inducing factor 1 in E.coli / 中国免疫学杂志

Objective:Nuclear apoptosis-inducing factor 1(NAIF1) is a novel apoptosis gene cloned in laboratory. To analyze the binding proteins of NAIF1 by pulldown method, the fusion expression vector of truncated human nuclear apoptosis-inducing factor 1〔NAIF1(73-327)〕 was constructed, were expressed and purified the recombinant GST-NAIF1(73-327) fusion protein in E.coli.Methods:The cDNA encoding human NAIF1(73-327) was amplified by PCR and cloned into pGEX-KG vector. The GST-NAIF1(73-327) fusion protein was expressed in E.coli and purified by affinity chromatography. The purified protein was detected by SDS-PAGE, Western blot and ESI-Q-TOF-MS/MS.Results:A prokaryotic expression vector of GST-NAIF1(73-327) was constructed and the GST-NAIF1(73-327) fusion protein was expressed in E.coli at high level. SDS-PAGE analyses indicated that the purified protein was about 53 kD. Western blot and MS/MS analyses verified the recombinant fusion protein.Conclusion:An efficient method for obtaining recombinant GST-NAIF1(73-327) fusion protein had been established and it could be used for further studies on the structure and function of NAIF1.