pCEP4/hIL-17真核表达载体的构建及表达

目的构建pCEP4/hIL-17载体及在真核细胞中表达hIL-17/mFc融合蛋白;初步研究IL-17生物学特性。方法采用RT-PCR的方法克隆hIL-17CDS段基因序列;将测序正确的hIL-17序列插入pCEP4质粒构建pCEP4/IL-17真核表达载体,转染中华仓鼠卵巢(CHO)细胞后,筛选阳性表达细胞株;并用RT-PCR、ELISA和Western blot等法鉴定IL-17基因的mRNA和蛋白表达;流式细胞术分析纯化的蛋白对Raji细胞表达的hIL-17受体的结合能力;以体外实验验证其促炎症作用。结果成功构建了pCEP4/hIL-17重组载体,并在CHO细胞中稳定表达;所获得hIL-17重组蛋白能稳定结合Raji细胞上的IL-17受体;体外刺激HeLa细胞,能明显促进IL-6等炎症因子的分泌。结论稳定表达hIL-17重组蛋白的CHO细胞系的建立,为进一步研究hIL-17的生物学功能奠定了良好的基础。

Construction and expression of pCEP4/hIL-17 eukaryotic expression vector

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.