pCEP4/hIL-17真核表达载体的构建及表达

Aim To construct pCEP4/hIL-17 recombinant expression vector and express it stably in eukaryotic cells. To investigate the biological activity in vitro. Methods The CDS region of human IL-17 gene was cloned by RT-PCR. After identification by sequencing, the hIL-17 gene was inserted into expression vector pCEP4 to construct the recombinant vector pCEP4/hIL-17, then transfected into Chinese Hamster Ovary (CHO) cells by Lipofectamine 2000. The transgenic CHO cell line stably expressing rhIL-17 protein was selected in the presence of Hygromycin B. After FCS-free cultivation and sub-cloning, the IL-17/mFc gene and protein expression was confirmed by RT-PCR, ELISA and Western blot analysis. The ability of combination with IL-17 receptor was investigated with Raji cells by flow cytometrical analysis (FACS) and its stimulation of the secretion of cytokines was measured in vitro. Results The recombinant pCEP4/hIL-17 and its transgenic CHO cells stably expressing rhIL-17 protein were obtained successfully. FACS analysis showed its high affinity with its receptor and it can stimulate HeLa, a human uterine cervix cancer cell line to excrete IL-6 in vitro. Conclusion The establishment of transgenic CHO cell line stably expressing rhIL-17 protein may pave the way for further study in biological functions of hIL-17.