Liu Gao-qin, Li Long-biao, Ju Song-guang, Zhang Wen-peng, Qian Yi-yong, Zhu Xue-fei, Lu Pei-rong, Zhang Xue-guang
Department of Ophthalmology, The First Affiliated Hospital of Soochow University, Suzhou, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2012 Mar;28(3):255-9.
To construct pCEP4/hIL-17B recombinant expression vector and express it stably in eukaryotic cells and investigate the biological activity in vitro.
The CDS region of human IL-17B gene was cloned by RT-PCR. After identification by sequencing, the hIL-17B gene was inserted into expression vector of pCEP4 to construct the recombinant vector pCEP4/hIL-17B, then transfected into 293T cells. The transgenic 293T cell line stably expressing rhIL-17B protein was selected in the presence of Hygromycin B. After FCS-free cultivation and sub-cloning, The IL-17B/mFc gene and protein expression was confirmed by RT-PCR, ELISA and Western blot analysis. To investigate the ability of combination with IL-17B receptor on human leukemic monocytic cell line, THP-1, by Flow cytometrical analysis (FACS) and of stimulation to secret cytokines in vitro.
The recombinant pCEP4/hIL-17B and its transgenic 293T cells stably expressing rhIL-17B protein were obtained successfully. FACS analysis showed its high affinity with its receptor and it can stimulated THP-1 cell line to excrete IL-1β and TNF-α in vitro and consistently caused a dose-dependent influx of neutrophil into the peritoneal cavity by intraperitoneal injection in vivo.
The obtainment of transgenic 293T cell line stably expressing rhIL-17B protein paved the way for further study on biological functions of hIL-17B.
