细胞粘附分子P-selectin功能片段在CHO细胞表面的锚定表达  

Eukaryotic Expression of P-selectin Functional Segment on the Membrane of CHO

作　　者：娄晓芳[1] 王双[1] 杜威世[1] 于蕊[1] 俞炜源[1] 孙志伟[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《生物工程学报》2008年第7期1162-1167,共6页Chinese Journal of Biotechnology

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。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 a necessary basis for the following study of selection the antibodies targeting P-selectin.

关 键 词：P—selectin 糖基化磷脂酰肌醇 粘附分子 

分 类 号：Q2[生物学—细胞生物学]