摘要
目的建立稳定表达糖基化磷脂酰肌醇锚定型人B71(GPIB71)的CHO/DHFR-细胞表达体系。方法用Lipofectin介导法将真核细胞高效表达载体pCDNA3GPIB71和pSV40DHFR共转染CHO/DHFR-细胞,经免疫荧光流式细胞术检测,并提取膜蛋白进行Westenblot检测。结果经G418筛选和MTX加压扩增,获得高效表达目的蛋白的细胞株。膜蛋白经WesternBlot检测具有生物活性。结论GPIB71在CHO/DHFR-细胞中高效表达,为临床应用与研究奠定了基础。
Objective To establish the stable CHO/DHFR^- expression system for the glycosylphosphatidylinositol （GPI） anchored B7-1 （GPI-B7-1）. Methods The high-efficiency eukaryotic expression vectors pCDNA3-GPI-B7-1 and pSV40-DHFR were cotransfected into CHO/DHFR^- cells by Lipofectin method, and then identified by direct immunofluorescence （flow-cytometry） assay. The fusion protein isolated from the surface of CHO/DHFR^- cells was identified by Western blotting and incubated with tumor cells membranes. Results The cell clones expressing the interest protein were obtained by using G418 selection and MTX induction. The biological activities of the expressed protein were determined by Western blotting, Conclusion The GPI-B7-1 gene is expressed efficiently in CHO/DHFR- cells, which lays a foundation for the further research on its clinical application.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2005年第6期532-534,539,共4页
Immunological Journal
基金
卫生部默沙东科研基金资助项目(003111711)
