细胞粘附分子P-selectin功能片段在CHO细胞表面锚定表达.pdfVIP

生 物 工 程 学 报 Chin J Biotech 2008, July 25; 24(7): 1162-1167 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2008 Institute of Microbiology, CAS CSM, All rights reserved 研究报告 细胞粘附分子P-selectin 功能片段在CHO 细胞表面的锚 定表达 娄晓芳, 王双, 杜威世, 于蕊, 俞炜源, 孙志伟 军事医学科学院生物工程研究所, 北京 100071 摘 要: 为了筛选出针对细胞粘附分子P-selectin 的特异性抗体, 克隆了P-selectin 功能性基因片段, 使其通过糖基化磷 脂酰肌醇(GPI)锚定表达于真核细胞的细胞膜上, 以用作筛选的抗原。提取人血小板的总RNA, RT-PCR 扩增出P-selectin 目的基因片段, 同时用重叠延伸PCR 的方法合成细胞膜锚定信号肽 GPI 基因; 将二者按上下游顺序插入含有弱化 neo 基因的真核表达载体pMCEw2 中; 并将构建的重组质粒pMCEw2-GPI-P-selectin 转染CHOdhfr- 细胞, G418 筛选获得阳性 细胞株, 利用ELISA 、免疫印迹和免疫荧光法检测P-selectin 的表达。实验证实了P-selectin 在细胞膜上获得稳定表达。 为进一步进行抗P-selectin 的特异性抗体的筛选提供了必要前提。 关键词: P-selectin, 糖基化磷脂酰肌醇, 粘附分子 Eukaryotic Expression of P-selectin Functional Segment on the Membrane of CHO Xiaofang Lou, Shuang Wang, Weishi Du, Rui Yu, Weiyuan Yu, and Zhiwei Sun Institute of biotechnology , Academy of Military Medical Science , Beijing 100071, China Abstract: Cell adhesive molecular P-selectin was cloned, expressed and anchored on CHO cell membrane through GPI for selection specific antibodies. Total human platelet RNA was extracted and the functional segment of P-selectin gene was cloned by RT-PCR. The P-selectin functional segment gene was cloned into a eukaryotic expression vector pMCEw2-GPI containing an attenuated neo gene together with a downstream GPI, which was synthesized by overlapping PCR. The recombinant plasmid pMCEw2-GPI-P- selectin was then transfected to CHOdhfr− cells and screened with G418. ELISA, western-blot and immunofluorescence were carried out to detect the stability of P-selection expression on cell membrane. These results provided