%0 Journal Article
%T Expression of humane IL-35-IgG4 (Fc) fusion protein in CHO/DG44 cells<br>人IL35-IgG4(Fc)融合蛋白在CHO/DG44细胞中的稳定表达
%A Jing Tang
%A Wenda Gao
%A Qing Zhang
%A Dawei Zhang
%A Yang Chen
%A Bo He
%A Quansheng Liu
%A <br>唐静
%A 高闻达
%A 张青
%A 张大为
%A 陈洋
%A 何波
%A 刘全胜
%J 生物工程学报
%D 2009
%I 
%X We constructed the eukaryotic expression vector of human IL-35-IgG4 (Fc)-pOptiVEC?-TOPO? by gene recombination technique and expressed the fusion protein human IL-35-IgG4 (Fc) in CHO/DG44 cells. The two components of the newly discovered cytokine human IL-35, EBI3 and IL-12p35, were amplified by PCR from the cDNA library derived from the KG-I cells after LPS induction. The two PCR-amplified cDNA fragments of human IL-35 were linked by over-lapping PCR and then cloned into the IgG4 (Fc)-pOptiVEC?-TOPO? vector. The constructed plasmid with the recombinant cDNA IL-35-IgG4 (Fc) was verified by restriction enzyme digestion analysis, PCR and DNA sequencing. The verified plasmid with the recombinant cDNA was transfected into CHO/DG44 cells using Lipofectamine? 2000. The success of the transfection was examined and confirmed by RT-PCR. After selection in a-MEM (-) medium, the IL-35-Ig G4 (Fc) positive CHO/DG44 clones were chosen and the media from these positive clones were collected to be used to purify the fusion protein. The positive CHO/DG44 clones were further cultured in increasing concentrations of MTX and the expression levels of the fusion protein IL-35-Ig G4 (Fc) were repetitively induced by MTX-induced gene amplification. The IL-35-Ig G4 (Fc) fusion protein was purified from the media collected from the positive CHO/DG44 clones by protein G affinity chromatography and then identified by SDS-PAGE and Western blotting. The results showed that one protein band was found to match well with the predicted relative molecular mass of human IL-35-IgG4 (Fc) and this protein could specifically bind to anti-human IgG4 (Fc) monoclonal antibody. In conclusion, our study successfully established an IL-35-IgG4 (Fc) positive DG44 cell line which could stably express IL-35-IgG4 (Fc) fusion protein.
%K IL-35-IgG4 (Fc) fusion protein
%K over-lapping PCR
%K stable transfection
%K CHO/DG44cell lines
%K MTX-induced gene amplification<br>IL-35-IgG4(Fc)融合蛋白
%K 重叠PCR
%K 稳定转染
%K CHO/DG44细胞
%K MTX诱导的基因扩增
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=A66E90C274451689E69F6F0291467824&aid=06A25AC9F6064B0CADD8969515A5E069&yid=DE12191FBD62783C&vid=C5154311167311FE&iid=CA4FD0336C81A37A&sid=2FECAA96FE989A15&eid=B5DB394EA5A78F20&journal_id=1000-3061&journal_name=生物工程学报&referenced_num=0&reference_num=22