中国免疫学杂志2016，Vol.32Issue(10)：1507-1511,1518,6.DOI:10.3969/j.issn.1000-484X.2016.10.021
人Cosmc胞外段蛋白在昆虫细胞Sf-9中的表达与纯化研究
Expression and purification of recombinant human Cosmc extracellular domain in Sf-9 insect cells
摘要
Abstract
Objective:In the present study,Bac-to-Bac baculovirus expression system was used to obtain recombinant human Cosmc extracellular domain protein,which can lay the foundation for the research about the structure and function of Cosmc protein in vitro,and simultaneously provide ideas for the research of O-glycosylation and related diseases. Methods: The Cosmc extracellular domain ( Cosmc-ED) gene was cloned into a transfer vector pFastBac1 to form the recombinant donor plasmid pFastBac1-Cosmc ED, which was transformed into competent cells DH10Bac. By using blue-white selection and PCR analysis,we could obtain recombinant shuttle vector rBacmid-Cosmc ED. Then, the recombinant gene DNA of rBacmid-Cosmc ED was used to transfect Sf-9 mediated by cationic lipid formulation,and the recombinant baculovirus bacmid was obtained,which was further used to infect the serum-free cell Sf-9 to express Cosmc-ED in the supernatant. Then the protein of interest was detected by SDS-PAGE and Western blot and purified with Ni-NTA affinity column. Results:SDS-PAGE and Western blot analysis showed a specific band about 33 kD,consistent with the interest protein. Mass spectrometry results further prove that the protein was Cosmc extracellular domain protein. Conclusion: Human Cosmc-ED protein can be successfully expressed in Sf-9 insect cells and laid basis for subsequent studies.关键词
人Cosmc 胞外段/昆虫细胞/杆状病毒表达系统/表达纯化Key words
Human Cosmc extracellular domain/Insect cells/Baculovirus expression system ( BEVS)/Expression and purifi-cation分类
医药卫生引用本文复制引用
谢秋玲,陶悦,张小秦,莫茜,金燕樑..人Cosmc胞外段蛋白在昆虫细胞Sf-9中的表达与纯化研究[J].中国免疫学杂志,2016,32(10):1507-1511,1518,6.基金项目
本文受上海市浦东新区科技发展基金创新资金资助( No. PKJ2012-Y51)。 （ No. PKJ2012-Y51）
